U.S. patent application number 16/421793 was filed with the patent office on 2020-05-21 for circulating mirnas as markers for breast cancer.
This patent application is currently assigned to Ruprecht-Karls-UIniversitat Heidelberg. The applicant listed for this patent is Ruprecht-Karls-Universitat Heidelberg. Invention is credited to Barbara BURWINKEL, Katarina Cuk, Dharanija Madhavan, Manuela Zucknick.
Application Number | 20200157631 16/421793 |
Document ID | / |
Family ID | 48669978 |
Filed Date | 2020-05-21 |
View All Diagrams
United States Patent
Application |
20200157631 |
Kind Code |
A1 |
BURWINKEL; Barbara ; et
al. |
May 21, 2020 |
CIRCULATING miRNAs AS MARKERS FOR BREAST CANCER
Abstract
The present invention is concerned with a method for diagnosing
breast cancer in a subject comprising the steps of determining in a
sample of a subject suspected to be afflicted with said breast
cancer the amount of at least one miRNA or the amounts of at least
the miRNAs of a combination of miRNAs selected from the group
consisting of: (i) miR-801, (ii) miR-801 and miR-148b, (iii)
miR-801 and miR-376c, (iv) miR-801 and miR-409-3p, (v) miR-801,
miR-376c and miR-148b, (vi) miR-801, miR-409-3p and miR-376c, (vii)
miR-801, miR-409-3p and miR-148b, miR-801, miR-376c, miR-409-3p and
miR-148b, (ix) miR-148b, (x) miR-409-3p, (xi) miR-376c, (xii)
miR-376c and miR-409-3p, (xiii) miR-148b and miR-376c, (xiv)
miR-148b and miR-409-3p, (xv) miR-148b, miR-376c and miR-409-3p,
(xvi) miR-127-3p, (xvii) miR-148b, (xvii) miR-376a, (xix) miR-376c,
(xx) miR-409-3p, (xxi) miR-652, (xxii) miR-127-3p, miR-148b,
miR-376a, miR-376c, miR-409-3p, miR-652, and miR-801, (xxiii)
miR-127-3p, miR-148b, miR-652, and miR-801, (xxiv) miR-376a,
miR-148b, miR-652, and miR-801, (xxv) miR-376c, miR-148b, miR-652,
and miR-801, and (xxvi) miR-409-3p, miR-148b, miR-652, and miR-801
and comparing said amount with a reference or comparing said
amounts with references, whereby breast cancer is to be diagnosed.
The present invention is also concerned with methods for diagnosing
metastasizing breast cancer in a subject and for determining the
circulating tumor cell (CTC) status in a subject comprising the
steps of (a) determining in a sample of a subject suspected to be
afflicted with said metastasizing breast cancer the amount of at
least one miRNA selected from the group consisting of: miR-801,
miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-375,
miR-142-3p, and miR-768-3p, and (b) comparing said amount with a
reference or comparing said amounts with references. Furthermore
the present invention is concerned with the use of the miRNAs of
the invention for diagnosing breast cancer, metastasizing breast
cancer, or for determining the CTC status in a subject. Moreover,
the present invention is concerned with devices and kits for
carrying methods of the invention.
Inventors: |
BURWINKEL; Barbara;
(Heidelberg, DE) ; Cuk; Katarina; (Heidelberg,
DE) ; Zucknick; Manuela; (Oslo, NO) ;
Madhavan; Dharanija; (Heidelberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ruprecht-Karls-Universitat Heidelberg |
Heidelberg |
|
DE |
|
|
Assignee: |
Ruprecht-Karls-UIniversitat
Heidelberg
Heidelberg
DE
|
Family ID: |
48669978 |
Appl. No.: |
16/421793 |
Filed: |
May 24, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14409953 |
Dec 19, 2014 |
10316367 |
|
|
PCT/EP2013/062994 |
Jun 21, 2013 |
|
|
|
16421793 |
|
|
|
|
61662816 |
Jun 21, 2012 |
|
|
|
61813029 |
Apr 17, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/178 20130101;
C12Q 1/6886 20130101; C12Q 2600/16 20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886 |
Claims
1. A method for diagnosing breast cancer in a subject comprising
the steps of: (a) determining in a sample of a subject suspected to
be afflicted with said breast cancer the amount of at least one
miRNA or the amounts of at least the miRNAs of a combination of
miRNAs selected from the group consisting of: (i) miR-801, (ii)
miR-801 and miR-148b, (iii) miR-801 and miR-376c, (iv) miR-801 and
miR-409-3p, (v) miR-801, miR-376c and miR-148b, (vi) miR-801,
miR-409-3p and miR-376c, (vii) miR-801, miR-409-3p and miR-148b,
(viii) miR-801, miR-376c, miR-409-3p and miR-148b, (ix) miR-148b,
(x) miR-409-3p, (xi) miR-376c, (xii) miR-376c and miR-409-3p,
(xiii) miR-148b and miR-376c, (xiv) miR-148b and miR-409-3p, (xv)
miR-148b, miR-376c and miR-409-3p, (xvi) miR-127-3p, (xvii)
miR-148b, (xvii) miR-376a, (xix) miR-376c, (xx) miR-409-3p, (xxi)
miR-652, (xxii) miR-127-3p, miR-148b, miR-376a, miR-376c,
miR-409-3p, miR-652, and miR-801, (xxiii) miR-127-3p, miR-148b,
miR-652, and miR-801, (xxiv) miR-376a, miR-148b, miR-652, and
miR-801, (xxv) miR-376c, miR-148b, miR-652, and miR-801, and (xxvi)
miR-409-3p, miR-148b, miR-652, and miR-801; and (b) comparing said
amount with a reference or comparing said amounts with references,
whereby breast cancer is to be diagnosed.
2. The method of claim 1, wherein said sample is a body fluid
sample and wherein an amount determined in step (a) of said at
least one miRNA or amounts of miRNAs of a combination of miRNAs
higher than said reference value is/are indicative of a subject
being afflicted with breast cancer.
3. The method of claim 2, wherein said body fluid is selected from
the group consisting of: blood, serum, plasma, saliva, urine, and
fluids obtainable from the breast glands.
4. The method of claim 1, wherein the sample is a tumor sample and
wherein an amount determined in step (a) of said at least one miRNA
or amounts of miRNAs of a combination of miRNAs lower than said
reference value is/are indicative of a subject being afflicted with
breast cancer.
5. The method of claim 1, wherein said breast cancer is early
breast cancer.
6-10. (canceled)
11. A method for determining the circulating tumor cell (CTC)
status in a subject comprising the steps of: (a) determining in a
sample of a subject suspected to be afflicted with breast cancer
the amount of at least one miRNA selected from the group consisting
of: miR-801, miR-141, miR-142-3p, miR-200a, miR-200b, miR-200c,
miR-203, miR-210, miR-375, and miR-768-3p, and (b) comparing said
amount with a reference or comparing said amounts with references,
whereby the CTC status is to be determined.
12. The method of claim 11, wherein the amount of least one miRNA
selected from the group consisting of: miR-801, miR-203, and
miR-768-3p, or the amounts of at least two miRNAs selected from at
least two different groups of miRNAs are determined, said groups
being selected from: (i) a group consisting of miR-142-3p and
miR-768-3p, (ii) a group consisting of miR-203, (iii) a group
consisting of miR-375, (iv) a group consisting of miR-210 and
miR-801, (v) a group consisting of miR-141, miR-200a, miR-200b,
miR-200c.
13. The method of claim 11, wherein said at least two miRNAs
comprise (i) miR-141, miR-200b, miR-375, and miR-801, (ii) miR-141,
miR-200b, miR-375, miR-801, miR-203, and miR-768-3p, (iii) miR-141,
miR-200c, miR-210, miR-801, and miR-768-3p, (iv) miR-141, miR-200b,
miR-142-3p, and miR-768-3p, (v) miR-141, miR-200b, miR-210,
miR-375, miR-801, miR-142-3p, and miR-768-3p, (vi) miR-141,
miR-200b, miR-200c, miR-210, miR-375, miR-801, miR-142-3p, and
miR-768-3p, or (vi) miR-141, miR-200b, miR-200c, miR-210, miR-375,
miR-203, miR-801, miR-142-3p, and miR-768-3p.
14. The method of claim 11, wherein said combination comprising at
least two miRNAs is selected from the group consisting of (i)
miR-141 and miR-200b, (ii) miR-141, miR-200b, and miR-200c, (iii)
miR-141, miR-200b, miR-210, and miR-200c, (iv) miR-141, miR-200b,
miR-210, miR-768-3p, and miR-200c, (v) miR-141, miR-200b, and
miR-375, (vi) miR-141, miR-200b, miR-210, miR-375, and miR-203, and
(vi) miR200c and miR-210.
15. The method of claim 11, wherein said reference is an amount of
miRNA or amounts of miRNAs of a combination of miRNAs which is/are
derived from a subject or group of subjects with a known CTC
status.
16. A method for recommending a breast cancer therapy to a subject
comprising the method of claim 1, and the further step of
recommending a breast cancer therapy to the subject if breast
cancer has been diagnosed.
17. The method of claim 16, wherein said breast cancer therapy is
selected from the group consisting of: chemotherapy, anti-hormone
therapy, targeted therapy, immunotherapy, surgery, radiation
therapy, and cell based immunotherapy.
18-21. (canceled)
22. A kit for carrying out the method of claim 1, wherein said kit
comprises instructions for carrying out said method, a detection
agent for determining the amount of at least one miRNA selected
from the group consisting of: miR-801, miR-376c, miR-409-3p,
miR-148b, miR-127-3p, miR-376a, and miR-652 in a sample of a
subject suspected to be afflicted with breast cancer, and standards
for a reference.
23-24. (canceled)
25. A kit for carrying out the method of claim 11, wherein said kit
comprises instructions for carrying out said method, a detection
agent for determining the amount of at least one miRNA selected
from the group consisting of: miR-141, miR-200a, miR-200b,
miR-200c, miR-203, miR-210, miR-375, miR-142-3p, miR-768-3p, and
miR-801 in a sample of a subject suspected to be afflicted with
breast cancer, and standards for a reference.
26. The kit of claim 25, wherein the miRNA is selected from the
group consisting of: miR-801, miR-203, miR-142-3p, and
miR-768-3p.
27. A method for diagnosing a benign or malignant breast tumor in a
subject comprising the steps of: (a) determining in a sample of a
subject suspected to be afflicted with breast tumor the amount of
at least one miRNA or the amounts of at least the miRNAs of a
combination of miRNAs selected from the group consisting of: (i)
miR-148b, (ii) miR-652, (iii) miR-801, (iv) miR-148b and miR 652,
(v) miR-148b and miR-801, (vi) miR-652 and miR-801, and (vii)
miR-148b, miR-652, and miR-801; and (b) comparing said amount with
a reference or comparing said amounts with references, whereby a
benign or malignant breast tumor is to be diagnosed.
28. The method of claim 27, wherein said sample is a body fluid
sample and wherein an amount determined in step (a) of said at
least one miRNA or amounts of miRNAs of a combination of miRNAs
higher than said reference value is/are indicative of a subject
being afflicted with a breast tumor.
29. The method of claim 27, wherein said body fluid is selected
from the group consisting of: blood, serum, plasma, saliva, urine,
and fluids obtainable from the breast glands.
30-35. (canceled)
36. A method for recommending a breast cancer therapy to a subject
comprising the method of claim 11, and the further step of
recommending a breast cancer therapy to the subject if breast
cancer has been diagnosed.
37. The method of claim 36, wherein said breast cancer therapy is
selected from the group consisting of: chemotherapy, anti-hormone
therapy, targeted therapy, immunotherapy, surgery, radiation
therapy, and cell based immunotherapy.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 14/409,953, filed Dec. 19, 2014, now U.S. Pat. No.
10,316,367, which is the U.S. National Stage of International
Patent Application No. PCT/EP2013/062994, filed Jun. 21, 2013,
which claims priority from U.S. Provisional Patent Application Nos.
61/813,029, filed Apr. 17, 2013, and 61/662,816, filed Jun. 21,
2012. The contents of these applications are incorporated herein by
reference in their entireties.
[0002] The present invention is concerned with a method for
diagnosing breast cancer in a subject comprising the steps of
determining in a sample of a subject suspected to be afflicted with
said breast cancer the amount of at least one miRNA or the amounts
of at least the miRNAs of a combination of miRNAs selected from the
group consisting of: (i) miR-801, (ii) miR-801 and is miR-148b,
(iii) miR-801 and miR-376c, (iv) miR-801 and miR-409-3p, (v)
miR-801, miR-376c and miR-148b, (vi) miR-801, miR-409-3p and
miR-376c, (vii) miR-801, miR-409-3p and miR-148b, (viii) miR-801,
miR-376c, miR-409-3p and miR-148b, (ix) miR-148b, (x) miR-409-3p,
(xi) miR-376c, (xii) miR-376c and miR-409-3p, (xiii) miR-148b and
miR-376c, (xiv) miR-148b and miR-409-3p, (xv) miR-148b, miR-376c
and miR-409-3p, (xvi) miR-127-3p, (xvii) miR-148b, (xvii) miR-376a,
(xix) miR-376c, (xx) miR-409-3p, (xxi) miR-652, (xxii) miR-127-3p,
miR-148b, miR-376a, miR-376c, miR-409-3p, miR-652, and miR-801,
(xxiii) miR-127-3p, miR-148b, miR-652, and miR-801, (xxiv)
miR-376a, miR-148b, miR-652, and miR-801, (xxv) miR-376c, miR-148b,
miR-652, and miR-801, and (xxvi) miR-409-3p, miR-148b, miR-652, and
miR-801 and comparing said amount with a reference or comparing
said amounts with references, whereby breast cancer is to be
diagnosed. The present invention is also concerned with methods for
diagnosing metastasizing breast cancer in a subject and for
determining the circulating tumor cell (CTC) status in a subject
comprising the steps of (a) determining in a sample of a subject
suspected to be afflicted with said metastasizing breast cancer the
amount of at least one miRNA selected from the group consisting of:
miR-801, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-210,
miR-375, miR-142-3p, and miR-768-3p, and (b) comparing said amount
with a reference or comparing said amounts with references.
Furthermore the present invention is concerned with the use of the
miRNAs of the invention for diagnosing breast cancer, metastasizing
breast cancer, or for determining the CTC status in a subject.
Moreover, the present invention is concerned with devices and kits
for carrying methods of the invention.
[0003] Breast cancer is the most common type of cancer and cause of
death among women in industrialized countries, Worldwide
approximately 1.3 million women develop breast cancer each year.
Mortality rates have continued to decrease over the years due to
all the efforts and advances made in early diagnosis and treatment
(Jemal A, Bray F, Center M M, Ferlay J, Ward E, Forman D. Global
cancer statistics. CA Cancer J Clin 2011; 61:69-90). Nevertheless,
thousands of women die from this disease each year. In US women the
overall five-year survival is 98% when diagnosed at an early stage
as opposed to 23% when the disease has already spread to distant
organs. Thus, early breast cancer detection belongs to one of the
major challenges in the struggle against this disease. Mammographic
screening is currently applied as the diagnostic standard. However,
it has limitations due to its use of ionizing radiation and a false
positive rate of 8-10%, also depending on the age of the
individuals to be screened (Taplin S, Abraham L, Barlow W E, Fenton
J J, Berns E A, Carney P A, Cutter G R, Sickles E A, Carl D, Elmore
J G, Mammography facility characteristics associated with
interpretive accuracy of screening mammography. J Natl Cancer Inst
2008; 100:876-87).
[0004] Protein based circulating tumor markers like
carcinoembryonic antigen (CEA) and carbohydrate antigen 15-3 (CA
15-3) are widely used as prognostic markers, as well as in
monitoring breast cancer treatment success and follow-up (Uehara M,
Kinoshita T, Hojo T, Akashi-Tanaka 5, Iwamoto E, Fukutomi T,
Long-term prognostic study of carcinoembryonic antigen (CEA) and
carbohydrate antigen 15-3 (CA 15-3) in breast cancer. Int J Clin
Oncol 2008; 13:447-51; Harris L, Fritsche H, Mennel R, Norton L,
Ravdin P, Taube S, Somerfield M R, Hayes D F, Bast R C, Jr.
American Society of Clinical Oncology 2007 update of
recommendations for the use of tumor markers in breast cancer, J
Clin Oncol 2007; 25:5287-312) However, the sensitivity of these
markers is low. Therefore, new sensitive and specific as well as
minimally invasive markers are needed.
[0005] Metastatic breast cancer (MBC) is a major health issue,
worldwide. Current treatment strategies target primarily palliative
care with very few cases being cured. An alternate approach of
tackling MBC is development of screening methods and applying
biomarkers to identify high risk groups and therapy response, This
could facilitate decision making for clinicians and help them adopt
the appropriate treatment regime for the patients.
