U.S. patent application number 15/611017 was filed with the patent office on 2017-09-21 for checkpoint blockade and microsatellite instability.
This patent application is currently assigned to The Johns Hopkins University. The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Luis Diaz, Kenneth W. Kinzler, Dung Le, Nickolas Papadopoulos, Bert Vogelstein.
Application Number | 20170267760 15/611017 |
Document ID | / |
Family ID | 55955037 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170267760 |
Kind Code |
A1 |
Diaz; Luis ; et al. |
September 21, 2017 |
Checkpoint Blockade and Microsatellite Instability
Abstract
Blockade of immune checkpoints such as cytotoxic T-lymphocyte
antigen-4 (CTLA-4) and programmed death-1 (PD-1) shows promise in
patients with cancer. Inhibitory antibodies directed at these
receptors have been shown to break immune tolerance and promote
anti-tumor immunity. These agents work particularly well in
patients with a certain category of tumor. Such tumors may be
particularly susceptible to treatment because of the multitude of
neoantigens which they produce.
Inventors: |
Diaz; Luis; (Ellicott City,
MD) ; Vogelstein; Bert; (Baltimore, MD) ;
Kinzler; Kenneth W.; (Baltimore, MD) ; Papadopoulos;
Nickolas; (Towson, MD) ; Le; Dung;
(Lutherville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
|
|
Assignee: |
The Johns Hopkins
University
Baltimore
MD
|
Family ID: |
55955037 |
Appl. No.: |
15/611017 |
Filed: |
June 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15523451 |
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PCT/US15/60331 |
Nov 12, 2015 |
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15611017 |
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62190977 |
Jul 10, 2015 |
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62079357 |
Nov 13, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/40 20130101;
A61P 35/00 20180101; C07K 2317/24 20130101; A61P 15/00 20180101;
C12Q 2600/156 20130101; C12Q 2600/106 20130101; A61K 2039/55
20130101; C07K 2317/76 20130101; C07K 16/30 20130101; C07K 2317/00
20130101; C12Q 1/6886 20130101; C07K 16/2827 20130101; A61P 1/04
20180101; C07K 16/2803 20130101; A61P 13/08 20180101; C07K 16/2818
20130101; A61K 2039/505 20130101; C12Y 113/11052 20130101; A61P
1/16 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C12Q 1/68 20060101 C12Q001/68; A61K 39/395 20060101
A61K039/395; G01N 33/574 20060101 G01N033/574 |
Goverment Interests
[0001] This invention was made with government support under
CA43460 and CA62924 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A method for treating cancer, the method comprising: testing one
or more microsatellite markers in a tumor sample from a patient;
determining microsatellite instability (MSI) for the tumor based on
the testing; and identifying the patient as a candidate for
treatment with an immune checkpoint inhibitor when the tumor
exhibits an MSI phenotype.
2. The method of claim 1, further comprising administering the
immune checkpoint inhibitor to the patient.
3. The method of claim 1, wherein the immune checkpoint inhibitor
comprises an antibody.
4. The method of claim 3, wherein the antibody is selected from the
group consisting of: an anti-PD-1 antibody; an anti-IDO antibody;
anti-CTLA-4 antibody; an anti-PD-L1 antibody; and an anti-LAG-3
antibody.
5. The method of claim 1, wherein the one or more microsatellite
markers are selected from the group consisting of BAT-25, BAT-26,
MONO-27, NR-21, NR-24, Penta C, and Penta D.
6. The method of claim 1, wherein the tumor is determined to
display the MSI phenotype when the testing indicates at least 100
mutations in a genome of the tumor relative to normal tissue of the
patient.
7. The method of claim 1, wherein the cancer is selected from the
group consisting of: colon, gastric, endometrial,
cholangiocarcinoma, pancreatic, and prostate cancer.
8. The method of claim 1, wherein the immune checkpoint inhibitor
comprises an antibody selected from the group consisting of: a
humanized monoclonal antibody; sMK-3475; and an IgG4 antibody.
9. The method of claim 1, wherein a plurality of microsatellite
markers is tested.