[0006] Circulating tumor cells (CTC) have been proposed as an FDA
approved independent prognostic marker for metastasis, specifically
for progression-free survival and overall survival. A cardinal cut
off of greater than 5 CTCs per 7.5 ml of blood has been defined as
CTC positive (Cristofanilli M, Budd G T, Ellis M J, Stopeck A, et
al; Circulating tumor cells, disease progression, and survival in
metastatic breast cancer; N Engl J Med. 2004 Aug. 19;
351(8):781-91). However, it is important to note that a significant
fraction of patients with overt distant metastases are negative for
CTCs. This could be partly contributed to the phenomenon of
epithelial-mesenchymal transition in CTCs, in which case they can
be missed by enumeration techniques that exploit the expression of
epithelial markers such as EpCAM or cytokeratin-8, -18 and -19.
[0007] miRNAs are small, non-coding RNAs (.about.18-25 nucleotides
in length) that regulate gene expression on a post-transcriptional
level by degrading mRNA molecules or blocking their translation
(Bartel D P.: MicroRNAs: genomics, biogenesis, mechanism, and
function. Cell 2004; 116:281-97). Hence, they play an essential
role in the regulation of a large number of biological processes,
including cancer (Calin G A, Dumitru C D, Shimizu M, Bichi R, Zupo
S, Noch E, Aldler H, Rattan 5, Keating M, Rai K, Rassenti L, Kipps
T, et al. Frequent deletions and down-regulation of micro-RNA genes
miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl
Acad Sci USA 2002; 99:15524-9). Under the standard nomenclature
system, names are assigned to experimentally confirmed miRNAs. The
prefix "mir" is followed by a dash and a number. The uncapitalized
"mir-" refers to the pre-miRNA, while a capitalized "miR-" refers
to the mature form. miRNAs with nearly identical sequences bar one
or two nucleotides are annotated with an additional lower case
letter. Species of origin is designated with a three-letter prefix,
e.g. hsa for Homo sapiens (human). Two mature miRNAs originating
from opposite arms of the same pre-miRNA are denoted with a -3p or
-5p suffix.
[0008] Circulating miRNAs are defined as miRNAs present in the
cell-free component of body fluids like plasma, serum, and the
like. Lawrie et al. (Lawrie C H, Gal S, Dunlop H M, Pushkaran B,
Liggins A P, Pulford K, Banham A H, Pezzella F, Boultwood J,
Wainscoat J S, Hatton C S, Harris A L. Detection of elevated levels
of tumour-associated microRNAs in serum of patients with diffuse
large B-cell lymphoma. Br J Haematol 2008; 141:672-5) were among
the first to demonstrate the presence of miRNAs in bodily fluids.
Since then, circulating miRNAs have been reported as aberrantly
expressed in blood plasma or serum in different types of cancer,
e.g. prostate, colorectal or esophageal carcinoma (Brase J C,
Johannes M, Schlomm T, Faith M, Haese A, Steuber T, Beissbarth T,
Kuner R, Sultmann H. Circulating miRNAs are correlated with tumor
progression in prostate cancer, Int J Cancer 2011; 128:608-16;
Huang Z, Huang D, Ni S, Peng Z, Sheng W, Du X. Plasma microRNAs are
promising novel markers for early detection of colorectal cancer.
Int J Cancer 2010; 127:118-26; Zhang C, Wang C, Chen X, Yang C, Li
K, Wang J, Dai J, Hu Z, Zhou X, Chen L, Zhang Y, Li Y, et al.
Expression profile of microRNAs in serum: a fingerprint for
esophageal squamous cell carcinoma. Clin Chem 2010; 56:1871-9.).
Their most important advantages include the possibility to be
measured repeatedly in a minimally invasive manner as well as their
remarkable stability in plasma/serum, where they circulate mostly
outside of exosomes and are stable due to their binding to
Argonaute proteins (Mitchell P S, Parkin R K, Kroh E M, Fritz B R,
Wyman S K, Pogosova-Agadjanyan E L, Peterson A, Noteboom J,
O'Briant K C, Allen A, Lin D W, Urban N, et al. Circulating
microRNAs as stable blood-based markers for cancer detection. Proc
Natl Acad Sci U S A 2008; 105:10513-8; Turchinovich A, Weiz L,
Langheinz A, Burwinkel B. Characterization of extracellular
circulating microRNA. Nucleic Acids Res 2011; 39:7223-33; Arroyo J
D, Chevillet J R, Kroh E M, Ruf I K, Pritchard C C, Gibson D F,
Mitchell P S, Bennett C F, Pogosova-Agadjanyan E L, Stirewalt D L,
Tait J F, Tewari M. Argonaute2 complexes carry a population of
circulating microRNAs independent of vesicles in human plasma. Proc
Natl Acad Sci USA 2011; 108:5003-8).
[0009] There is thus an urgent need in the art for improved methods
for the detection of breast cancer and metastasizing breast cancer.
Moreover, there is a need for a reliable method for determining the
CTC status of patient. Since the methods would preferably be also
used in preventive screening of apparently healthy subjects, a low
grade of invasiveness would be preferred.
[0010] Therefore, the present invention relates to a method for
diagnosing breast cancer in a subject comprising the steps of: (a)
determining in a sample of a subject suspected to be afflicted with
said breast cancer the amount of at least one miRNA or the amounts
of at least the miRNAs of a combination of miRNAs selected from the
group consisting of: (i) miR-801, which appears to be a fragment of
RNU11/U11 small nuclear RNA, (miRBase (Griffiths-Jones S. NAR 2004
32(Database Issue):D109-D111; Kozomara A, Griffiths-Jones S. NAR
2011 39(Database Issue):D152-D157) (ii) miR-801 and miR-148b, (iii)
miR-801 and miR-376c, (iv) miR-801 and miR-409-3p, (v) miR-801,
miR-376c and miR-148b, (vi) miR-801, miR-409-3p and miR-376c, (vii)
miR-801, miR-409-3p and miR-148b, (viii) miR-801, miR-376c,
miR-409-3p and miR-148b, (ix) miR-148b, (x) miR-409-3p, (xi)
miR-376c, (xii) miR-376c and miR-409-3p, (xiii) miR-148b and
miR-376c, (xiv) miR-148b and miR-409-3p, (xv) miR-148b, miR-376c
and miR-409-3p, (xvi) miR-127-3p, (xvii) miR-148b, (xvii) miR-376a,
(xix) miR-376c, (xx) miR-409-3p, (xxi) miR-652, (xxii) miR-127-3p,
miR-148b, miR-376a, miR-376c, miR-409-3p, miR-652, and miR-801,
(xxiii) miR-127-3p, miR-148b, miR-652, and miR-801, (xxiv)
miR-376a, miR-148b, miR-652, and miR-801, (xxv) miR-376c, miR-148b,
miR-652, and miR-801, and (xxvi) miR-409-3p, miR-148b, miR-652, and
miR-801; and (b) comparing said amount with a reference or
comparing said amounts with references, whereby breast cancer is to
be diagnosed.
[0011] The method for diagnosing breast cancer, preferably, is an
in vitro method. Moreover, it may comprise steps in addition to
those explicitly mentioned above. For example, further steps may
relate, e.g., to isolating miRNAs from a sample in step a), to the
additional determination of other markers, or to the use of an
automatic device in step a) and/or in step b),
[0012] The term "diagnosing" as used herein refers to assessing the
probability according to which a subject is afflicted or will be
afflicted with a disease or condition referred to in this
specification. As will be understood by those skilled in the art,
such an assessment is usually not intended to be correct for 100%
of the subjects to be diagnosed. The term, however, requires that a
statistically significant portion of subjects can be correctly
diagnosed to be afflicted with the disease or condition. Whether a
portion is statistically significant can be determined without
further ado by the person skilled in the art using various well
known statistic evaluation tools, e.g., determination of confidence
intervals, and p-value determination, e.g. via binomial tests.
Details are found in Dowdy and Wearden, Statistics for Research,
John Wiley & Sons, New York 1983. Preferred confidence
intervals are at least 90%, at least 95%, at least 97%, at least
98% or at least 99%, The significance levels of statistical tests
are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the
probability envisaged by the present invention allows that the
diagnosis will be correct for at least 60%, at least 70%, at least
80%, or at least 90% of the subjects of a given cohort or
population. Preferably, the diagnostic method has a sufficiently
large sensitivity and specificity as described below. Preferably,
the sensitivity envisaged by the present invention allows that the
diagnosis of cases will be correct for at least 75%, at least 80%,
at least 85%, at least 90%, or at least 95% of the afflicted
subjects of a given cohort or population. Also, preferably, the
specificity envisaged by the present invention allows that the
diagnosis will be correct for at least 25%, at least 50%, at least
75%, at least 80%, at least 85%, at least 90%, or at least 95% of
the unafflicted subjects of a given cohort or population.
[0013] The term "breast cancer" (BC) as used herein relates to an
abnormal hyperproliferation of breast tissue cells in a subject.
Preferably, the breast cancer is a primary breast cancer, more
preferably with a tumor size classification in situ (IS) or pT3,
more preferably with a tumor size classification of pT1 or pT2.
[0014] The term "subject" as referred to herein encompasses
animals, preferably mammals, and, more preferably, humans. More
preferably, said subject was in the past afflicted with, is at
present afflicted with, is suspected to be afflicted with, or is at
risk to be afflicted with breast cancer. Subjects that are
afflicted with the said disease can be identified by the
accompanying symptoms known for the disease, These symptoms are
known in the art and described, e.g., in Breast Cancer Facts &
Figures 2011-2012. issued by the American Cancer Society, Inc.,
Atlanta. However, a subject suspected to be afflicted with the
aforementioned disease may also be an apparently healthy subject,
e.g., investigated by routine clinical screening, or may be a
subject being at risk for developing the aforementioned disease.
Risk groups (e.g. individuals with a genetic predisposition to
develop breast cancer) for the disease are known in the art and
described in, e.g., Dumitrescu R G, Cotarla I: Understanding breast
cancer risk--where do we stand in 2005? Journal of Cellular and
Molecular Medicine (2005); 9(1):208-221: Bradbury A R, Olopade O I:
Genetic susceptibility to breast cancer. Reviews in Endocrine and
Metabolic Disorders (2007); 8(3):255-267. Preferably, the subject
is female. More preferably, the subject is a woman at most 50 years
old.
[0015] The term "sample", as used herein, refers to a sample of a
body fluid, to a sample of separated cells or to a sample from a
tissue or an organ or to a sample of wash/rinse fluid obtained from
an outer or inner body surface. Samples can be obtained by
well-known techniques and include, preferably, scrapes, swabs or
biopsies from the digestive tract, liver, pancreas, anal canal, the
oral cavity, the upper aerodigestive tract and the epidermis. Such
samples can be obtained by use of brushes, (cotton) swabs, spatula,
rinse/wash fluids, punch biopsy devices, puncture of cavities with
needles or surgical instrumentation. More preferably, samples are
samples of body fluids, e.g., preferably, blood, plasma, serum,
urine, saliva, lacrimal fluid, and fluids obtainable from the
breast glands, e.g. milk. More preferably, the samples of body
fluids are free of cells of the subject. Tissue or organ samples
may be obtained from any tissue or organ by, e.g., biopsy or other
surgical procedures, Separated cells may be obtained from the body
fluids or the tissues or organs by separating techniques such as
filtration, centrifugation or cell sorting. Preferably, cell,
tissue or organ samples are obtained from those body fluids, cells,
tissues or organs which are known or suspected to contain the
miRNAs of the present invention. More preferably, samples are
obtained from those body fluids, cells, tissues or organs described
herein below to contain the miRNAs of the present invention.
Preferably, the sample is a blood sample, more preferably a plasma
sample, most preferably a plasma sample processed as described
herein below. Preferably, in case the sample is a tumor sample, the
miRNA is not miR-801.
[0016] The term "miRNA" or "microRNA" is understood by the skilled
artisan and relates to a short ribonucleic acid (RNA) molecule
found in eukaryotic cells and in body fluids of metazoan organisms.
It is to be understood that the present invention preferably also
encompasses pri-miRNAs, and the pre-miRNAs of the miRNAs of the
present invention. Thus preferably, a miRNA-precursor consists of
25 to several thousand nucleotides, more preferably 40 to 130
nucleotides, even more preferably 50 to 120 nucleotides, or, most
preferably 60 to 110 nucleotides. Preferably, a miRNA consists of 5
to 100 nucleotides, more preferably 10 to 50 nucleotides, even more
preferably 12 to 40 nucleotides, or, most preferably 18 to 26
nucleotides. Preferably, the miRNAs of the present invention are
miRNAs of human origin, i.e. they are encoded in the human genome.
Also preferably, the term miRNA relates o the "guide" strand which
eventually enters the RNA-induced silencing complex (RISC) as well
as to the "passenger" strand complementary thereto. Preferably, the
miRNA or miRNAs used in the method for diagnosing breast cancer
is/are selected from the list consisting of miR-801 (SEQ ID NO: 1,
miRBase (Griffiths-Jones S., NAR 2004 32(Database Issue):D109-D111;
Kozomara A, Griffiths-Jones S., NAR 2011 39 (Database
Issue):D152-D157) ID MI0005202: 5'-GAUUGCUCUGCGUGCGGAAUCGAC-3'),
miR-148b (SEQ ID NO: 2, miRBase ID MI0000811, more preferably
MIMAT0000759: 5'-UCAGUGCAUCACAGAACUUUGU-3'; new ID in miRBase
release 18; hsa-miR-148b-3p), miR-376c, preferably miR-376c-3p (SEQ
ID NO; 3, miRBase ID MI0000776, more preferably MIMAT0000720:
5'-AACAUAGAGGAAAUUCCACGU-3'; formerly known as hsa-miR-368),
miR-409-3p (SEQ ID NO: 4, miRBase ID MI0001735, more preferably
MIMAT0001639: 5'-GAAUGUUGCUCGGUGAACCCCU-3'), miR-203 (SEQ ID NO: 5,
miRBase ID MI0000283, more preferably MIMAT0000264::
5'-GUGAAAUGUUUAGGACCACUAG-3'), miR-768-3p (SEQ ID NO: 6, miRBase ID
MI0005117: 5'-UCACAAUGCUGACACUCAAACUGCUGAC-3'), miR-142-3p (SEQ ID
NO: 7, miRBase ID MI0000458, more preferably MIMAT0000434:
5'-UGUAGUGUUUCCUACUUUAUGGA-3'), miR-141 (SEQ ID NO: 8, miRBase ID
MI0000457, more preferably MIMAT0000432:
5'-UAACACUGUCUGGUAAAGAUGG-3''; new ID in miRBase release 18:
hsa-miR-141-3p), miR-200b (SEQ ID NO: 9, miRBase ID MI0000342, more
preferably MIMAT0000318: 5'-UAAUACUGCCUGGUAAUGAUGA-3'; new ID in
miRBase release 18; hsa-miR-200b-3p), miR-200c (SEQ ID NO: 10,
miRBase ID MI0000650, more preferably MIMAT0000617;
5'-UAAUACUGCCGGGUAAUGAUGGA-3'; new ID in miRBase release 18:
hsa-miR-200c-3p), miR-210 (SEQ ID NO: 11, miRBase ID MI0000286,
more preferably MIMAT0000267: 5'-CUGUGCGUGUGACAGCGGCUGA-3'),
miR-375 (SEQ ID NO; 12, miRBase ID MI0000783, more preferably
MIMAT0000728: 5'-UUUGUUCGUUCGGCUCGCGUGA-3'), miR-200a (SEQ ID NO:
13, miRBase ID MI0000737, more preferably MIMAT0000682;
5'-UAACACUGUCUGGUAACGAUGU-3'; new ID in miRBase release 18:
hsa-miR-200a-3p), miR-127-3p (SEQ ID NO: 14, miRBase ID MI0000472,
more preferably MIMAT0000446: 5'-UCGGAUCCGUCUGAGCUUGGCU-3'),
miR-376a (miRBase ID MI0000784, more preferably SEQ ID NO: 15,
miRBase ID MIMAT0000729: 5'- AUCAUAGAGGAAAAUCCACGU-3'; new ID in
miRBase release 19: hsa-miR-376a-3p), miR-652 (miRBase ID
MI0003667, more preferably SEQ ID NO: 16, miRBase ID MIMAT0003322:
5'-AAUGGCGCCACUAGGGUUGUG-3''; new ID in miRBase release 19:
hsa-miR-652-3p), hsa-miR-18a (miRBase ID MI0000072, more preferably
SEQ ID NO: 17, miRBase ID MIMAT0000072:
5'-UAAGGUGCAUAGUGCAGAUAG-3'), hsa-miR-34a* (also known as
hsa-miR-34a-3p; miRBase ID MI0000268, more preferably SEQ ID NO;
18, miRBase ID MIMAT0004557; 5'-CAAUCAGCAAGUAUACUGCCCU-3'),
hsa-miR-93* (also known as hsa-miR-93-3p; miRBase ID MI0000095,
more preferably SEQ ID NO: 19, miRBase ID MIMAT0004509:
5'-ACUGCUGAGCUAGCACUUCCCG-3'), hsa-miR-138-1* (also known as
hsa-miR-138-1-3p; miRBase ID MI0000476, more preferably SEQ ID NO:
20, miRBase ID MIMAT0004607. 5'-GCUACUUCACAACACCAGGGCC-3'),
hsa-miR-145 (also known as hsa-miR-145-5p; miRBase ID MI0000461,
more preferably SEQ ID NO: 21, miRBase ID MIMAT0000437:
GUCCAGUUUUCCCAGGAAUCCCU-a), hsa-miR-190b (miRBase ID MI0005545,
more preferably SEQ ID NO: 22, miRBase ID MIMAT0004929;
5'-UGAUAUGUUUGAUAUUGGGUU-3'), hsa-miR-320 (also known as
hsa-miR-320a; miRBase ID MI0000542, more preferably SEQ ID NO: 23,
miRBase ID MIMAT0000510: 5'-AAAAGCUGGGUUGAGAGGGCGA-3'), hsa-miR-328
(miRBase ID MI0000804, more preferably SEQ ID NO: 24, miRBase ID
MIMAT0000752: 5'-CUGGCCCUCUCUGCCCUUCCGU-3'), hsa-miR-339-3p
(miRBase ID MI0000815, more preferably SEQ ID NO: 25, miRBase ID
MIMAT0004702: 5'-UGAGCGCCUCGACGACAGAGCCG-3'), hsa-miR-485-3p
(miRBase ID MI0002469, more preferably SEQ ID NO: 26, miRBase ID
MIMAT0002176: 5'-GUCAUACACGGCUCUCCUCUCU-3'), hsa-miR-579 (miRBase
ID MI0003586, more preferably SEQ ID NO: 27, miRBase ID
MIMAT0003244: 5'-UUCAUUUGGUAUAAACCGCGAUU-3'), and hsa-miR-875-5p
(miRBase ID MI0005541, more preferably SEQ ID NO: 28, miRBase ID
MIMAT0004922: 5'-UAUACCUCAGUUUUAUCAGGUG-3').
[0017] The term "combination of miRNAs" relates to combinations of
the miRNAs of the present invention. It is to be understood that a
specific combination of miRNAs may be used for diagnosing breast
cancer (BC) or for diagnosing metastasizing breast cancer (MBC), or
both. Preferred combinations for diagnosing BC are
miR-801+miR-148b, miR-801+miR-376c, miR-801+miR-409-3p,
miR-801+miR-376c+miR-148b, miR-801+miR-409-3p+miR-376c,
miR-801+miR-409-3p+miR-148b, miR-801+miR-376c+miR-409-3p+miR-148b,
miR-376c+miR-409-3p, miR-148b+miR-376c, miR-148b+miR-409-3p, and
miR-148b+miR-376c+miR-409-3p. Most preferred combinations for
diagnosing BC are
miR-127-3p+miR-148b+miR-376a+miR-376c+miR-409-3p+miR-652+miR-801,
miR-127-3p+miR-148b+miR-652+miR-801,
miR-376a+miR-148b+miR-652+miR-801,
miR-376c+miR-148b+miR-652+miR-801, and miR-409-3p+miR-148b,
miR-652+miR-801.
[0018] The amount of a miRNA can be determined in a sample of a
subject by techniques well known in the art. Depending on the
nature of the sample, the amount may be determined by PCR based
techniques for quantifying the amount of a polynucleotide or by
other methods like mass spectrometry or (next generation)
sequencing or one of the methods described in the examples (Cissell
K A, Deo S K, Trends in microRNA detection. Anal Bioanal Chem.
2009; 394(4)1109-1116 or de Planell-Saguer M, Rodicio M C.
Analytical aspects of microRNA in diagnostics: a review. Anal Chim
Acta 2011 Aug. 12; 699(2):134-52).
[0019] The term "determining the amounts of at least the miRNAs of
a combination of miRNAs", as used herein, preferably relates to
determining the amount of each of the miRNAs of the combination
separately in order to be able to compare the amount of each miRNA
of the combination to a reference specific for said miRNA.
[0020] "Comparing" as used herein encompasses comparing the amount
of the miRNA referred to herein which is comprised by the sample to
be analyzed with an amount of the said miRNA in a suitable
reference sample as specified elsewhere herein in this description.
It is to be understood that comparing as used herein refers to a
comparison of corresponding parameters or values, e.g., an absolute
amount of the miRNA as referred to herein is compared to an
absolute reference amount of said miRNA; a concentration of the
miRNA as referred to herein is compared to a reference
concentration of said miRNA; an intensity signal obtained from the
miRNA as referred to herein in a test sample is compared to the
same type of intensity signal of said miRNA in a reference sample.
The comparison referred to in the methods of the present invention
may be carried out manually or computer assisted. For a computer
assisted comparison, the value of the determined amount may be
compared to values corresponding to suitable references which are
stored in a database by a computer program. The computer program
may further evaluate the result of the comparison by means of an
expert system. Accordingly, the result of the identification
referred to herein may be automatically provided in a suitable
output format.
[0021] The term "reference", "reference value", or "reference
amount" as used herein refers to an amount of miRNA, which allows
assessing if being afflicted with BC or MBC or not being afflicted
with BC or MBC is to be assumed for the subject from which the
sample is derived. A suitable reference value may be determined
from a reference sample to be analyzed together, i.e.
simultaneously or subsequently, with the sample.
[0022] Reference amounts can, in principle, be calculated for a
group or cohort of subjects as specified herein based on the
average or median values for a given miRNA by applying standard
methods of statistics. In particular, accuracy of a test such as a
method aiming to diagnose an event, or not, is best described by
its receiver-operating characteristics (ROC) (see especially Zweig
1993, Clin. Chem. 39:561-577). The ROC graph is a plot of all of
the sensitivity versus specificity pairs resulting from
continuously varying the decision threshold over the entire range
of data observed. The clinical performance of a diagnostic method
depends on its accuracy, i.e. its ability to correctly allocate
subjects to a certain prognosis or diagnosis. The ROC plot
indicates the overlap between the two distributions by plotting the
sensitivity versus 1-specificity for the complete range of
thresholds suitable for making a distinction. On the y-axis is
sensitivity, or the true-positive fraction, which is defined as the
ratio of number of true-positive test results to the sum of number
of true-positive and number of false-negative test results. This
has also been referred to as positivity in the presence of a
disease or condition. It is calculated solely from the affected
subgroup. On the x-axis is the false-positive fraction, or
1-specificity, which is defined as the ratio of number of
false-positive results to the sum of number of true-negative and
number of false-positive results. It is an index of specificity and
is calculated entirely from the unaffected subgroup. Because the
true- and false-positive fractions are calculated entirely
separately, by using the test results from two different subgroups,
the ROC plot is independent of the prevalence of the event in the
cohort. Each point on the ROC plot represents a
sensitivity/-specificity pair corresponding to a particular
decision threshold. A test with perfect discrimination (no overlap
in the two distributions of results) has an ROC plot that passes
through the upper left corner, where the true-positive fraction is
1.0, or 100% (perfect sensitivity), and the false-positive fraction
is 0 (perfect specificity). The theoretical plot for a test with no
discrimination (identical distributions of results for the two
groups) is a 45.degree. diagonal line from the lower left corner to
the upper right corner. Most plots fall in between these two
extremes. If the ROC plot falls completely below the 45.degree.
diagonal, this is easily remedied by reversing the criterion for
"positivity" from "greater than" to "less than" or vice versa.
Qualitatively, the closer the plot is to the upper left corner, the
higher the overall accuracy of the test. Dependent on a desired
confidence interval, a threshold can be derived from the ROC curve
allowing for the diagnosis or prediction for a given event with a
proper balance of sensitivity and specificity, respectively.
Accordingly, the reference to be used for the methods of the
present invention can be generated, preferably, by establishing a
ROC for said cohort as described above and deriving a threshold
amount there from. Dependent on a desired sensitivity and
specificity for a diagnostic method, the ROC plot allows deriving
suitable thresholds. Preferably, the reference amounts lie within
the range of values that represent a sensitivity of at least 75%
and a specificity of at least 45%, or a sensitivity of at least 80%
and a specificity of at least 40%, or a sensitivity of at least 85%
and a specificity of at least 33%, or a sensitivity of at least 90%
and a specificity of at least 25%.
[0023] Preferably, the reference amount as used herein is derived
from samples of subjects obtained before treatment, but for which
it is known if their donors were being afflicted with BC or MBC or
not. This reference amount level may be a discrete figure or may be
a range of figures. Evidently, the reference level or amount may
vary between individual species of miRNA. The measuring system
therefore, preferably, is calibrated with a sample or with a series
of samples comprising known amounts of each specific miRNA. It is
understood by the skilled person that in such case the amount of
miRNA can preferably be expressed as arbitrary units (AU). Thus,
preferably, the amounts of miRNA are determined by comparing the
signal obtained from the sample to signals comprised in a
calibration curve. The reference amount applicable for an
individual subject may vary depending on various physiological
parameters such as age or subpopulation. Thus, a suitable reference
amount may be determined by the methods of the present invention
from a reference sample to be analyzed together, i.e.
simultaneously or subsequently, with the test sample. Moreover, a
threshold amount can be preferably used as a reference amount. A
reference amount may, preferably, be derived from a sample of a
subject or group of subjects being afflicted with BC or MBC which
is/are known to be afflicted with BC or MBC. A reference amount
may, preferably, also be derived from a sample of a subject or
group of subjects known to be not afflicted with BC or MBC. It is
to be understood that the aforementioned amounts may vary due to
statistics and errors of measurement. A deviation, i.e. a decrease
or an increase of the miRNA amounts referred to herein is,
preferably, a statistically significant deviation, i.e. a
statistically significant decrease or a statistically significant
increase.
[0024] In a preferred embodiment of the method for diagnosing
breast cancer, the amount of miRNA and the reference amount are
determined in a sample of a body fluid, preferably blood, plasma,
serum, saliva, or a fluid obtainable from the breast glands, more
preferably plasma processed as detailed herein below and the
reference amounts are, preferably, those which are the average or
mean amounts found in subjects being afflicted with BC for a given
population or cohort of subjects. More preferably, the reference
amounts are reference ranges which represent the 75th, the 80th,
the 85th, the 90th, the 91st, the 92nd, the 93rd, the 94th, the
95th, the 96th, the 97th, the 98th, or the 99th percentile of
amounts found in subjects being afflicted with BC for a given
population or cohort of subjects. Also preferably, the reference
amounts are reference ranges which represent the average or mean
values +/-1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 standard deviations of
amounts found in subjects being afflicted with BC for a given
population or cohort of subjects. In such case, it has been found
that an amount of miRNA equal to or increased relative to the
reference amount or reference range is, preferably, indicative of a
subject being afflicted with BC while a decreased amount of miRNA
is indicative for a subject not being afflicted with BC. As is
detailed herein in the examples, preferably, a decreased amount of
miRNA is indicative of a subject not being afflicted with BC, and
an increased amount of miRNA is indicative of a subject being
afflicted with BC. Meaning, preferably, that a subject with a high
amount of miRNA in a sample specified in this paragraph has a high
probability to be afflicted with BC, and that a subject with a low
amount of miRNA has a low probability to be afflicted with BC.
[0025] In another preferred embodiment of the method for diagnosing
breast cancer, the amount of miRNA and the reference amount are
determined in a sample of a body fluid, preferably blood, plasma,
serum, saliva, or a fluid obtainable from the breast glands, more
preferably plasma processed as detailed herein below and the
reference amounts are, preferably, those which are the average or
mean amounts found in subjects known not to be afflicted with BC,
i.e. control subjects, for a given population or cohort of
subjects. More preferably, the reference amounts are reference
ranges which represent the 75th, the 80th, the 85th, the 90th, the
91st, the 92nd, the 93rd, the 94th, the 95th, the 96th, the 97th,
the 98th, or the 99th percentile of amounts found in subjects known
not to be afflicted with BC, i.e, control subjects, for a given
population or cohort of subjects. Also preferably, the reference
amounts are reference ranges which represent the average or mean
values +/-1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 standard deviations of
amounts found in subjects known not to be afflicted with BC, i.e.
control subjects, for a given population or cohort of subjects. In
such case, it has been found that an amount of miRNA increased
relative to the reference amount or reference range is, preferably,
indicative of a subject being afflicted with BC while an amount
essentially equal to the reference amount or within the reference
range of miRNA is indicative for a subject not being afflicted with
BC. Meaning, preferably, that a subject with a high amount of miRNA
in a sample specified in this paragraph has a high probability to
be afflicted with BC, and that a subject with an amount of miRNA
corresponding to the reference amount or lying within the reference
range has a low probability to be afflicted with BC.
[0026] In a more preferred embodiment of the method for diagnosing
breast cancer, the amount of miRNA and the reference amount are
determined in a sample of a body fluid, preferably blood, plasma,
serum, saliva, or a fluid obtainable from the breast glands, more
preferably plasma processed as detailed herein below and the
reference amounts are, preferably, those which represent the
maximal value of the sum of the method's sensitivity and
specificity levels as specified by the ROC curve which is obtained
for the comparison of a given population of cohort of subjects
being afflicted with BC with a given population of cohort of
subjects not being afflicted with BC, i.e. control subjects.
[0027] In another preferred embodiment of the method for diagnosing
breast cancer, the amount of miRNA and the reference amount are
determined in a sample from a tumor, preferably a breast tumor or a
metastasis thereof, and the reference amounts are, preferably,
those which are the average, mean, or median amounts found in
subjects or samples known not to be afflicted with BC, i.e. control
subjects or tissues, for a given population or cohort of subjects.
More preferably, the reference amounts are reference ranges which
represent the 75.sup.th, the 80.sup.th, the 85.sup.th, the
90.sup.th, the 91.sup.st, the 92.sup.nd, the 93.sup.rd, the
94.sup.th, the 95.sup.th, the 96.sup.th, the 97.sup.th, the
98.sup.th, or the 99.sup.th percentile of amounts found in subjects
known not to be afflicted with BC, i.e. control subjects, for a
given population or cohort of subjects. Also preferably, the
reference amounts are reference ranges which represent the average
or mean values +/-1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 standard
deviations of amounts found in subjects known not to be afflicted
with BC, i.e. control subjects, for a given population or cohort of
subjects. In such case, it has been found that an amount of miRNA
decreased relative to the reference amount or reference range is,
preferably, indicative of a subject being afflicted with BC while
an amount essentially equal to the reference amount or within the
reference range of miRNA is indicative for a subject not being
afflicted with BC. Meaning, preferably, that a subject with a low
amount of miRNA in a sample specified in this paragraph has a high
probability to be afflicted with BC and that a subject with an
amount of miRNA corresponding to the reference amount or lying
within the reference range has a low probability to be afflicted
with BC.
[0028] In another preferred embodiment of the method for diagnosing
breast cancer, the amount of miRNA and the reference amount are
determined in a sample from a tumor, preferably a breast tumor or a
metastasis thereof, and the reference amounts are, preferably,
those which are the average, mean, or median amounts found in
subjects known to be afflicted with BC for a given population or
cohort of subjects. More preferably, the reference amounts are
reference ranges which represent the 75th, the 80th, the 85th, the
90th, the 91st, the 92nd, the 93rd, the 94th, the 95th, the 96th,
the 97th, the 98th, or the 99th percentile of amounts found in
subjects known to be afflicted with BC for a given population or
cohort of subjects. Also preferably, the reference amounts are
reference ranges which represent the average or mean values +/-1,
1.5, 2, 2.5, 3, 3.5, 4, or 4.5 standard deviations of amounts found
in subjects known to be afflicted with BC for a given population or
cohort of subjects. In such case, it has been found that an amount
of miRNA decreased or essentially equal to relative to the
reference amount or reference range is, preferably, indicative of a
subject being afflicted with BC while an amount above the reference
range of miRNA is indicative for a subject being afflicted with
BC.
[0029] In a more preferred embodiment of the method for diagnosing
breast cancer, the amount of miRNA and the reference amount are
determined in a sample from a tumor, preferably a breast tumor or a
metastasis thereof, and the reference amounts are, preferably,
those which represent the maximal value of the sum of the method's
sensitivity and specificity levels as specified by the ROC curve
which is obtained for the comparison of a given population of
cohort of subjects being afflicted with BC prior to the treatment
with a given population of cohort of subjects not being afflicted
with BC, i.e. control subjects.