10. The method of claim 9, wherein the plurality of microsatellite
markers is each selected from the group consisting of BAT-25,
BAT-26, MONO-27, NR-21, NR-24, Penta C, and Penta D.
11. The method of claim 9, wherein the plurality of microsatellite
markers comprises BAT-25, BAT-26, MONO-27, NR-21, NR-24, Penta C,
and Penta D.
12. The method of claim 1, wherein testing the one or more
microsatellite markers includes one technique selected from the
group consisting of an immunohistochemistry (IHC) assay, a PCR
test, and next generation sequencing.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the area of cancer. In
particular, it relates to cancer therapy.
BACKGROUND OF THE INVENTION
[0003] Microsatellite instability (MSI) is the accumulation of
sequencing errors in microsatellites. This occurs in tumors with
deficiency in DNA mismatch repair. MSI is present in Lynch Syndrome
which is an inherited cancer syndrome that predisposes patients to
colon, endometrial, gastric cancer, ovarian, small intestine,
liver, hepatobiliary, upper urinary tract, brain, and prostate
cancer. MSI is also present in 10-20% of sporadic colorectal,
gastric, prostate, lung, ampullary, and endometrial cancers.
Between 0.3% and 13% of pancreatic cancers are reported to be MSI
as well.
[0004] The importance of intact immune surveillance in controlling
outgrowth of neoplastic transformation has been known for decades.
Accumulating evidence shows a correlation between
tumor-infiltrating lymphocytes (TILs) in cancer tissue and
favorable prognosis in various malignancies. In particular, the
presence of CD8+ T-cells and the ratio of CD8+ effector
T-cells/FoxP3+ regulatory T-cells seems to correlate with improved
prognosis and long-term survival in solid malignancies such as
ovarian, colorectal and pancreatic cancer, hepatocellular
carcinoma, malignant MEL and RCC. TILs can be expanded ex vivo and
re-infused, inducing durable objective tumor responses in cancers
such as melanoma.
[0005] The PD-1 receptor-ligand interaction is a major pathway
hijacked by tumors to suppress immune control. The normal function
of PD-1, expressed on the cell surface of activated T-cells under
healthy conditions, is to down-modulate unwanted or excessive
immune responses, including autoimmune reactions. The ligands for
PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be
induced in various tumors. Binding of either PD-1 ligand to PD-1
inhibits T-cell activation triggered through the T-cell receptor.
PD-L1 is expressed at low levels on various non-hematopoietic
tissues, most notably on vascular endothelium, whereas PD-L2
protein is only detectably expressed on antigen-presenting cells
found in lymphoid tissue or chronic inflammatory environments.
PD-L2 is thought to control immune T-cell activation in lymphoid
organs, whereas PD-L1 serves to dampen unwarranted T-cell function
in peripheral tissues. Although healthy organs express little (if
any) PD-L1, a variety of cancers were demonstrated to express
abundant levels of this T-cell inhibitor. High expression of PD-L1
on tumor cells (and to a lesser extent of PD-L2) has been found to
correlate with poor prognosis and survival in various cancer types,
including renal cell carcinoma (RCC), pancreatic carcinoma,
hepatocellular carcinoma, ovarian carcinoma and non-small cell lung
cancer (NSCLC). Furthermore, PD-1 has been suggested to regulate
tumor-specific T cell expansion in patients with malignant MEL. The
observed correlation of clinical prognosis with PD-L1 expression in
multiple cancers suggests that the PD-1/PD-L1 pathway plays a
critical role in tumor immune evasion and should be considered as
an attractive target for therapeutic intervention.
[0006] Blockade of immune checkpoints such as cytotoxic
T-lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) is
showing promise in patients with cancer. CTLA-4 and PD-1 are
upregulated on activated T cells and provide inhibitory signals to
T cells undergoing activation. Inhibitory antibodies directed at
these receptors have been shown to break immune tolerance and
promote anti-tumor immunity. MK-3475 is a humanized monoclonal IgG4
antibody against PD-1 and is showing activity in multiple tumor
types including melanoma and non-small cell lung cancer (NSCLC).