[0030] The definitions made above apply mutatis mutandis to the
following:
[0031] The present invention also relates to a method for
diagnosing metastasizing breast cancer in a subject comprising the
steps of: (a) determining in a sample of a subject suspected to be
afflicted with said metastasizing breast cancer the amount of at
least one miRNA selected from the group consisting of: miR-141,
miR-142-3p, miR-200a, miR-200b, miR-200c, miR-203, miR-210,
miR-375, miR-768-3p, and miR-801, and (b) comparing said amount
with a reference or comparing said amounts with references, whereby
metastasizing breast cancer is to be diagnosed.
[0032] The present invention further relates to a method for
determining the circulating tumor cell (CTC) status in a subject
comprising the steps of: (a) determining in a sample of a subject
suspected to be afflicted with breast cancer the amount of at least
one miRNA selected from the group consisting of: miR-801, miR-141,
miR-142-3p, miR-200a, miR-200b, miR-200c, miR-203, miR-210,
miR-375, and miR-768-3p, and (b) comparing said amount with a
reference or comparing said amounts with references, whereby the
CTC status is to be determined.
[0033] The method for diagnosing metastasizing breast cancer and
the method for determining the CTC status in a subject, preferably,
are in vitro methods. Moreover, the methods may comprise steps in
addition to those explicitly mentioned above. For example, further
steps may relate, e.g., to isolating miRNAs from a sample in step
a), to the additional determination of other markers, to the use of
an automatic device in step a) and/or in step b), or to the
diagnosis of breast cancer prior to applying the method.
[0034] As used herein, the term "metastatic breast cancer" (MBC)
relates to a breast cancer wherein cancer cells grow as a
metastasis at least one secondary site, i.e. a non-adjacent organ
or part of the body of a subject.
[0035] The term "circulating tumor cell" or "CTC" is understood by
the skilled artisan and relates to a tumor cell detached from the
primary or metastatic tumor and circulating in the bloodstream. It
is to be understood that the number of CTC is a prognostic marker
for disease and therapy outcome in breast cancer, e.g. for overall
survival. The term "CTC status" relates to the presence or absence
of more than a reference amount of CTC in a sample. Preferably, the
reference amount of CTC is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,
7, or 7.5 CTC/7.5 ml blood, 5 CTC/7.5 ml blood being more
preferred, In subjects where a blood sample comprises more than
said reference amount of CTC, the CTC status is unfavorable,
indicating a low probability of successful treatment and a low
progression-free and overall survival probability. Conversely, in
subjects where a blood sample comprises less than said reference
amount of CTC, the CTC status is favorable, indicating a high
probability of successful treatment and a high progression-free and
overall survival probability. Advantageously, it has been found in
the present invention that the amounts of the miRNAs used for
determining the CTC status of a subject as defined herein below are
indicative of the CTC status of a subject. Thus, determining the
CTC status in a subject as used herein relates to determining the
amount or amounts of said miRNA or miRNAs and thus obtaining an
indication of the subject's CTC status. Preferably, the status can
be diagnosed to be "favorable" or "unfavorable".
[0036] Preferably, the miRNA or miRNAs used in the method for
diagnosing metastasizing breast cancer or in the method for
determining the CTC status in a subject is/are selected from the
list consisting of miR-801, miR-141, miR-142-3p, miR-200a,
miR-200b, miR-200c, miR-203, miR-210, miR-375, and miR-768-3p;
miR-801, miR-203, and miR-768-3p being most preferred.
[0037] Preferred combinations of miRNAs for diagnosing MBC or for
determining the CTC status of a subject are at least two miRNAs
selected from at least two different groups of miRNAs, said groups
being selected from: (i) a group consisting of miR-142-3p and
miR-768-3p, (ii) a group consisting of miR-203, (iii) a group
consisting of miR-375, (iv) a group consisting of miR-210 and
miR-801, (v) a group consisting of miR-141, miR-200a, miR-200b,
miR-200c. Preferred combinations for diagnosing MBC are
miR-141+miR-200b+miR-200c+miR-210+miR-768-3p,
miR-141+miR-210+miR-801+miR-142-3p+miR-768-3p,
miR-141+miR-200c+miR-2104-miR-768-3p,
miR-141+miR-200b+miR-210+miR-375+miR-801+miR-142-3p+miR-768-3p,
miR-141+miR-200b+miR-375+miR-801,
miR-141+miR-200b+miR-375+miR-801+miR-203+miR-768-3p,
miR-141+miR-142-3p+miR-200b+miR-200c+miR-210+miR-375+miR-203+miR-801+miR--
768-3p, miR-200c+miR-210+miR-768-3p, or
miR-141+miR-200c+miR-210+miR-801+miR-768-3p. Preferred combinations
for determining the CTC status of a subject are miR-141 miR-200b,
miR-141+miR-200b, miR-142-3p, miR-768-3p,
miR-141+miR-200b+miR-142-3p+miR-768-3p, miR-141+miR-200b+miR-375,
miR-141+miR-200b+miR-375+miR-210 miR-203, or
miR-141+miR-200b+miR-142-3p+miR-768-3p.
[0038] In a preferred embodiment of the method for diagnosing
metastasizing breast cancer, the amount of miRNA and the reference
amount are determined in a sample of a body fluid, preferably
blood, plasma, serum, saliva, or a fluid obtainable from the breast
glands, more preferably plasma processed as detailed herein below
and the reference amounts are, preferably, those which are the
average or mean amounts found in subjects known not to be afflicted
with MBC, i.e. control subjects, for a given population or cohort
of subjects. More preferably, the reference amounts are reference
ranges which represent the 75th, the 80th, the 85th, the 90th, the
91st, the 92nd, the 93rd, the 94th, the 95th, the 96th, the 97th,
the 98th, or the 99th percentile of amounts found in subjects known
not to be afflicted with MBC, i.e. control subjects, for a given
population or cohort of subjects. Also preferably, the reference
amounts are reference ranges which represent the average or mean
values +/-1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 standard deviations of
amounts found in subjects known not to be afflicted with MBC, i.e.
control subjects, for a given population or cohort of subjects. In
such case, it has been found that an amount of miR-203, miR-375,
miR-210, miR-801, miR-141, miR-200a, miR-200b, or miR-200c
increased relative to the reference amount or reference range is,
preferably, indicative of a subject being afflicted with MBC while
an amount essentially equal to the reference amount or within the
reference range is indicative for a subject not being afflicted
with MBC. Meaning, preferably, that a subject with a high amount of
miR-203, miR-375, miR-210, miR-801, miR-141, miR-200a, miR-200b, or
miR-200c in a sample specified in this paragraph has a high
probability to be afflicted with BC, and that a subject with an
amount of miRNA corresponding to the reference amount or lying
within the reference range has a low probability to be afflicted
with BC. Conversely, it has been found that an amount of miR-768-3p
decreased relative to the reference amount or reference range is,
preferably, indicative of a subject being afflicted with MBC while
an amount of miR-768-3p equal to the reference amount or within the
reference range is indicative for a subject not being afflicted
with MBC. Meaning, preferably, that a subject with a low amount of
miR-768-3p in a sample specified in this paragraph has a high
probability to be afflicted with MBC, and that a subject with an
amount of miRNA corresponding to the reference amount or lying
within the reference range has a low probability to be afflicted
with MBC.
[0039] In a preferred embodiment of the method for diagnosing
metastasizing breast cancer, the amount of miRNA and the reference
amount are determined in a sample of a body fluid, preferably
blood, plasma, serum, saliva, or a fluid obtainable from the breast
glands, more preferably plasma processed as detailed herein below
and the reference amounts are, preferably, those which are the
average or mean amounts found in subjects known to be afflicted
with MBC for a given population or cohort of subjects. More
preferably, the reference amounts are reference ranges which
represent the 75th, the 80th, the 85th, the 90th, the 91st, the
92nd, the 93rd, the 94th, the 95th, the 96th, the 97th, the 98th,
or the 99th percentile of amounts found in subjects known to be
afflicted with MBC for a given population or cohort of subjects.
Also preferably, the reference amounts are reference ranges which
represent the average or mean values +/-1, 1.5, 2, 2,5, 3, 3,5, 4,
or 4,5 standard deviations of amounts found in subjects known to be
afflicted with MBC for a given population or cohort of subjects. In
such case, it has been found that an amount of miR-203, miR-375,
miR-210, miR-801, miR-141, miR-200a, miR-200b, or miR-200c
decreased relative to the reference amount or reference range is,
preferably, indicative of a subject being afflicted with MBC while
an amount essentially equal to the reference amount or within the
reference range is indicative for a subject being afflicted with
MBC. Meaning, preferably, that a subject with a high amount of
miR-203, miR-375, miR-210, miR-801, miR-141, miR-200a, miR-200b, or
miR-200c or an amount or lying within the reference range in a
sample specified in this paragraph has a high probability to be
afflicted with MBC, and that a subject with an amount of miRNA
lower than the reference has a low probability to be afflicted with
MBC. Conversely, it has been found that an amount of miR-768-3p
increased or essentially equal relative to the reference amount or
reference range is, preferably, indicative of a subject being
afflicted with MBC while an amount of miR-768-3p lower than the
reference range is indicative for a subject not being afflicted
with MBC. Meaning, preferably, that a subject with a high amount of
miR-768-3p in a sample specified in this paragraph has a high
probability to be afflicted with MBC, and that a subject with a low
amount of miRNA relative to the reference amount has a low
probability to be afflicted with MBC.
[0040] In a more preferred embodiment of the method for diagnosing
metastasizing breast cancer, the amount of miRNA and the reference
amount are determined in a sample of a body fluid, preferably
blood, plasma, serum, saliva, or a fluid obtainable from the breast
glands, more preferably plasma processed as detailed herein below
and the reference amounts are, preferably, those which represent
the maximal value of the sum of the method's sensitivity and
specificity levels as specified by the ROC curve which is obtained
for the comparison of a given population of cohort of subjects
being afflicted with MBC with a given population of cohort of
subjects not being afflicted with MBC. Also preferably, the
reference amounts lie within the range of values that represent a
sensitivity of at least 75% and a specificity of at least 65%, or a
sensitivity of at least 80% and a specificity of at least 55%, or a
sensitivity of at least 85% and a specificity of at least 45%, or a
sensitivity of at least 90% and a specificity of at least 35%.
[0041] In a preferred embodiment of the method for determining the
CTC status in a subject, the amount of miRNA and the reference
amount are determined in a sample of a body fluid, preferably
blood, plasma, serum, saliva, or a fluid obtainable from the breast
glands, more preferably plasma processed as detailed herein below
and the reference amounts are, preferably, those which are the
average or mean amounts found in subjects known to have a favorable
CTC status, for a given population or cohort of subjects. More
preferably, the reference amounts are reference ranges which
represent the 75th, the 80th, the 85th, the 90th, the 91st, the
92nd, the 93rd, the 94th, the 95th, the 96th, the 97th, the 98th,
or the 99th percentile of amounts found in subjects known to have a
favorable CTC status, for a given population or cohort of subjects.
Also preferably, the reference amounts are reference ranges which
represent the average or mean values +/-1, 1.5, 2, 2.5, 3, 3.5, 4,
4.5 standard deviations of amounts found in subjects known to have
a favorable CTC status, for a given population or cohort of
subjects. In such case, it has been found that an amount of
miR-203, miR-375, miR-210, miR-801, miR-141, miR-200a, miR-200b, or
miR-200c increased relative to the reference amount or reference
range is, preferably, indicative of a subject having an unfavorable
CTC status while an amount essentially equal to the reference
amount or within the reference range is indicative for a subject
having a favorable CTC status.
[0042] In another preferred embodiment of the method for
determining the CTC status in a subject, the amount of miRNA and
the reference amount are determined in a sample of a body fluid,
preferably blood, plasma, serum, saliva, or a fluid obtainable from
the breast glands, more preferably plasma processed as detailed
herein below and the reference amounts are, preferably, those which
are the average or mean amounts found in subjects known to have an
unfavorable CTC status, for a given population or cohort of
subjects. More preferably, the reference amounts are reference
ranges which represent the 75th, the 80th, the 85th, the 90th, the
91st, the 92nd, the 93rd, the 94th, the 95th, the 96th, the 97th,
the 98th, or the 99th percentile of amounts found in subjects known
to have an unfavorable CTC status, for a given population or cohort
of subjects. Also preferably, the reference amounts are reference
ranges which represent the average or mean values 1, 1.5, 2, 2.5,
3, 3.5, 4, 4.5 standard deviations of amounts found in subjects
known to have an unfavorable CTC status, for a given population or
cohort of subjects. In such case, it has been found that an amount
of miR-203, miR-375, miR-210, miR-801, miR-141, miR-200a, miR-200b,
or miR-200c essentially equal to the reference amount or within the
reference range is, preferably, indicative of a subject having an
unfavorable CTC status while an amount decreased relative to the
reference range is indicative for a subject having a favorable CTC
status.
[0043] In a more preferred embodiment of the method for determining
the CTC status in a subject, the amount of miRNA and the reference
amount are determined in a sample of a body fluid, preferably
blood, plasma, serum, saliva, or a fluid obtainable from the breast
glands, more preferably plasma processed as detailed herein below
and the reference amounts are, preferably, those which represent
the maximal value of the sum of the method's sensitivity and
specificity levels as specified by the ROC curve which is obtained
for the comparison of a given population of cohort of subjects
known to have an unfavorable CTC status with a given population of
cohort of subjects known to have a favorable CTC status. Also
preferably, the reference amounts lie within the range of values
that represent a sensitivity of at least 75% and a specificity of
at least 75%, or a sensitivity of at least 80% and a specificity of
at least 60%, or a sensitivity of at least 85% and a specificity of
at least 50%, or a sensitivity of at least 90% and a specificity of
at least 45%.
[0044] The present invention also relates to a method for
recommending a breast cancer therapy to a subject comprising first
diagnosing breast cancer in a subject by a method described herein,
followed by the further step of recommending a breast cancer
therapy to the subject if breast cancer has been diagnosed.
[0045] The method for recommending a breast cancer therapy may
comprise steps in addition to those explicitly mentioned above. For
example, further steps may relate, e.g., to isolating miRNAs from a
sample, to the additional determination of other markers, to the
use of an automatic device in the determination steps, or to the
diagnosis of breast cancer prior to applying the method.
[0046] As used herein, the term "therapy" refers to all measures
applied to a subject to ameliorate the diseases or disorders
referred to herein or the symptoms accompanied therewith to a
significant extent. Said therapy as used herein also includes
measures leading to an entire restoration of the health with
respect to the diseases or disorders referred to herein. It is to
be understood that therapy as used in accordance with the present
invention may not be effective in all subjects to be treated.
However, the term shall require that a statistically significant
portion of subjects being afflicted with a disease or disorder
referred to herein can be successfully treated. Whether a portion
is statistically significant can be determined without further ado
by the person skilled in the art using various well known statistic
evaluation tools discussed herein above.
[0047] The term "breast cancer therapy", as used herein, relates to
applying to a subject afflicted with breast cancer, including
metastasizing breast cancer, measures to remove cancer cells from
the subject, to inhibit growth of cancer cells, to kill cancer
cells, or to cause the body of a patient to inhibit the growth of
or to kill cancer cells. Preferably, breast cancer therapy is
chemotherapy, anti-hormone therapy, targeted therapy,
immunotherapy, or any combination thereof. It is, however, also
envisaged that the cancer therapy is radiation therapy or surgery,
alone or combination with other therapy regimens. It is understood
by the skilled person that the selection of the breast cancer
therapy depends on several factors, like age of the subject, tumor
staging, and receptor status of tumor cells. It is, however, also
understood by the person skilled in the art, that the selection of
the breast cancer therapy can be assisted by the methods of the
present invention: if, e.g. BC is diagnosed by the method for
diagnosing BC, but no MBC is diagnosed by the method for diagnosing
MBC, surgical removal of tumor may be sufficient. If, e.g. BC is
diagnosed by the method for diagnosing BC and MBC is diagnosed by
the method for diagnosing MBC, therapy measures in addition to
surgery, e.g. chemotherapy and/or targeted therapy, may be
appropriate. Likewise, if, e.g. BC is diagnosed by the method for
diagnosing BC, and an unfavorable CTC status is determined by the
method for determining the CTC status, e.g. a further addition of
immunotherapy to the therapy regimen may be required.
[0048] As used herein, the term "chemotherapy" relates to treatment
of a subject with an antineoplastic drug. Preferably, chemotherapy
is a treatment including alkylating agents (e.g. cyclophosphamide),
platinum (e.g. carboplatin), anthracyclines (e.g. doxorubicin,
epirubicin, idarubicin, or daunorubicin) and topoisomerase II
inhibitors (e.g. etoposide, irinotecan, topotecan, camptothecin, or
VP16), anaplastic lymphoma kinase (ALK)-inhibitors (e.g. Crizotinib
or AP26130), aurora kinase inhibitors (e.g.
N[4-[4-(4-Methylpiperazin-1-yl)-6-[(5-methyl-1H-pyrazol-3-yl)amino]pyrimi-
din-2-yl]sulfanylphenyl]cyclopropanecarboxamide (VX-680)),
antiangiogenic agents (e.g. Bevacizumab), or Iodine
131-1-(3-iodobenzyl)guanidine (therapeutic
metaiodobenzylguanidine), histone deacetylase (HDAC) inhibitors,
alone or any suitable combination thereof. It is to be understood
that chemotherapy, preferably, relates to a complete cycle of
treatment, i.e, a series of several (e.g. four, six, or eight)
doses of antineoplastic drug or drugs applied to a subject
separated by several days or weeks without such application.
[0049] The term "anti-hormone therapy" relates to breast cancer
therapy by blocking hormone receptors, e.g. estrogen receptor or
progesterone receptor, expressed on tumor cells, or by blocking the
biosynthesis of estrogen. Blocking of hormone receptors can
preferably be achieved by administering compounds, e.g. tamoxifen,
binding specifically and thereby blocking the activity of said
hormone receptors. Blocking of estrogen biosynthesis is preferably
achieved by administration of aromatase inhibitors like, e.g.
anastrozole or letrozole. It is known to the skilled artisan that
anti-hormone therapy is only advisable in cases where tumor cells
are expressing hormone receptors.
[0050] The term "targeted therapy", as used herein, relates to
application to a patient a chemical substance known to block growth
of cancer cells by interfering with specific molecules known to be
necessary for tumorigenesis or cancer or cancer cell growth.
Examples known to the skilled artisan are small molecules like,
e.g. PARP-inhibitors (e.g. Iniparib), or monoclonal antibodies
like, e.g., Trastuzumab.
[0051] The term "immunotherapy" as used herein relates to the
treatment of cancer by modulation of the immune response of a
subject. Said modulation may be inducing, enhancing, or suppressing
said immune response, The term "cell based immunotherapy" relates
to a breast cancer therapy comprising application of immune cells,
e.g. T-cells, preferably tumor-specific NK cells, to a subject.
[0052] The terms "radiation therapy" or "radiotherapy" is known to
the skilled artisan. The term relates to the use of ionizing
radiation to treat or control cancer. The skilled person also knows
the term "surgery", relating to operative measures for treating
breast cancer, e.g. excision of tumor tissue.
[0053] In a preferred embodiment, the miRNAs of the present
invention are used for diagnosing breast cancer, i.e., preferably,
the amount of said miRNAs is determined and the value obtained is
compared to a reference amount as specified herein above. Measuring
the amount of a miRNA is preferably accomplished by, e.g.,
quantitative real-time PCR (qRT-PCR), or mass spectrometry,
[0054] In another preferred embodiment, the amount of miRNAs of the
present invention is determined using a detection agent. As used
herein, the term "detection agent" relates to an agent specifically
interacting with, and thus recognizing, a miRNA of the present
invention. Preferably, said detection agent is a polynucleotide or
an oligonucleotide. Preferably, the detection agent is labeled in a
way allowing detection of said detection agent by appropriate
measures. Labeling can be done by various techniques well known in
the art and depending of the label to be used. Preferred labels to
be used are fluorescent labels comprising, inter alia,
fluorochromes such as fluorescein, rhodamin, or Texas Red. However,
the label may also be an enzyme or an antibody. It is envisaged
that an enzyme to be used as a label will generate a detectable
signal by reacting with a substrate. Suitable enzymes, substrates
and techniques are well known in the art. An oligonucleotide to be
used as label may specifically recognize a target molecule which
can be detected directly (e.g., a target molecule which is itself
fluorescent) or indirectly (e.g., a target molecule which generates
a detectable signal, such as an enzyme). The labeled detection
agents of the sample will be contacted to the sample to allow
specific interaction of the labeled detection agent with the miRNAs
in the sample. Washing may be required to remove nonspecifically
bound detection agent which otherwise would yield false values.
After this interaction step is complete, a researcher will place
the detection device into a reader device or scanner. A device for
detecting fluorescent labels, preferably, consists of some lasers,
preferably a special microscope, and a camera. The fluorescent
labels will be excited by the laser, and the microscope and camera
work together to create a digital image of the sample. These data
may be then stored in a computer, and a special program will be
used, e.g., to subtract out background data. The resulting data
are, preferably, normalized, and may be converted into a numeric
and common unit format. The data will be analyzed to compare
samples to references and to identify significant changes. It is to
be understood that the labeled detection agent need not necessarily
detect the specific miRNA molecule isolated from the sample; the
detection agent may also detect the amplification product obtained
from said miRNA molecule, e.g., preferably, by PCR, qPCR, or
qRT-PCR.
[0055] It is, however, also envisaged that the detection agent is
used without a label. Preferably, the detection agent is bound to a
solid surface and the sample, comprising miRNAs from a sample which
have been labeled are contacted to with said surface-bound
detection agent.
[0056] The present invention further relates to the use of at least
one miRNA selected from the group consisting of: miR-801, miR-376c,
miR-409-3p, miR-148b, miR-203, miR-142-3p, miR-141, miR-200a,
miR-200b, miR-200c, miR-210, miR-375, miR-768-3p, miR-127-3p,
miR-376a, and miR-652 in a sample of a subject suspected to be
afflicted with breast cancer or a detection agent which
specifically detects said at least one miRNA for diagnosing breast
cancer. Preferably, the miRNA is selected from the list consisting
of miR-801, miR-376c, miR-409-3p, miR-148b, miR-203, miR-142-3p,
miR-768-3p, miR-127-3p, miR-376a, and miR-652.
[0057] The present invention also relates to the use of at least
one miRNA selected from the group consisting of: miR-801, miR-376c,
miR-409-3p, miR-148b, miR-203, miR-142-3p, miR-141, miR-200a,
miR-200b, miR-200c, miR-210, miR-375 miR-768-3p, miR-127-3p,
miR-376a, and miR-652 in a sample of a subject suspected to be
afflicted with breast cancer or a detection agent which
specifically detects said at least one miRNA for recommending a
breast cancer therapy. Preferably, the miRNA is selected from the
list consisting of miR-801, miR-376c, miR-409-3p, miR-148b,
miR-203, miR-142-3p, miR-768-3p, miR-127-3p, miR-376a, and
miR-652.
[0058] The present invention further relates to the use of at least
one miRNA selected from the group consisting of: miR-141,
miR-142-3p, miR-200a, miR-200b, miR-200c, miR-203; miR-210;
miR-375, miR-768-3p, and miR-801, in a sample of a subject
suspected or known to be afflicted with breast cancer or a
detection agent which specifically detects said at least one miRNA
for diagnosing metastasizing breast cancer or for determining the
CTC status of a subject. Preferably, the miRNA is selected from the
list consisting of miR-801, miR-376c, miR-409-3p, miR-148b,
miR-203, miR-142-3p, and miR-768-3p. More preferably, the miRNA is
selected from the list consisting of miR-801, miR-203, miR-142-3p,
and miR-768-3p.
[0059] The present invention also relates to a device for
diagnosing breast cancer comprising: (a) an analyzing unit
comprising a detection agent for determining the amount of at least
one miRNA selected from the group consisting of: miR-801, miR-376c,
miR-409-3p, miR-148b, miR-203, miR-142-3p, miR-141, miR-200a,
miR-200b, miR-200c, miR-210, miR-375, miR-768-3p, miR-127-3p,
miR-376a, and miR-652 in a sample of a subject suspected to be
afflicted with breast cancer; and (b) an evaluation unit comprising
a data processor having tangibly embedded an algorithm for carrying
out a comparison of the amount determined by the analyzing unit
with a reference and which is capable of generating an output file
containing a diagnosis established based on the said comparison.
More preferably, the miRNA is selected from the list consisting of
miR-801, miR-376c, miR-409-3p, miR-148b, miR-203, miR-142-3p,
miR-768-3p, miR-127-3p, miR-376a, and miR-652.
[0060] The present invention further relates to a device for
diagnosing metastasizing breast cancer or for determining the CTC
status of a subject comprising: (a) an analyzing unit comprising a
detection agent for determining the amount of at least one miRNA
selected from the group consisting of; miR-141, miR-200a, miR-200b,
miR-200c, miR-203, miR-210, miR-375, miR-142-3p, miR-768-3p, and
miR-801 in a sample of a subject suspected to be afflicted with
breast cancer; and (b) an evaluation unit comprising a data
processor having tangibly embedded an algorithm for carrying out a
comparison of the amount determined by the analyzing unit with a
reference and which is capable of generating an output file
containing a diagnosis established based on the said comparison.
More preferably, the miRNA is selected from the list consisting of
miR-801, miR-203, miR-142-3p, and miR-768-3p.
[0061] The term "device" as used herein relates to a system of
means comprising at least the aforementioned means operatively
linked to each other as to allow the diagnosis. Preferred means for
determining the amount of the miRNAs of the present invention, and
means for carrying out the comparison are disclosed above in
connection with the methods of the invention. How to link the means
in an operating manner will depend on the type of means included
into the device, For example, where means for automatically
determining the amount of the miRNAs of the present invention are
applied, the data obtained by said automatically operating means
can be processed by, e.g., a computer program in order to establish
a diagnosis. Preferably, the means are comprised by a single device
in such a case. Said device may accordingly include an analyzing
unit for the measurement of the amount of the miRNAs of the present
invention in a sample and an evaluation unit for processing the
resulting data for the diagnosis. Preferred means for detection are
disclosed in connection with embodiments relating to the methods of
the invention above. In such a case, the means are operatively
linked in that the user of the system brings together the result of
the determination of the amount and the diagnostic value thereof
due to the instructions and interpretations given in a manual. The
means may appear as separate devices in such an embodiment and are,
preferably, packaged together as a kit. The person skilled in the
art will realize how to link the means without further inventive
skills. Preferred devices are those which can be applied without
the particular knowledge of a specialized clinician, e.g., test
stripes or electronic devices which merely require loading with a
sample. The results may be given as output of parametric diagnostic
raw data, preferably, as absolute or relative amounts. It is to be
understood that these data will need interpretation by the
clinician. However, also envisaged are expert system devices
wherein the output comprises processed diagnostic raw data the
interpretation of which does not require a specialized clinician.
Further preferred devices comprise the analyzing units/devices
(e.g., biosensors, arrays, solid supports coupled to ligands
specifically recognizing the miRNAs of the present invention,
Plasmon surface resonance devices, NMR spectro-meters,
mass-spectrometers etc.) or evaluation units/devices referred to
above in accordance with the methods of the invention.
[0062] The present invention further relates to a kit for carrying
out a method for diagnosing BC, wherein said kit comprises
instructions for carrying out said method, a detection agent for
determining the amount of at least one miRNA selected from the
group consisting of: miR-801, miR-376c, miR-409-3p, miR-148b,
miR-203, miR-142-3p, miR-141, miR200a, miR-200b, miR-200c, miR-210,
miR-375, miR-768-3p, miR-127-3p, miR-376a, and miR-652 in a sample
of a subject suspected to be afflicted with breast cancer, and
standards for a reference. Preferably, the miRNA is selected from
the list consisting of miR-801, miR-376c, miR-409-3p, miR-148b,
miR-203, miR-142-3p, miR-768-3p, miR-127-3p, miR-376a, and
miR-652.
[0063] The present invention also relates to a kit for carrying out
a method for diagnosing MBC or for determining the CTC status in a
subject, wherein said kit comprises instructions for carrying out
said method, a detection agent for determining the amount of at
least one miRNA selected from the group consisting of: miR-141 ,
miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-375,
miR-142-3p, miR-768-3p, and miR-801 in a sample of a subject
suspected to be afflicted with metastatic breast cancer, and
standards for a reference. Preferably, the miRNA is selected from
the list consisting of miR-801, miR-203, miR-142-3p, and
miR-768-3p.
[0064] The term "kit" as used herein refers to a collection of the
aforementioned compounds, means or reagents of the present
invention which may or may not be packaged together. The components
of the kit may be comprised by separate vials (i.e. as a kit of
separate parts) or provided in a single vial. Moreover, it is to be
understood that the kit of the present invention is to be used for
practicing the methods referred to herein above. It is, preferably,
envisaged that all components are provided in a ready-to-use manner
for practicing the methods referred to above. Further, the kit
preferably contains instructions for carrying out the said methods.
The instructions can be provided by a user's manual in paper- or
electronic form. For example, the manual may comprise instructions
for interpreting the results obtained when carrying out the
aforementioned methods using the kit of the present invention.
[0065] Also, the present invention relates to a method for
diagnosing a breast tumor in a subject comprising the steps of: (a)
determining in a sample of a subject suspected to be afflicted with
breast tumor the amount of at least one miRNA or the amounts of at
least the miRNAs of a combination of miRNAs selected from the group
consisting of: (i) miR-148b, (ii) miR-652, (iii) miR-801, (iv)
miR-148b and miR 652, (v) miR-148b and miR-801, (vi) miR-652 and
miR-801, and (vii) miR-148b, miR-652, and miR-801; and (b)
comparing said amount with a reference or comparing said amounts
with references, whereby a breast tumor is to be diagnosed.
[0066] As used herein, the term "breast tumor" relates to an
abnormal hyperproliferation of breast tissue cells in a subject,
which may be a benign (non-cancerous) tumor or a malign (cancerous)
tumor. Benign breast tumors, preferably, include fibroadenomas,
granular cell tumors, intraductal papillomas, and phyllodes tumors.
A malign tumor, preferably, is a breast cancer as specified herein
above.
[0067] The present invention also relates to a method of
determining treatment success in a subject afflicted with
metastatic breast cancer, comprising (a) determining in a sample of
a subject receiving or having received treatment against
metastasizing breast cancer the amount of at least one miRNA
selected from the group consisting of: miR-801, miR-141, miR-200a,
miR-200b, miR-200c, miR-203, miR-210, and miR-375; and (b)
comparing said amount with a reference or comparing said amounts
with references, thereby determining treatment success.
[0068] The term "treatment success", as used herein, preferably
relates to an amelioration of the diseases or disorders referred to
herein or the symptoms accompanied therewith to a significant
extent. More preferably, the term relates to a complete cure of
said subject, i.e. to the prevention of progression and/or relapse
of metastasizing breast cancer for at least five years.
Accordingly, "determining treatment success" relates to assessing
the probability according to which a subject was successfully
treated. Preferably, the term relates to predicting progression
free survival and/or overall survival of the subject, more
preferably for a specific period of time. The term "predicting
progression free survival" relates to determining the probability
of a subject surviving without relapse and/or progression of
metastatic breast cancer for a specific period of time.
Accordingly, the term "predicting overall survival" relates to
determining the probability according to which a subject will
survive for a specific period of time. Preferably, said period of
time is at least 12 months, more preferably at least 24 months,
[0069] It is understood by the skilled person that the reference
used in the method of determining treatment success is a specific
reference, which may be different from the references used in the
other methods of the present invention. Also, the skilled person
knows how to obtain a suitable reference according to the methods
specified herein above. Preferably, the reference is derived from a
sample of the same subject obtained before treatment. More
preferably, the reference is derived from one or more subjects
known to have successfully been treated. Alternatively, the
reference may be derived from one or more subjects known to have
not successfully been treated. Most preferably, the reference
corresponds to the lower tertile, lower quartile, lower fifth, or
lower sixth of values obtained from a cohort of individuals after
having been received treatment against metastasizing breast
cancer.
[0070] Preferably, the method of determining treatment success
according to the present invention comprises determining the
aforesaid miRNA or miRNAs in a sample of a subject receiving
treatment, i.e. in a sample obtained from a subject receiving
treatment at the time the sample is obtained. It is understood by
the skilled person that a subject receiving treatment, preferably,
is a subject having received the first dose of active principle
and/or surgery at least one day, more preferably at least one week,
even more preferably at least two weeks, or, most preferably, at
least one month before the sample is obtained. More preferably, the
method of determining treatment success according to the present
invention comprises determining the aforesaid miRNA or miRNAs in a
sample of a subject having received at least one treatment cycle,
most preferably a complete treatment cycle.
[0071] All references cited in this specification are herewith
incorporated by reference with respect to their entire disclosure
content and the disclosure content specifically mentioned in this
specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIGS. 1A-1D to 6A-6P show data obtained from patient cohort
I, whereas FIGS. 7 to 9A-9C show data obtained from patient cohort
H.
[0073] FIGS. 1A-1D: Circulating miRNAs validated as being
upregulated in the plasma of breast cancer patients compared to
healthy controls. Box and whisker plots of cel-miR-39 normalized Ct
values for miR-148b (A), miR-376c (B), miR-409-3p (C) and miR-801
(D) with their corresponding two-sided Wilcoxon rank sum test
p-values.