Previously, activity of a different PD-1 blocking antibody,
BMS-936558, a fully humanized monoclonal IgG4 antibody, also showed
activity in melanoma, NSCLC, and a complete response in a single
patient with colorectal cancer.
[0007] MK-3475 (previously known as SCH 900475) is a potent and
highly-selective humanized mAb of the IgG4/kappa isotype designed
to directly block the interaction between PD-1 and its ligands,
PD-L1 and PD-L2. MK-3475 contains the S228P stabilizing mutation
and has no antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement-dependent cytotoxicity (CDC) activity. MK-3475 strongly
enhances T lymphocyte immune responses in cultured blood cells from
healthy human donors, cancer patients, and primates. In T-cell
activation assays using human donor blood cells, the EC50 was in
the range of 0.1 to 0.3 nM. MK-3475 also modulates the level of
interleukin-2 (IL-2), tumor necrosis factor alpha (TNF.alpha.),
interferon gamma (IFN.gamma.), and other cytokines. The antibody
potentiates existing immune responses only in the presence of
antigen and does not nonspecifically activate T-cells.
[0008] The programmed death 1 (PD-1) pathway is a negative feedback
system repressing Th1 cytotoxic immune responses that, if
unregulated, could damage the host.sup.1-3. It is upregulated in
many tumors and their surrounding microenvironment. Blockade of
this pathway with antibodies to PD-1 or its ligands has led to
remarkable clinical responses in some patients with many different
cancer types, including melanomas, non-small cell lung cancer,
renal cell carcinoma, bladder cancer and Hodgkin's
lymphoma.sup.4-10. The expression of ligands to PD-1 (PD-L1 or
PD-L2) on the surface of tumor cells or immune cells is important
but not a definitive predictive biomarker for response to PD-1
blockade.sup.4,6-8,11.
[0009] We were intrigued that, in reports of the effects of PD-1
blockade in human tumors, only one of 33 colorectal cancer (CRC)
patients responded to this treatment, in contrast to substantial
fractions of patients with melanomas, renal cell cancers, and lung
tumors..sup.10,12. What was different about this single patient? We
hypothesized that this patient had MMR-deficiency, because
MMR-deficiency occurs in a small fraction of advanced
CRCs,.sup.13,14 somatic mutations found in tumors can be recognized
by the patient's own immune system,.sup.15 and MMR-deficient
cancers have 10- to 100-fold more somatic mutations than
MMR-proficient CRC..sup.16-18 Moreover, MMR-deficient cancers
contain prominent lymphocyte infiltrates, consistent with an immune
response.sup.19-22. And two of the tumor types that were most
responsive to PD-1 blockade in a study by Topalian et al..sup.10
had high numbers of somatic mutations as a result of exposure to
cigarette smoke (lung cancers) or UV radiation
(melanomas).sup.23,24. Our hypothesis was correct: the tumor of the
single CRC patient who responded to PD-1 blockade was
MMR-deficient.sup.25. We therefore hypothesized that MMR-deficient
tumors are more responsive to PD-1 blockade than are MMR-proficient
tumors.
[0010] To test this hypothesis, we initiated a phase 2 clinical
trial to evaluate immune checkpoint blockade in patients whose
tumors had or did not have MMR-deficiency. Since MMR deficiency in
tumors arises through two routes.sup.26-28, we recruited patients
with Hereditary Non-Polyposis Colorectal Cancer (HNPCC, also known
as Lynch Syndrome), which results from an inherited germline defect
in one of four MMR genes followed by a second inactivating somatic
change in the remaining wild-type allele. We also recruited
patients with sporadic MMR-deficient tumors, where both alleles of
a MMR gene are inactivated by somatic mutations or by epigenetic
silencing.sup.29. In either case, the neoplasms that arise harbor
hundreds or thousands of mutations.sup.16,18.
[0011] There is a continuing need in the art to improve cancer
treatments so that the lives of patients are not curtailed and so
that the quality of life is not diminished.
SUMMARY OF THE INVENTION
[0012] According to one embodiment of the invention a method of
treating a cancer patient is provided. The cancer patient has a
high mutational burden, such as found in microsatellite instable
cancer (MSI). An immune checkpoint inhibitory antibody is
administered to the cancer patient.
[0013] According to another embodiment of the invention a method of
treating a cancer patient is provided. A sample from a cancer
patient is tested for one or more microsatellite markers selected
from the group consisting of BAT-25, BAT-26, MONO-27, NR-21, NR-24,
Penta C, and Penta D, and determined to have microsatellite
instability. The cancer is selected from the group consisting of:
colon, gastric, endometrial, cholangiocarcinoma, pancreatic, and
prostate cancers. An anti-PD-1 antibody is administered to the
cancer patient.
[0014] According to another embodiment of the invention a method is
provided for categorizing a tumor of a human. A sample from the
human is tested to evaluate stability of one or more microsatellite
markers. Microsatellite instability is determined in the sample.
The tumor is identified as a good candidate for treatment with an
immune checkpoint inhibitory antibody.
[0015] According to yet another embodiment of the invention a
method is provided for categorizing a tumor of a human. A sample
from the human is tested to evaluate stability of one or more
microsatellite markers. Microsatellite stability in the sample is
determined. The tumor is identified as a bad candidate for
treatment with an immune checkpoint inhibitory antibody.
[0016] These and other embodiments which will be apparent to those
of skill in the art upon reading the specification provide the art
with methods for treating micro satellite instable cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1A-1B. Clinical Responses to pembrolizumab. (FIG. 1A)
Biochemical Responses. Serum protein biomarker levels were measured
with each cycle and the values represent percent change from
baseline. Patients were included if baseline tumor marker values
were greater than the upper limit of normal. CA-125 was used for a
patient with endometrial cancer; CA19-9 was used for one
cholangiocarcinoma and one ampullary cancer; and CEA was used for
all other patients. Green, red, and black lines represent patients
with MMR-deficient CRCs, MMR-proficient CRCs, and MMR-deficient
non-CRC, respectively. (FIG. 1B) Radiographic responses. Tumor
responses were measured at regular intervals and values show the
best fractional change of the sum of longest diameters (SLD) from
the baseline measurements of each measurable tumor.
[0018] FIGS. 2A-2D. Clinical benefit to pembrolizumab according to
MMR status. Kaplan-Meier curves are shown for (FIG. 2A)
progression-free survival in the colorectal cancer cohorts, (FIG.
2B) overall survival in the colorectal cancer cohorts, (FIG. 2C)
progression-free survival of patients with MMR-deficient cancers
other than colorectal (median PFS=5.4 months; 95% CI, 3% to not
estimable), and (FIG. 2D) overall survival of patients with
MMR-deficient cancers other than colorectal. In both cohorts with
MMR-deficient tumors (CRC and non-CRC), median overall survival was
not reached. Patients in the cohort with MMR-proficient cancers had
a median PFS of 2.2 months (95% CI 1.4 to 2.8%) and a median OS of
5.0 months (95% CI 3.0 to not estimable).
[0019] FIG. 3 (Figure S2.) Spider plot of radiographic response.
Tumor responses were measured at regular intervals and values show
percent change of the sum of longest diameters (SLD) from the
baseline measurements of each measurable tumor. Patients were only
included if baseline and on study treatment scans were available.
Green and red represent patients with MMR-deficient and proficient
CRCs, respectively. Blue represents patients with MMR-deficient
cancers other than CRC.
[0020] FIGS. 4A-4B (Figure S3). MMR-proficient and deficient CRCs
have comparable time on treatment and duration of metastatic
disease prior to study enrollment. Kaplan-Meier estimates of (FIG.
4A) time on therapy immediately prior to study enrollment (HR 0.81,
95% CI 0.38 to 1.752, p=0.60) and (FIG. 4B) duration of metastatic
disease prior to enrollment (HR 1.13, 95% CI 0.49 to 2.62, p=0.78)
on this pembrolizumab study were comparable between the
MMR-deficient and proficient CRC cohorts. The short duration on
prior therapy is expected in a treatment refractory CRC
population.