[0074] FIGS. 2A-2E: Evaluation of the diagnostic potential of
miR-148b, miR-376c, miR-409-3p and miR-801 in the plasma of breast
cancer patients. (A-D) ROC curves of logistic regression models for
individual miRNAs miR-148b, miR-376c, miR-409-3p and miR-801, (E) A
combined ROC curve of the logistic regression model for the least
redundant and most informative diagnostic panel consisting of
miR-148b, miR-409-3p and miR-801 with their corresponding area
under the curve (AUC) values.
[0075] FIGS. 3A-3D: Expression of miR-148b, miR-376c, miR-409-3p
and miR-801 in benign versus malignant breast tissue. Box and
whisker plots with RNU6B normalized relative expression levels of
miR-148b (A), miR-376c (B), miR-409-3p (C) and miR-801 (D) in
benign and malignant breast tissue with their corresponding
two-sided Wilcoxon rank sum test p-values
[0076] FIGS. 4A-4B: Validation of candidate miRNAs. (A) Box and
whisker plots of the expression of 10 candidate miRNAs, represented
as Ct values, across 61 CTC-positive, 72 CTC-negative MBC cases and
76 controls. (B) Spearman partial correlations between miRNAs based
on their expression with the corresponding correlations values
depicted along the lines (p<0.00001 for all miR pairs that are
connected by lines).
[0077] FIGS. 5A-5H: ROC analysis. (A-E) Leave-one-out
cross-validated ROC curves for logistic regression models based on
individual miRNAs for all three comparisons. (F-H) Leave-one-out
cross-validated ROC curves for multiparametric panel based on
penalised LASSO logistic regression model. (F) CTC-positive vs
CTC-negative: miR-141, miR-200b, miR-142-3p, miR-768-3p (83%
specificity at 80% sensitivity); (G) CTC-positive vs control:
miR-141, miR-200b, miR-200c, miR-210, miR-375, miR-801, miR-142-3p,
miR-768-3p (91% specificity at 90% sensitivity); (H) CTC-negative
vs control: miR-141, miR-200b, miR-200c, miR-210, miR-375, miR-203,
miR-801, miR-142-3p, miR-768-3p (65% specificity at 80%
sensitivity). AUC--Area under the curve
[0078] FIGS. 6A-6P: Kaplan-Meier curves of each miRNAs (miR-141,
miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-375, and
miR-801) for progression free survival (PFS) (A-H) and overall
survival (OS) (I-P). Stratified curves are shown for two groups
lower quartile, and upper three quartiles. Kaplan-Meier curves show
that lower the Ct value, i.e., higher the expression of the miRNA,
lower is the probability of progression- free, and overall
survival.
[0079] FIG. 7: Wilcoxon rank sum tests with continuity correction
confirmed that miR-127-3p (p<0.001), miR-148b (p<0.0001),
miR-376a (p=0.03), miR-376c (p=0.03), miR-409-3p (p=0.005), miR-652
(p<0.0001) and miR-801 (p<0.0001) were significantly
upregulated in breast cancer patients compared to healthy controls
as shown in box and whisker plots with Ct values for these
circulating miRNAs. Box and whisker plots of Ct values for miR-148b
(p=0.02), miR-652 (p=0.01) and miR-801 (p=0.003) demonstrate that
these miRNAs are upregulated in the plasma of benign breast tumor
patients compared to healthy controls as well.
[0080] FIGS. 8A-8I: Evaluation of the diagnostic potential of
miR-127-3p, miR-148b, miR-376a, miR-376c, miR-409-3p, miR-652 and
miR-801 in the plasma of patients with breast tumors, ROC curves of
logistic regression models for individual circulating miRNAs
miR-127-3p, miR-148b, miR-376a, miR-376c, miR-409-3p, miR-652 and
miR-801 show potential to discriminate between healthy controls and
breast tumor patients with area under the curve (AUC) values of up
to 0.75 (A-G). A combined ROC curve of the logistic regression
model for the combination of all seven aforementioned circulating
miRNAs for women of all ages (H) or only those up to 50 years of
age (I), indicate good discriminatory potential with AUC values of
0.81 and 0.86, respectively.
[0081] FIGS. 9A-9C: Expression of miR-127-3p, miR-376a and miR-652
in benign versus malignant breast tissue. Box and whisker plots
with RNU6B normalized relative expression levels of miR-127-3p (A)
and miR-652 (B) in benign and malignant breast tissue with the
corresponding two-sided Wilcoxon rank sum test p-values for A-C.
Due to low miR-376a levels in the investigated tissue samples the
normalization strategy was not applicable and un-normalized Ct
values are presented for miR-376a (C).
[0082] FIG. 10: Box and whisker plots showing a significant
decrease in the levels of the eight miRNAs measured in metastatic
breast cancer patients before and after one round of therapy.
[0083] FIGS. 11A-11I: Kaplan-Meier curves of miRNAs indicated for
progression free survival (PFS) in metastatic breast cancer
patients after therapy. Kaplan-Meier curves of miRNA amounts
stratified based on the Ct values as lower quartile (or 25
percentile) and rest. Kaplan-Meier curves show that the lower the
Ct value, i.e., the higher the expression of the miRNA, the lower
is the probability of progression-free, and overall survival,
[0084] FIGS. 12A-12I: Kaplan-Meier curves of miRNAs indicated for
overall survival (OS) in metastatic breast cancer patients after
therapy. Kaplan-Meier curves of miRNA amounts stratified based on
the Ct values as lower quartile (or 25 percentile) and rest.
Kaplan-Meier curves show that the lower the Ct value, i.e., the
higher the expression of the miRNA, the lower is the probability of
progression-free, and overall survival.
[0085] The following Examples shall merely illustrate the
invention. They shall not be construed, whatsoever, to limit the
scope of the invention.
EXAMPLES
I. Cohort I
Example 1: Breast Cancer Patients and Healthy Controls
[0086] This study was approved by the Ethical Committee of the
Medical Faculty in Heidelberg. For cohort I, blood samples were
collected from 133 female metastatic breast cancer patients, 127
female primary breast cancer patients and 80 healthy female
volunteers who served as controls. All cases and controls were
Caucasian. In metastatic breast cancer patients, circulating tumour
cells (CTC) were enumerated by evaluating it with the
CellSearch.RTM. system (Veridex, LLC, Raritan, N.J.). Based on the
CTC numbers, patients were classified as CTC-positive (.gtoreq.5
intact CTCs/7.5 ml blood) or CTC-negative (no intact, apoptotic or
enucleated CTCs). Patients had received one or more lines of
therapy for their metastatic disease prior to enrolment into the
study. For primary breast cancer patients, blood samples were
collected at the time-point of diagnosis before they underwent any
therapeutic procedures, such as surgery, radiation or systemic
therapy. Patient histopathology results were confirmed by surgical
resection of the tumors and clinico-pathological features defined
by operative findings. For neoadjuvant primary breast cancer
patients, histopathological characteristics and tumor stage were
assessed based on histobiopsy results and imaging techniques.
Control blood samples were collected from healthy women with no
history of malignant diseases, no blood donations received in the
previous 3 years and no reported current inflammatory condition
based on self-report. Malignant tissue samples were collected from
non-neoadjuvant primary breast cancer patients (n=24) during
surgery, They were snap-frozen in liquid nitrogen and stored at
-80.degree. C. within 15 min of harvesting. For women with benign
findings (n=8) tissue was collected during the diagnostic
histobiopsy.
Example 2: Blood Processing and miRNA Isolation from Plasma
[0087] EDTA blood samples were collected from cases and control
individuals and processed for plasma within 2 hours of collection.
To avoid contamination with epithelial cells from the initial skin
puncture the first blood tube collected during phlebotomy was not
processed for plasma. Blood was centrifuged at 1300 g for 20
minutes at 10.degree. C. The supernatant (plasma) was transferred
into microcentrifuge tubes followed by a second high-speed
centrifugation step at 15500 g for 10 minutes at 10.degree. C. to
remove cell debris and fragments, The plasma was aliquoted into
cryo vials, snap-frozen in liquid nitrogen and stored at
-80.degree. C. until use.
[0088] Total RNA (including miRNAs) was extracted from 400 .mu.L of
plasma. Denaturation and phase separation were conducted using
TRIzol LS (Invitrogen, Germany) according to manufacturer's
protocol, with a minor modification; 10 fmol of a C. elegans
miR-39/miR-238 mixture was spiked-in. The aqueous phase was
transferred into another tube, 1.5 volumes of absolute ethanol were
added and the mixture was applied to miRNeasy Mini kit columns
(Qiagen, Germany). After washing miRNAs were eluted in 30 .mu.L of
RNase-free water.
Example 3: miRNA Profiling of Plasma with TaqMan Low Density Arrays
(TLDA)
[0089] Profiling was carried out on 11 CTC positive metastatic
(>20 CTCs/7.5 ml blood), 9 CTC negative metastatic, 10 early
stage primary breast cancer patients and 10 healthy controls using
TaqMan Low Density Arrays (Human microRNA Cards A and B v2.0) from
Applied Biosystems according to manufacturer's protocol, These
arrays measured the expression of 667 human miRNAs from miRBase
version v.10. In brief, a fixed volume of miRNAs (3 .mu.l) was
reverse transcribed using the TaqMan MicroRNA Reverse Transcription
Kit and TaqMan MicroRNA Megaplex RT Human Pool Sets A (v2.1) &
B (v2.0). cDNA was pre-amplified for 12 cycles with Megaplex PreAmp
Human primer Pools A (v2.1) & B (v2.0), respectively, and
loaded into the TLDA array card ports. Real-time PCR was carried
out with an Applied Biosystems 7900HT thermocycler under the
following conditions: 50.degree. C. for 2 min, 94.5.degree. C. for
10 min, followed by 40 cycles of 97.degree. C. for 30 sec and
59.7.degree. C. for 1 min. Raw data was exported using SDS Relative
Quantification Software version 2.2.2 (Applied Biosystems) with
automatic baseline and threshold settings.
[0090] Raw Ct values of the initial plasma screening step with TLDA
arrays were analyzed using the statistical computational
environment R version 2.11 (http://www.r-project.org/) and the R
package HTqPCR from Bioconductor (v1.2.0) (R Development Core Team
(2010). R: A language and environment for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria. ISBN
3-900051-07-0). miRNAs with Ct values smaller than 15 or larger
than 35 across all the samples were filtered out from further
analysis, along with all miRNAs with inter-quartile ranges
IQR<1.5 (invariant miRNAs), after which quantile normalization
and averaging of duplicates followed. After limma analysis to
identify miRNAs that were differentially expressed between cases
and controls, the results were adjusted for multiple testing by
controlling the false discovery rate (FDR) according to the method
of Benjamini-Hochberg (Smyth G K. Linear models and empirical bayes
methods for assessing differential expression in microarray
experiments, Stat Appl Genet Mol Biol 2004; 3:Article 3; Benjamini
Y, Hochberg Y. Controlling the False Discovery Rate: A Practical
and Powerful Approach to Multiple Testing. J. Roy. Statistical
Society, Series B (Methodological) 1995; 57:289-301.)
Example 4: Validation of Selected Marker Candidates
[0091] Reverse transcription (RT) reactions were performed using
TaqMan miRNA Reverse Transcription Kit (Applied Biosystems,
Germany) and miRNA-specific RT primers for hsa-miR-141,
hsa-miR-142-3p, hsa-miR-148b, hsa-miR-200a, hsa-miR-200b,
hsa-miR-200c, hsa-miR-203, hsa-miR-210, hsa-miR-375, hsa-miR-376c,
hsa-miR-409-3p, hsa-miR-768-3p and hsa-miR-801 (Applied Biosystems,
Germany). Singleplex (primary breast cancer) or multiplex
(metastatic breast cancer) reactions were carried out in a volume
of 7.5 .mu.l or 15 .mu.l, respectively. Each reaction comprised
1.times.RT buffer, 1 mM dNTPs, 0.3.times.miRNA-specific RT primers,
0.25 U RNase inhibitor, 3.3 U Multiscribe Reverse Transcriptase and
a fixed volume of miRNA template (2 or 1 .mu.l, respectively). For
benign and malignant breast cancer tissue samples the reactions
were carried out in 15 .mu.l and comprised the following:
1.times.RT buffer, 1 mM dNTPs, 0.6.times.miRNA-specific and RNU6B
RT primers, 0.25 U RNase inhibitor, 3.3 U Multiscribe Reverse
Transcriptase and 5 ng RNA. Blinding of samples and a randomized,
simultaneous investigation of cases and controls on reaction plates
was intended to minimize bias and batch effects during validation.
RT was carried out in a G-STORM GS2 FOR cycler (Alphametrix,
Germany) under the following conditions: 16.degree. C. for 30 min,
42.degree. C. for 30 min and 85.degree. C. for 5 min, followed by a
hold at 4.degree. C.
[0092] TaqMan real-time PCR reactions were performed in triplicates
in scaled-down reactions comprising 2.5 .mu.L TaqMan 2.times.
Universal PCR Master Mix with No AmpErase UNG (Applied Biosystems,
Germany), 0.25 .mu.L 20.times.miRNA-specific primer/probe mix
(Applied Biosystems, Germany) and 2.25 .mu.L of the reverse
transcription product (diluted 1:4). Real-time FOR was carried out
in a LightCycler 480 thermocycler (Roche, Germany) under the
following conditions: 95.degree. C. for 10 min, then 50 cycles of
95.degree. C. for 15 s, 60.degree. C. for 30 s and 72.degree. C.
for 30 s, followed by a hold at 4.degree. C.
[0093] Raw data from validation studies in blood plasma was
normalized to spiked-in cel-miR-39 as described in Kroh et al.
(Kroh E M, Parkin R K, Mitchell P S, Tewari M. Analysis of
circulating microRNA biomarkers in plasma and serum using
quantitative reverse transcription-FOR (qRT-FOR). Methods 2010;
50:298-301). Raw Ct values from breast tissue samples were
normalized to RNU6B as described in User Bulletin #2: ABI PRISM
7700 Sequence Detection System (Applied Biosystems).
Example 5: Comparison of Early Breast Cancer Cases with
Controls
[0094] Wilcoxon rank sum tests with continuity correction were used
to identify miRNAs that were differentially expressed between cases
and controls in the validation set (127 primary breast cancer
patients and 80 controls). To evaluate the breast cancer detection
potential, receiver operating characteristic (ROC) curves were
constructed and the areas under the curves (AUC) calculated. Based
on ROC curves with 95% confidence intervals, lowest specificities
at pre-defined sensitivities (75% to 90%) were computed for the
most informative and least redundant model of miRNAs. Based on ROC
curves, lowest specificities at pre-defined sensitivities (75% to
90%) were computed for the most informative and least redundant
model of miRNAs as the lower bounds of the 95% confidence intervals
(Tom Fawcett (2006) "An introduction to ROC analysis". Pattern
Recognition Letters 27, 861-874. DOI: 10.1016/j.patrec.2005.10.010;
using R package pROC v1.3.2).
Example 6: Comparison of Metastatic Breast Cancer Cases (CTC
Positive and CTC Negative) with Controls
[0095] Wilcoxon rank sum tests were applied to assess the
significance of differences between the CTC positive and CTC
negative, CTC positive and controls, and, CTC negative and
controls. Leave-one-out cross-validated receiver operating
characteristic (ROC) curves were built for logistic regression
models based on individual miRNAs. Penalised LASSO logistic
regression model (with penalty parameter tuning performed by
10-fold cross-validation) was used to compute the least redundant
and most informative panel of miRNAs that can discriminate two
groups. Corresponding area under the curve (AUC) was calculated for
each model. Based on ROC curves with 95% confidence intervals,
lowest specificities at pre-defined sensitivities (75% to 90%) were
computed for the most informative and least redundant model of
miRNAs.
Example 7: miRNA Profiling Revealed Putative Marker Candidates for
Breast Cancer Detection in Plasma
[0096] In an initial screening step using TLDA arrays we analyzed
plasma miRNA profiles of 10 early stage breast cancer patients as
well as 10 healthy controls. The patients all had an invasive
ductal carcinoma, which was ER/PR+ and HER2- with an AJCC TNM stage
I or II (Hayes D F, Allred C, Anderson B O, Ashley P, Barlow W,
Berry D, Carlson R W, Gelman R, Hilsenbeck 5, Hortobagyi G N,
Kattan M, Lester S C et al. Breast cancer, In: Edge S B, Byrd D R,
Compton C C, Fritz A G, Greene F L, Trotti A. AJCC cancer staging
manual (seventh edition). New York: Springer, 2010:345-376.),
Patients were age-matched to healthy controls. The mean and median
ages of patients were 54 and 51 years respectively, while they were
53 and 54.5 years for controls.