[0021] FIG. 5 (Figure S4.) Waterfall plot of biochemical response.
Serum protein biomarker levels were measured with each cycle and
the values represent best percent change from baseline. Patients
were included if baseline tumor marker values were greater than the
upper limit of normal. CA-125 was used for a patient with
endometrial cancer; CA19-9 was used for 1 cholangiocarcinoma and 1
ampullary cancer; and CEA was used for all other patients. Green
and red represent patients with MMR-deficient and proficient CRCs,
respectively. Blue represents patients with MMR-deficient cancers
other than CRC.
[0022] FIGS. 6A-6B (Figure S5.) Somatic mutations in MMR-deficient
and proficient tumors. Total somatic mutations per tumor identified
by exome sequencing of tumor and matched normal DNA (FIG. 6A) and
correlation with objective responses (FIG. 6B) (non-parametric
Wilcoxon test, p=0.007 and Jonckheere-Terpstra test for trend,
p=0.02).
[0023] FIG. 7 (Figure S6). Immunohistochemistry of CD8 and PD-L1
Expression. The invasive front (yellow dashed line) from a
MMR-deficient CRC (subject #16, top) and MMR-proficient CRC
(subject #3, bottom). The yellow dashed line separates tumor (T)
and normal (N) tissue. There is marked expression of PD-L1 (blue
arrows) and CD8 (brown dots) in the MMR-deficient tumor (top
panels) patient while there is very little expression of either
marker in the MMR-proficient tumor (bottom panels). Representative
images of tumor infiltrating lymphocytes (TIL) in another
MMR-deficient CRC (subject #19, top) and MMR-proficient CRC
(subject #3, bottom) immunolabeled with an antibody to CD8 (brown
dots). Note the infiltration of CD8 cells in the MMR-deficient
tumor. Invasive front original magnification 10.times. and TIL
20.times..
[0024] FIG. 8 (Figure S7.) CD8 and PD-L1 Expression in the
MMR-deficient and MMR-proficient tumor microenvironment. T cell
density units are cells/mm2 of tumor. Invasive front refers to the
immune cells (TILs and macrophages) at the junction of the tumor
and normal tissue. P-values obtained using an unpaired t-test.
[0025] FIG. 9 (Figure S8.) CD8 expression and clinical benefit to
pembrolizumab. Correlation between the intratumoral CD8.sup.+ T
cell density (cells/mm2) and objective response
(Jonckheere-Terpstra test for trend, p=0.02).
[0026] FIG. 10 (Table S1.) Comparison of immune-related and RECIST
response criteria (adapted from Wolchok et al. Clin Can Res 2009;
15:7412-20.)
[0027] FIG. 11 (Table S2.) Immune-Related response to treatment
[0028] FIG. 12 (Table S4.) Correlation of total somatic mutations
and mutation associated neoantigens (MANA) with clinical
outcomes
[0029] FIG. 13 (Table S5.) Correlation of immune markers with
clinical outcome
DETAILED DESCRIPTION OF THE INVENTION
[0030] The inventors have found that immune checkpoint inhibitors
work best in tumors with high mutation burdens. Furthermore, tumors
deficient in mismatch repair are particularly susceptible to a
particular form of immunotherapy because this phenotype results in
ongoing accumulation of mutations at a high frequency. The
inventors have developed a treatment for cancer patients that
display the microsatellite instability phenotype or other high
mutational burden. The treatment involves an inhibitory antibody
for an immune checkpoint. Such checkpoints include PD-1, IDO,
CTLA-4, PD-L1, and LAG-3. Other immune checkpoints can be used as
well. Antibodies can be administered by any means that is
convenient, including but not limited to intravenous infusion, oral
administration, subcutaneous administration, sublingual
administration, ocular administration, nasal administration,
etc.
[0031] Microsatellite instability (MSI) tumors are deficient in DNA
mismatch repair which leads to a high rate of spontaneous mutations
and the potential for the expression of neo-antigens. Furthermore,
similar to melanoma, in MSI positive colon cancers, there is often
prominent lymphocyte infiltration. Any tumors that are MSI or
otherwise high mutational burden may be treated according to the
invention. They may be tested for the attribute of MSI according to
any method known in the art, including but not limited that
described in example 1 below. Any of one or more MSI markers can be
tested to determine an MSI phenotype. Samples may be tested for
high mutational burden by identifying tumors with at least 100, at
least 200, at least 300, at least 400, at least 500, at least 600,
at least 700, at least 800, at least 900, at least 1000, at least
1100, at least 1200, at least 1300, at least 1400, at least 1500,
or at least 1600 mutations per tumor genome. High mutational burden
means a large number of somatic mutations in the tumor relative to
normal tissues of the individual. An average number of somatic
mutations in a non-MSI tumor is about 70 somatic mutations.
[0032] Any type of tumor that displays the MSI phenotype or a high
mutational burden may be tested and/or treated according to the
invention. These include without limitation cancers of the colon,
gastric, endometrial, cholangiocarcinoma, pancreatic, and prostate
cancer. Tumors of the ampulla, biliary, brain, including glioma,
breast, lung, skin, esophagus, liver, kidney, ovaries, sarcoma,
uterus, cervix, bladder, testes, oral cavity, tongue, and small and
large bowel may also be tested and/or treated.
[0033] Testing of MSI can be accomplished by any means known in the
art. One or more of the following markers may be tested: five
nearly monomorphic mononucleotide repeat markers (BAT-25, BAT-26,
MONO-27, NR-21 and NR-24) and two highly polymorphic
pentanucleotide repeat markers (Penta C and Penta D). In one
commercial system which can be used, fluorescently labeled primers
(marker panel) are used for co-amplification of all seven of the
above named markers. Fragments are detected after amplification for
assignment of genotype/phenotype.
[0034] Samples that can be tested for MSI include tumor tissue as
well as body fluids that contain nucleic acids shed from tumors.
Testing for tumor DNA in such tissues and body fluids is well
known.
[0035] Types of antibodies which can be used include any that are
developed for the immune checkpoint inhibitors. These can be
monoclonal or polyclonal. They may be single chain fragments or
other fragments of full antibodies, including those made by
enzymatic cleavage or recombinant DNA techniques. They may be of
any isotype, including but not limited to IgG, IgM, IgE. The
antibodies may be of any species source, including human, goat,
rabbit, mouse, cow, chimpanzee. The antibodies may be humanized or
chimeric. The antibodies may be conjugated or engineered to be
attached to another moiety, whether a therapeutic molecule or a
tracer molecule. The therapeutic molecule may be a toxin, for
example.
[0036] The data from the small phase 2 trial of pembrolizumab to
treat tumors with and without deficiency of MMR supports the
hypothesis that MMR-deficient tumors are more responsive to PD-1
blockade than are MMR-proficient tumors. MMR-deficiency occurs in
many cancers, including those of the colorectum, uterus, stomach,
biliary tract, pancreas, ovary, prostate and small
intestine.sup.18,34-42. Patients with MMR-deficient tumors of these
types also benefit from anti-PD-1 therapy, as may patients whose
tumors contain other DNA repair deficiencies, such as those with
mutations in POLD, POLE, or MYH..sup.18,43,44
[0037] The hypothesis that MMR-deficient tumors stimulate the
immune system is not a new idea.sup.45, and has been supported by
the dense immune infiltration and Th1-associated cytokine-rich
environment observed in MMR-deficient tumors..sup.19-22,46 A recent
study refined these classic observations by showing that the
MMR-deficient tumor microenvironment strongly expressed several
immune checkpoint ligands including PD-1, PD-L1, CTLA-4, LAG-3 and
IDO, indicating that their active immune microenvironment is
counterbalanced by immune inhibitory signals that resists tumor
elimination.sup.47. That the immune infiltrate associated with
MMR-deficient carcinomas was directed at neoantigens was the most
likely explanation for both the old and new findings. The
correlation of higher mutational load and higher response rate to
anti-CTLA-4 in melanoma.sup.41 and anti-PD-1 in lung cancer.sup.48
provide further support for the idea that MANA recognition is an
important component of the endogenous anti-tumor immune
response.