[0097] After filtering, normalization of raw array data and
averaging of duplicates (as described in Example 3 above) a total
of 139 variant miRNAs remained for statistical analysis. A heat map
of the results of hierarchical cluster analysis of the correlations
across samples and principal components analysis identified one
control sample as an outlier, which was then removed from further
statistical analysis. Limma analysis revealed 13 circulating miRNAs
with statistically significant differences in expression between
cases and controls before adjustment for multiple testing. Seven
miRNAs were downregulated (miR-139-3p, miR-193a-3p, miR-206,
miR-519a, miR-526b*, miR-571c and miR-571) and six upregulated
(miR-148b, miR-184, miR-376c, miR-409-3p, miR-424 and miR-801) in
the plasma of early stage breast cancer patients. A list of these
miRNAs can be found in Table 1 together with their (i) p-values,
(ii) p-values adjusted for multiple testing according to the method
of Benjamini-Hochberg (indicating false discovery rates, FDRs),
(iii) mean Ct values for both investigated groups and (iv)
differences in mean Ct values (.DELTA.Ct) between the control and
cases group.
TABLE-US-00001 TABLE 1 Circulating miRNAs differentially expressed
in the plasma of early stage breast cancer cases compared to
healthy controls in TLDA array analysis. Candidates chosen for
validation are in bold and finally validated miRNAs are underlined.
adj. p-value mean Ct mean Ct miRNA p-value (FDR) (controls) (cases)
.DELTA.Ct* hsa-miR-571 <0.001 0.095 30.4 37.9 -7.42 hsa-miR-801
0.002 0.155 30.8 28.5 2.24 hsa-miR-139-3p 0.007 0.259 29.8 34.9
-5.06 hsa-miR-376c 0.008 0.259 31.9 30.5 1.47 hsa-miR-193a-3p 0.009
0.259 38.3 40.0 -1.71 hsa-miR-424 0.014 0.282 38.3 35.3 3.06
hsa-miR-409-3p 0.013 0.282 33.8 32.3 1.56 hsa-miR-184 0.019 0.304
39.4 36.6 2.75 hsa-miR-206 0.020 0.304 30.1 31.7 -1.61 hsa-miR-148b
0.027 0.376 31.7 30.6 1.02 hsa-miR-526b* 0.032 0.407 36.6 38.6
-2.00 hsa-miR-519a 0.039 0.447 35.8 38.1 -2.27 hsa-miR-517c 0.048
0.498 36.4 38.5 -2.14 *.DELTA.Ct = mean Ctcontrols - mean
Ctcases
Example 8: miR-148b, miR-376c, miR-409-3p and miR-801 are
Upregulated in Plasma of Breast Cancer Patients
[0098] The following criteria were applied to choose the best
candidates for marker validation studies in plasma: unadjusted
p<0.05 and mean Ct<33 in at least one investigated group (as
miRNA expression should be stably detectable in at least one group)
(Table 1). The application of these criteria resulted in seven
candidates for validation: miR-139-3p, miR-148b, miR-206, miR-376c,
miR-409-3p, miR-571 and miR-801.
[0099] To find the appropriate sample size necessary to detect fold
changes as small as two-fold, power simulations were carried out.
Statistical power was estimated based on observed standard
deviations in the preliminary small-scale validation experiments.
In all tested scenarios in which total sample size was .gtoreq.200
and included at least 80 controls statistical power was very high
(>93%).
[0100] A total of 127 breast cancer and 80 healthy control plasma
samples were analyzed for their expression of the aforementioned
seven marker candidates. After a preliminary small-scale validation
on 50 samples, miR-139-3p showed a clearly non-significant p-value
(p=0.60) and miR-571 could not be detected in either group. For
these reasons, validation was continued only with the five
remaining miRNAs. A comparison of case and control groups using a
Wilcoxon rank sum test with continuity correction resulted in four
circulating miRNAs validated as being upregulated in the plasma of
breast cancer patients. These miRNAs were miR-148b (p<0.001),
miR-376c (p<0.0001), miR-409-3p (p<0.0001) and miR-801
(p<0.001), whereas miR-206 (p=0.26) did not reach statistical
significance (FIGS. 1A-1D).
Example 9: Diagnostic Potential of miR-148b, miR-376c, miR-409-3p
and miR-8 in Plasma
[0101] ROC curve analysis was performed to evaluate the diagnostic
potential of miR-148b, miR-376c, miR-409-3p and miR-801 for breast
cancer detection in blood plasma. The discriminatory power between
tumor and control samples is depicted by the areas under the curves
(AUC). Individually, miR-148b had an AUC of 0.65, miR-376c of 0.66,
miR-409-3p of 0.66 and miR-801 of 0.64 (FIGS. 2A-2D).
[0102] We found that miR-148b, miR-376c and miR-409-3p expressions
correlate to each other with Spearman rank correlation coefficients
as follows (all p<0.00001): (i) p=0.64 between miR-148b and
miR-409-3p, (ii) p=0,66 between miR-148b and miR-376c and (iii)
p=0.91 between miR-376c and miR-409-3p. The correlation coefficient
between miR-148b and miR-801 expressions is also considerable
(p=0.35), but other correlations were not substantial. By
investigating different combinations of miR-148b, miR-376c,
miR-409-3p and miR-801 we found that a combined ROC curve with
miR-148b, miR-409-3p and miR-801 gave the most informative and
least redundant miRNA panel with an AUC of 0.69 (FIG. 2E). The
discriminatory power of all four significantly upregulated miRNAs
(AUC=0.69) did not outperform this panel and other miRNA
combinations performed only slightly poorer.
Example 10: miR-148b, miR-376c and miR-409-3p are Downregulated in
Malignant Primary Breast Cancer Tissue
[0103] A total of 24 primary breast cancer surgery tissue samples
and 8 benign breast biopsies were analyzed for their miR-148b,
miR-376c, miR-409-3p and miR-801 expression levels. A comparison of
these two sample groups showed that, in contrast to plasma,
miR-148b (p=0.007), miR-376c (p<0.0001) and miR-409-3p (p-0.002)
were downregulated in malignant breast cancer tissue in comparison
to benign breast tissue samples (FIGS. 3A-3C). In the case of
miR-801 (p-0.80) no significant differences in expression levels
were detected (FIG. 3D).
Example 11: Circulating miRNA Profiles of CTC-Positive and
CTC-Negative MBC are Significantly Different
[0104] 30 plasma samples consisting of 11 CTC-positive
(CTC=20/7.5ml blood), 9 CTC-negative cases, and 10 controls were
profiled by low-density TagMan arrays. After filtering,
normalization of raw array data and averaging of duplicates (as
described in Example 3 above), 216 unique and variably expressed
miRNAs remained, which were used for hierarchical clustering and
limma analysis. Surprisingly, we observed that the differences in
profiles between CTC-positive and CTC-negative MBC patients were
larger than those between CTC-negative and healthy controls.
Clustering of samples revealed that CTC-positive cases formed one
cluster, while CTC-negative cases and controls formed two
sub-clusters of another branch. Concomitantly, limma analysis
returned more miRNAs significant for the comparison of CTC-positive
cases (19 miRNAs) than for CTC-negative cases (4 miRNAs) with
controls. Analysis of CTC-positive against CTC-negative cases
engendered 12 up-and 3 downregulated miRNAs in the CTC-positive
group. Stringent cut-offs were applied to ensure reduction in false
positives and feasibility of testing (controlling the false
discovery rate (FDR) according to the method of Benjamini-Hochberg
at a level of 0.1). Consequently, seventeen miRNAs were selected
for the validation study: miR-99a, miR-133b, miR-139-3p, miR-141,
miR-1423p, miR-193b*, miR-200a, miR-200b, miR-200c, miR-203,
miR-206, miR-210, miR-375, miR-571, miR-630, miR-768-3p and
miR-801.
Example 12: Eight Circulating miRNAs Significantly Upregulated in
CTC-Positive MBC Compared to CTC-Negative MBC or Controls
[0105] After preliminary testing, five out of the seventeen
candidates (miR-133b, miR-139-3p, miR-193b*, miR-206, miR-99a)
could not be analyzed due to low expression, while miR-571 and
miR-630 could not be detected by TagMan miRNA assays, The remaining
ten miRNAs, including four members of the miR-200 family (miR-141,
miR-200a, miR-200b, miR-200c), along with miR-142-3p, miR-203,
miR-210, miR-375, miR-768-3p and miR-801, were analyzed in an
expanded sample set of 133 MBC cases and 76 controls.
[0106] Wilcoxon rank sum tests with continuity correction confirmed
that miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR210,
miR-375 and miR-801 were significantly upregulated in CTC-positive
in comparison to CTC-negative cases (fold change (FC) of 2.41 to
26.17, p<0.00001 for all miRNAs). Based on the trend of our
array results, the differences in circulating miRNAs between these
subgroups and controls were additionally explored. These eight
miRNAs were also found to have significantly increased expression
in positive cases (FC of 3.36 to 36.25, p<0.00001 for all
miRNAs). However, only four out of these eight miRNAs were
significantly upregulated in CTC-negative cases (miR-141, miR-200c,
miR-210, miR-801; FC of 1.39 to 2.14, p<0.05 for all miRNAs).
Although miR-768-3p had only a negligible decrease when comparing
CTC-positive and CTC-negative cases, it was found to be
significantly downregulated in CTC-positive (p=0.006, FC=0.68), and
CTC-negative cases (p=0.003, FC=0.77). No significant changes in
expression were found in miR-142-3p in any of the comparisons.
These results are represented in FIG. 4(A) and Table 2. Analysis of
the correlation in expression of these ten miRNAs discerned a high
correlation among the members of the miR-200 family (p>0.3,
P<0.00001), between miR-210 and miR-801 (p=0.53, P<0.00001),
and between miR-142-3p and miR-768-3p (p=0.41, P<0.00001) (FIG.
4(B)),
TABLE-US-00002 TABLE 2 Validation of candidate miRNAs. Results of
Wilcoxon rank sum tests with median fold change (FC =
2.sup.-.DELTA.Ct), corresponding two sided p value, and
leave-one-out cross-validated area under the curve (AUC) estimates
for the 10 candidate miRNAs. CTC-positive versus CTC-positive
versus CTC-negative versus CTC-negative Control Control FC P AUC FC
P AUC FC P AUC miR-141 26.17 8.27E-13 0.85 36.25 1.69E-16 0.90 1.39
6.75E-03 0.59 miR-200a 15.24 6.85E-13 0.85 15.78 7.34E-15 0.88 1.04
3.05E-01 0.47 miR-200b 11.63 9.53E-15 0.88 13.18 9.65E-17 0.91 1.13
8.52E-01 0.03 miR-200c 9.38 5.73E-13 0.86 14.22 1.54E-17 0.92 1.52
1.22E-02 0.59 mi R-203 4.06 6.37E-06 0.71 3.36 5.89E-06 0.71 0.83
2.47E-01 0.49 miR-210 2.41 2.77E-07 0.74 5.17 2.29E-14 0.87 2.14
2.57E-07 0.73 miR-375 4.96 5.98E-10 0.80 3.89 2.22E-09 0.79 0.78
1.27E-01 0.52 miR-801 2.83 2.54E-06 0.72 4.99 5.91E-13 0.85 1.77
2.87E-05 0.67 miR-142-3p 1.16 4.44E-01 0.17 0.96 6.73E-01 0.45 0.83
1.72E-01 0.52 miR-768-3p 0.88 6.76E-01 0.35 0.68 6.12E-03 0.61 0.77
2.96E-02 0.58
Example 13: Circulating mRNAs Differentiate CTC-Positive from
CTC-Negative MBC
[0107] Leave-one-out cross-validated ROC analysis predicted the
ability of the investigated miRNAs to differentiate GIG-positive
from GIG-negative cases, and CTC-positive cases from controls with
high AUCs (FIG. 5A-E; Table 2). For GIG-positive versus
CTC-negative cases, although a multivariable model comprising
miR-141 and miR-200b was predicted (0.87), the AUC of miR-200b
alone (0.88) was found to be marginally greater than that of the
model. Combination of miR-141, miR-200b and miR-375 performed with
equal accuracy (AUC=0.88). With an equal sensitivity and
specificity as the models (80% and 83% respectively, FIG. 5F), we
reckon miR-200b alone might be sufficient for distinguishing
GIG-positive from CTC-negative cases. For GIG-positive cases versus
controls, the predicted multivariable model with miR-141, miR-200b,
miR-200c, miR-210 and miR-768-3p had a very high AUG of 0.95 (90%
sensitivity and 91% specificity, FIG. 5G). Even though,
individually the miRNAs could not differentiate GIG-negative cases
from controls with high certainty, the model predicted combination
of three miRNAs, miR-200c, miR-210 and miR-768-3p, had an
appreciable AUG of 0.78 (80% sensitivity and 65% specificity) (FIG.
5H).
Example 14: Circulating miRNAs Correlate with CTC Counts
[0108] The eight miRNAs that were significantly upregulated in the
CTC-positive and CTC-negative comparison, also evidenced a strong
correlation to CTC counts. Spearman correlation analysis
demonstrated lower Ct values, and thus higher miRNA expression,
correlated with higher number of CTCs (p<0.00001). In contrast,
miR-142-3p and miR-768-3p had very poor correlation to CTC numbers
(p of -0.13 and -0.05 respectively). miR-16, which is considered as
an endogenous control for breast cancer tissue, also had poor and
no significant correlation to CTC numbers (p=-0.06, p=0.47) (Table
3).
TABLE-US-00003 TABLE 3 Correlation of miRNA and CTC counts.
Spearman rank correlation of miRNA expression (Ct value) and number
of CTCs. CTC counts rho value P miR-141 -0.55 2.29E-14 miR-200a
-0.56 6.12E-15 miR-200b -0.61 <2.2E-16 miR-200c -0.57 1.98E-15
miR-203 -0.43 8.98E-09 miR-210 -0.45 1.40E-09 miR-375 -0.53
3.72E-13 miR-801 -0.39 2.04E-07 miR-142-3p -0.13 0.1 miR-768-3p
-0.05 0.52 miR-16 -0.06 0.47
Example 15: Analysis of miRNA with Survival Data
[0109] Patients with metastatic breast cancer (MBC) were recruited
into the study, and blood was collected. The patients were
followed-up and monitored for progression by radiological methods
(Eg. CT scans). Patients were classified as follows: [0110] 1.
Progressive disease: Increase in size of tumor or spread of tumor
to other regions [0111] 2. Stable disease: No change in size of
tumor [0112] 3. Partial remission: Decrease in size of tumor [0113]
4. Complete remission: Tumor not discernible by radiological
imaging
[0114] Progression free survival (PFS) was defined as the time from
enrollment into the study to progressive disease in months; Overall
survival (OS) was defined as the time from enrollment into study to
death in months; miRNA quantification: Eight miRNAs, namely
miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-375,
and miR-801, that were significantly upregulated in CTC positive
when compared to CTC negative were quantified in each sample as
described in Example 4.
[0115] Statistical analysis: Correlation of miRNA expression to PFS
and OS were assessed by both Cox proportional hazard model and
logrank test. Cox models assume proportional hazards and a linear
relationship between miRNA expression and PFS hazard, and
calculates the log Hazard ratio and the corresponding P value (Cox,
D. R. and D. Oakes. Analysis of Survival Data. London: Chapman and
Hall. 1984). In the logrank test the relationship between miRNA
expression and PFS hazard can be explained by a categorization into
the two groups (those with Ct>median CT, and those with
Ct<median Ct). Contrary to the Cox model, we do not assume a
linear relationship between miRNA expression values and PFS (N.
Mantel. Evaluation of survival data and two new rank order
statistics arising in its consideration. Cancer Chemotherapy
Reports. 1996 50 (3): 163-70).
TABLE-US-00004 TABLE 4 P values from log rank test estimating the
association between miRNA levels and progression-free (PFS) and
overall survival (OS). PFS OS miR-141 4.58E-02 6.77E-06 miR-200a
6.69E-05 1.24E-07 miR-200b 1.74E-05 3.72E-09 miR-200c 3.06E-05
1.06E-09 miR-203 9.20E-02 7.28E-03 miR-210 1.07E-01 2.30E-04
miR-375 1.45E-03 3.96E-05 miR-801 1.51E-02 2.45E-05 CTC 1.70E-03
4.49E-07
Example 16: Prediction of Therapy Success in MBC Patients
[0116] It was described above that eight miRNAs (miR-141, miR-200a,
mir-200b, miR-200c, miR-203, miR-210, miR-375, miR-801) that could
serve as prognostic markers in metastatic breast cancer (MBC)
patients. It was further analysed if the above miRNAs could be
useful in monitoring therapy response. For this, these 8 miRNAs
were measured in 76 patient samples (plasma) after one round of
therapy, wherein appropriate treatment for each patient was
selected from chemotherapy, radiotherapy, and/or hormone therapy
(FIG. 10).