[0038] Based on the results of the current and previous studies, we
suggest that the greatly (>20-fold) increased number of
mutation-associated neoantigens resulting from MMR deficiency (FIG.
12 (Table S4); also available on line at New England Journal of
Medicine; incorporated by reference herein) is the basis for the
enhanced anti-PD-1 responsiveness of this genetically defined
subset of cancers. Though our estimates for the number of
mutation-associated neoantigens in tumors is based only on in
silico predictions of binding-affinity, this suggestion is
consistent with the observation that MMR-proficient tumors have far
less infiltration of lymphocytes than MMR-deficient tumors (FIG. 7
(S6), FIG. 8 (S7) and FIG. 13 (Table S5); available on line at New
England Journal of Medicine; incorporated by reference herein).
Recent studies.sup.49,50 show that only a tiny proportion of
predicted neo-epitopes are actually presented on the cell surface
with MHC and are targets of endogenous T cell responses. It seems
likely, though that the number of predicted mutation-associated
neoantigens is proportionate to the number of actual
mutation-associated neoantigens, and tumors with a high number of
actual mutation-associated neoantigens are more likely to stimulate
the immune system to react against the tumor. Alternative
mechanisms underlying the difference in anti-PD-1 responsiveness
between MMR-deficient and MMR-proficient tumors should also be
considered. For example, different signaling pathways activated in
MMR-deficient and MMR-proficient tumors may result in differences
in secretion of soluble factors that could result in differential
activation of the PD-1 pathway within the tumor
microenvironment.sup.26-28. Genetic differences could effect
epigenetic differences that alter the expression of
tumor-associated self-antigens that in turn could alter the
antigenicity of the tumor. Experimental analyses of
antigen-specific immune responses as well as changes in immune
microenvironments should help to define the relative contribution
of these factors to the striking responsiveness of MMR-deficient
tumors to PD-1 antibodies.
[0039] Several notable observations were made during the course of
this study. First, changes in serum protein biomarkers, like CEA,
corresponded with clinical benefit after a single dose of therapy.
Declines in CEA levels preceded objective radiographic evidence by
several months; perhaps other biomarkers such as circulating tumor
DNA (ctDNA) may also be beneficial as surrogate markers of early
response..sup.51,52 Second, our results suggest that the evaluation
of tumor genomes can help guide immunotherapy. They support the
view that the number and type of alterations may prove useful for
judging the potential utility of immune checkpoint inhibitors, even
in MMR-proficient cancers.sup.41,48,53 Most importantly, our
results demonstrate a new approach for the treatment of a specific
class of tumors based solely on genetic status: i.e., without
regard to underlying tumor type.
[0040] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
Example 1
MSI Testing
[0041] MSI testing is already standardized and performed in
CLIA-certified laboratories without need for assay development.
Archived tumor samples or newly obtained biopsies will be used for
determining MSI. MSI status will be performed locally by CLIA
certified immunohistochemistry (IHC) or PCR based tests for
eligibility. Evaluable patients will be confirmed using the MSI
Analysis System from Promega at Johns Hopkins. This test will
determine MSI status through the insertion or deletion of repeating
units in the five nearly monomorphic mononucleotide repeat markers
(BAT-25, BAT-26, MONO-27, NR-21 and NR-24). At least 2 MSI loci are
required to be evaluable in Cohorts A and C. Patients may be
assigned to a new cohort and/or replaced based on the Promega test
results.
Example 2
Methods
[0042] Patients
[0043] Treatment-refractory progressive metastatic cancer patients
for this phase 2 study were recruited from three participating
centers (Table 1). Three cohorts were evaluated: Cohort A was
composed of patients with MMR-deficient colorectal adenocarcinomas;
Cohort B was composed of patients with MMR-proficient colorectal
adenocarcinomas; and Cohort C was composed of patients with
MMR-deficient cancers of types other than colorectal.