[0117] Correlation of miRNA amounts and also circulating tumour
cells (CTC) after therapy to progression free survival (PFS) and
overall survival (OS) was established by log-rank test after
stratifying data as lower quartile and rest (miRNAs) or
CTC-positive and CTC-negative (CTC), and the corresponding
Kaplan-meier curves are presented in FIG. 11 and FIG. 12.
[0118] miR-200b performed the best among all miRNAs; comparison of
the cox regression model with miR-200b or CTCs by ANOVA test is
shown in Table 6:
TABLE-US-00005 TABLE 5 P values from log rank test estimating the
association between miRNA levels after therapy and progression-free
(PFS) and overall survival (OS). PFS OS miR-141 0.018 6.65E-08
miR-200a 0.024 3.13E-04 miR-2006 0.003 9.00E-07 miR-200c 0.007
5.92E-04 miR-203 0.094 6.60E-03 miR-210 0.021 1.64E-03 miR-375
0.146 5.34E-04 miR-801 0.482 3.81E-03 CTC 0.018 6.65E-08
TABLE-US-00006 TABLE 6 correlation analysis and ANOVA test
progression-free survival overall survival miRNA200b CTC miRNA200b
CTC P-value 0.005 0.28 5.2E-05 0.019 Regression 8.847 0.285 1.87
1.05 coefficient ANOVA <2.2E-16 <2.2E-16
[0119] Apparently, the miRNAs of the present invention outperform
CTC as a marker for PFS and/or OS.
II. Cohort II
[0120] Cohort II consisted of 120 primary breast cancer patients,
30 women with benign breast tumors and 60 healthy female volunteers
who served as controls. Except where otherwise noted, samples were
obtained and processed as described for cohort I.
Example 17: miRNA Profiling Revealing Further Putative Marker
Candidates for Breast Cancer Detection in Plasma
[0121] The initial screening step using TLDA arrays was similar to
example 7, using data from 10 early stage breast cancer patients as
well as 10 healthy controls.
[0122] Quality control plots (Pearson's correlations across samples
and principal components analysis) identified one control sample
(B024) as an outlier, which was then removed from further
statistical analysis. Raw Ct values from TLDAs were quantile
normalized so that the values from different runs would have the
same distribution and could be easily compared. Following quantile
normalization and filtering of undetermined miRNAs (Ct>35 across
all the samples), data from TLDA microfluidic cards A and cards B
were combined for further analysis. Duplicate miRNA measurements
were averaged and a total of 402 miRNAs remained for statistical
analysis. Early stage breast cancer patients and healthy controls
were compared using Limma analysis and 38 circulating miRNAs were
found to be significantly different between these two groups. A
list of the significant miRNAs can be found in Table 7 together
with their (i) p-values, (ii) p-values adjusted for multiple
testing according to the method of Benjamini-Hochberg (indicating
false discovery rates, FDRs), (iii) mean Ct values for both
investigated groups and (iv) differences in mean Ct values
(.DELTA.Ct) between the control and cases group.
TABLE-US-00007 TABLE 7 Circulating miRNAs deregulated in the plasma
of early stage breast cancer patients compared to healthy controls
in TLDA analysis (Limma test). Circulating miRNAs selected for
further investigation are in bold and finally validated miRNAs are
underlined. False mean Ct mean Ct miRNA p-value discovery rate
(controls) (cases) .DELTA.Ct* hsa-miR-148b 0.0005 0.07 31.6 30.4
1.2 hsa-miR-328 0.0004 0.07 28.6 27.9 0.7 hsa-miR-376c 0.0002 0.07
32.0 30.4 1.6 hsa-miR-652 0.0008 0.08 31.9 30.7 1.2 hsa-miR-320
0.001 0.11 24.0 24.8 -0.8 hsa-miR-145 0.004 0.23 28.1 27.1 1.0
hsa-miR-339-3p 0.004 0.23 30.9 30.2 0.7 hsa-miR-193a-3p 0.007 0.28
38.3 40.0 -1.7 hsa-miR-206 0.007 0.28 29.9 31.7 -1.8 hsa-miR-801
0.007 0.28 30.6 28.4 2.2 hsa-miR-139-3p 0.010 0.38 29.8 34.9 -5.1
hsa-miR-221 0.015 0.38 28.2 27.7 0.5 hsa-miR-376a 0.014 0.38 32.9
31.5 1.4 hsa-miR-138-1* 0.013 0.38 28.0 28.7 -0.7 hsa-miR-190b
0.015 0.38 33.9 32.9 1.0 hsa-miR-409-3p 0.013 0.38 34.0 32.3 1.7
hsa-miR-424 0.016 0.39 38.3 35.3 3.0 hsa-miR-184 0.020 0.42 39.4
36.7 2.7 hsa-miR-875-5p 0.019 0.42 34.0 33.1 0.9 hsa-miR-93* 0.024
0.46 30.2 31.2 -1.0 hsa-miR-526b* 0.024 0.46 36.6 38.6 -2.0
hsa-let-7c 0.036 0.52 30.0 30.6 -0.6 hsa-miR-18a 0.038 0.52 29.2
28.5 0.7 hsa-miR-29a 0.049 0.52 26.8 27.3 -0.5 hsa-miR-29c 0.043
0.52 30.3 30.8 -0.5 hsa-miR-127-3p 0.047 0.52 33.1 31.2 1.9
hsa-miR-190 0.042 0.52 36.4 34.2 2.2 hsa-miR-323-3p 0.049 0.52 31.0
30.5 0.5 hsa-miR-485-3p 0.040 0.52 32.0 31.3 0.7 hsa-miR-519a 0.031
0.52 35.7 38.1 -2.4 hsa-miR-548d-3p 0.040 0.52 40.0 38.8 1.2
hsa-miR-579 0.035 0.52 32.3 33.1 -0.8 hsa-miR-598 0.041 0.52 31.4
32.0 -0.6 hsa-miR-200a* 0.042 0.52 37.6 40.0 -2.4 hsa-miR-148b*
0.034 0.52 38.8 36.9 1.9 hsa-miR-34a* 0.049 0.52 31.3 32.3 -1.0
hsa-miR-941 0.042 0.52 37.8 40.0 -2.2 hsa-miR-188-5p 0.047 0.52
28.7 29.2 -0.5 *.DELTA.Ct = mean Ct.sub.controls - mean
Ct.sub.cases
Example 18: miR-127-3p, miR-148b, miR-376a, miR-376c, miR-409-3p,
miR-652 and miR-801 are Upregulated in Plasma of Breast Cancer
Patients
[0123] The following criteria were applied to choose the best
candidates for marker validation studies in plasma: unadjusted
p<0.05, mean Ct<33 in at least one investigated group (as
miRNA expression should be stably detectable in at least one group)
and (iii) |.DELTA.Ct|>1 (indicating that the miRNA amounts in
the patient and control plasma differ markedly) (Table 7). The
application of these criteria resulted in nine candidates for
validation: miR-127-3p, miR-139-3p, miR-148b, miR-206, miR-376a,
miR-376c, miR-409-3p, miR-652 and miR-801.
[0124] In validation studies, circulating miR-139-3p and miR-206
did not reach statistical significance, but an investigation of a
validation cohort (n=210) consisting of 30 women with benign and
120 with malignant breast tumors, as well as 60 healthy controls
showed that circulating miR-127-3p (P<0.001), miR-148b
(P-(0.0001), miR-376a (P=0.03), miR-376c (P=0.03), miR-409-3p
(P-0.005), miR-652 (P<0.0001) and miR-801 (P<0.0001) have
increased levels in the plasma of women with breast cancer when
compared to healthy controls (FIG. 7). Additionally, circulating
miR-148b (P=0.02), miR-652 (P=0.01) and miR-801 (P=0.003) differed
significantly even in the plasma of women with benign breast tumors
when compared to healthy women (FIG. 7).
Example 19: Diagnostic Potential of mir-127-3p, miR-148b, miR-376a,
miR-376c, miR-409-3p, miR-652 and miR-801 in Plasma
[0125] ROC curve analysis was performed to evaluate the diagnostic
potential of mir-127-3p, miR-148b, miR-376a, miR-376c, miR-409-3p,
miR-652 and miR-801 for breast cancer detection in blood plasma.
The discriminatory power between tumor and control samples is
depicted by the areas under the curves (AUC).
[0126] Individually, miR-127-3p had an AUC of 0.65 (95% CI:
0.57-0.73), miR-148b of 0.70 (95% CI: 0.62-0.78), miR-376a of 0.59
(95% CI: 0.51-0.67), miR-376c of 0.59 (95% CI: 0.51-0.67),
miR-409-3p of 0.62 (95% CI: 0.54-0.70), miR-652 of 0.75 (95% CI:
0.67-0.82) and miR-801 of 0.72 (95% CI: 0.65-0.80) (FIGS.
8A-8G).
[0127] The combination of all seven miRNAs yielded the best
discriminatory power with AUC=0.81 (95% Cl=0.75-0.88) for the
detection of breast tumors (FIG. 8H). In younger women (up to the
age of 50) these circulating miRNAs performed superiorly and had an
even higher accuracy (AUC-0.86; 95% CI-0.79-0.93) for breast tumor
detection (FIG. 8I).
Example 20: Correlation Between Plasma miRNAs
[0128] Inter-relationships between miRNA expressions were
investigated by computing Spearman rank correlation coefficients
(p), Four out of seven validated miRNAs (miR-127-3p, miR-376a,
miR-376c and miR-409-3p) originate from the same miRNA cluster
located on the chromosomal region 1402 and their plasma levels were
found to correlate to each other strongly. Table 8 provides a
detailed representation of the various miRNA inter-correlations.
Apart from the four miRNAs belonging to the same miRNA cluster,
circulating miR-148b correlated considerably with miR-127-3p and
miR-652, whereas miR-801 showed no significant correlations except
for a slight correlation to miR-148b levels.
Example 21: miR-127-3p, miR-376a and miR-652 are Downregulated in
Malignant Primary Breast Cancer Tissue
[0129] A total of 24 primary breast cancer surgery tissue samples
and 8 benign breast biopsies were analyzed for their miR-127-3p,
miR-376a and miR-652 expression levels. A comparison of these two
sample groups showed that, in contrast to plasma, miR-127-3p
(p=0.02), miR-376a (p=0.001) and miR-652 (p=0.03) were
downregulated in malignant breast cancer tissue in comparison to
benign breast tissue samples (FIGS. 9A-9C).
TABLE-US-00008 TABLE 8 Spearman correlation rank coefficients (p)
and confidence intervals (95% Cl), as well as P values for selected
miRNA pairs. miR-148b miR-376a miR-376c miR-409-3p mill-652 miR-801
miR- p = 0.62 p = 0.85 p = 0.92 p = 0.89 p = 0.48 p = 0.06 127-3p
95% Cl = 0.49-0.72 95% Cl = 0.79-0.89 95% Cl = 0.88-0.94 95% Cl =
0.84-0.92 95% Cl = 0.32-0.61 95% Cl = (-0.13)-0.24 P < 0.0001 P
< 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.55 miR-
xxx p = 0.55 p = 0.59 p = 0.56 p = 0.78 p = 0.24 148b 95% Cl =
0.41-0.67 95% Cl = 0.45-0.70 95% Cl = 0.42-0.68 95% Cl = 0.70-0.84
95% Cl = 0.06-0.41 P < 0.0001 P < 0.0001 P < 0.0001 P <
0.0001 P = 0.0077 miR- p = 0.55 xxx p = 0.87 p = 0.84 p = 0.43 p =
0.04 376a 95% Cl = 0.41-0.67 95% Cl = 0.81-0.91 95% Cl = 0.77-0.88
95% Cl = 0.27-0.57 95% Cl = (-0.22)-0.15 P < 0.0001 P <
0.0001 P < 0.0001 P < 0.0001 P = 0.69 miR- p = 0.59 p = 0.87
xxx p = 0.91 p = 0.43 p = 0.03 376c 95% Cl = 0.45-0.70 95% Cl =
0.81-0.91 95% Cl = 0.87-0.94 95% Cl = 0.26-0.57 95% Cl =
(-0.15)-0.22 P < 0.0001 P < 0.0001 P < 0.0001 P <
0.0001 P = 0.73 miR- p = 0.56 p = 0.84 p = 0.91 xxx p = 0.44 p =
0.07 409-3p 95% Cl = 0.42-0.68 95% Cl = 0.77-0.88 95% Cl =
0.87-0.94 95% Cl = 0.28-0.58 95% Cl = (-0.11)-0.25 P < 0.0001 P
< 0.0001 P < 0.0001 P < 0.0001 P = 0.44 miR- p = 0.78 p =
0.43 p = 0.43 p = 0.44 xxx p = 0.09 652 95% Cl = 0.70-0.84 95% Cl =
0.27-0.57 95% Cl = 0.26-0.57 95% Cl = 0.28-0.58 95% Cl =
(-0.10)-0.27 P < 0.0001 P < 0.0001 P < 0.0001 P <
0.0001 P = 0.34
Example 22: Prediction of Therapy Success in MBC Patients
[0130] It was described above that eight miRNAs (miR-141, miR-200a,
mir-200b, miR-200c, miR-203, miR-210, miR-375, miR-801) that could
serve as prognostic markers in metastatic breast cancer (MBC)
patients. It was further analysed if the above miRNAs could be
useful in monitoring therapy response. For this, these 8 miRNAs
were measured in 76 patient samples (plasma) after one round of
chemotherapy (FIG. 10).
[0131] Correlation of miRNA amounts and also circulating tumour
cells (CTC) after therapy to progression free survival (PFS) and
overall survival (OS) was established by log-rank test after
stratifying data as lower quartile and rest (miRNAs) or
CTC-positive and CTC-negative (CTC), and the corresponding
Kaplan-meier curves are presented in FIG. 11 and FIG. 12.
[0132] miR-200b performed the best among all miRNAs; comparison of
the cox regression model with miR-200b or CTCs by ANOVA test is
shown in Table 9:
TABLE-US-00009 TABLE 9 correlation analysis and ANOVA test
progression-free survival overall survival miRNA200b CTC miRNA200b
CTC P-value 0.005 0.28 5.2E-05 0.019 Regression 8.847 0.285 1.87
1.05 coefficient ANOVA <2.2E-16 <2.2E-16
[0133] Apparently, the miRNAs of the present invention outperform
CTC as a marker for PFS and/or OS.
Sequence CWU 1
1
28124RNAHomo sapiens 1gauugcucug cgugcggaau cgac 24222RNAHomo
sapiens 2ucagugcauc acagaacuuu gu 22321RNAHomo sapiens 3aacauagagg
aaauuccacg u 21422RNAHomo sapiens 4gaauguugcu cggugaaccc cu
22522RNAHomo sapiens 5gugaaauguu uaggaccacu ag 22628RNAHomo sapiens
6ucacaaugcu gacacucaaa cugcugac 28723RNAHomo sapiens 7uguaguguuu
ccuacuuuau gga 23822RNAHomo sapiens 8uaacacuguc ugguaaagau gg
22922RNAHomo sapiens 9uaauacugcc ugguaaugau ga 221023RNAHomo
sapiens 10uaauacugcc ggguaaugau gga 231122RNAHomo sapiens
11cugugcgugu gacagcggcu ga 221222RNAHomo sapiens 12uuuguucguu
cggcucgcgu ga 221322RNAHomo sapiens 13uaacacuguc ugguaacgau gu
221422RNAHomo sapiens 14ucggauccgu cugagcuugg cu 221521RNAHomo
sapiens 15aucauagagg aaaauccacg u 211621RNAHomo sapiens
16aauggcgcca cuaggguugu g 211723RNAHomo sapiens 17uaaggugcau
cuagugcaga uag 231822RNAHomo sapiens 18caaucagcaa guauacugcc cu
221922RNAHomo sapiens 19acugcugagc uagcacuucc cg 222022RNAHomo
sapiens 20gcuacuucac aacaccaggg cc 222123RNAHomo sapiens
21guccaguuuu cccaggaauc ccu 232221RNAHomo sapiens 22ugauauguuu
gauauugggu u 212322RNAHomo sapiens 23aaaagcuggg uugagagggc ga
222422RNAHomo sapiens 24cuggcccucu cugcccuucc gu 222523RNAHomo
sapiens 25ugagcgccuc gacgacagag ccg 232622RNAHomo sapiens
26gucauacacg gcucuccucu cu 222723RNAHomo sapiens 27uucauuuggu
auaaaccgcg auu 232822RNAHomo sapiens 28uauaccucag uuuuaucagg ug
22
* * * * *
References