[0044] Study Oversight
[0045] The protocol, which can be found at NEJM.org, was approved
by each site's institutional review boards, and the study was
conducted in accordance with the Declaration of Helsinki and the
International Conference on Harmonization Guidelines for Good
Clinical Practice. All the patients provided written informed
consent before study entry. The principal investigator (D.L.) and
study sponsor (L.A.D.) were responsible for oversight of the study.
Merck donated the study drug, reviewed the final drafts of the
protocol and of this manuscript. The clinical study was primarily
funded through philanthropic support.
[0046] Study Design
[0047] This phase 2 trial was conducted using a Green-Dahlberg
two-stage design and consisted of the three parallel cohorts
described above. The study agent, pembrolizumab (Merck), was
administered at 10 mg/kg intravenously every 14 days. Pembrolizumab
is a humanized monoclonal anti-PD-1 antibody of the IgG4/kappa
isotype that blocks the interaction between PD-1 and its ligands,
PD-L1 and PD-L2.
[0048] Safety assessments were performed before each treatment.
Assessments of total tumor burden via measurements of serum
biomarkers were performed at the start of each cycle. Radiologic
assessments were made at 12 weeks and every 8 weeks thereafter.
Further details concerning the clinical protocol are provided in
the Example 3.
[0049] Analysis of Mismatch Repair Status
[0050] Tumors with genetic defects in MMR pathways are known to
harbor thousands of somatic mutations, especially in regions of
repetitive DNA known as microsatellites. The accumulation of
mutations in these regions of the genome is termed microsatellite
instability (MSI).sup.26-28. MMR-status was assessed using the MSI
Analysis System from Promega in tumors, through the evaluation of
selected microsatellite sequences particularly prone to copying
errors when MMR is compromised.sup.26-28. See Supplementary
Appendix for additional details.
[0051] Genomic & Bioinformatic Analyses
[0052] Primary tumor samples and matched normal peripheral-blood
specimens were obtained from a subset of subjects with
MMR-deficient and others with MMR-proficient carcinomas where
sufficient tumor tissue was available for exome
sequencing.sup.30and HLA haplotyping. To assess the potential for
mutant peptide binding, somatic exome data combined with the
individual patient's MHC class I HLA haplotype was applied to the
an epitope prediction algorithm.sup.31,32. This algorithm provided
an estimate of the total number of mutation-associated neoantigens
in each tumor. Additional details are provided in the Supplementary
Appendix (available on line at New England Journal of Medicine;
incorporated by reference herein).
[0053] Statistical Analysis
[0054] The primary endpoints for Cohorts A and B were
immune-related objective response rate (irORR) and immune-related
progression-free survival (irPFS) rate at 20 weeks assessed using
immune-related response criteria (irRC).sup.33. The primary
endpoint for Cohort C was irPFS rate at 20 weeks. Immune-related
criteria (i.e, criteria used to evaluate immune-based therapies)
are based on radiographic responses, and unlike RECIST criteria,
capture extent of disease after disease progression; these criteria
are defined and compared to RECIST v1.1 in FIG. 10 (Table S1).
Response rate and PFS rate at 20 weeks were evaluated and reported
in this study using RECIST v1.1 and irRC (FIG. 10 (Table S1)). PFS
and overall survival was summarized by Kaplan-Meier method. Details
of the hypothesis, the decision rules to reject the null hypotheses
and early-stopping rules for efficacy and futility, and statistical
methods are provided in the Supplementary Appendix.
Example 3
Supplementary Methods
[0055] Patients
[0056] To be eligible for participation in this study, patients had
to be at least 18 years of age, have histologically confirmed
evidence of previously-treated, progressive carcinoma. All patients
underwent MMR status testing prior to enrollment. All patients had
at least one measurable lesion as defined by the Response
Evaluation Criteria in Solid Tumors (RECIST), version 1.1, an
Eastern Cooperative Oncology Group (ECOG) performance-status score
of 0 or 1, and adequate hematologic, hepatic, and renal function.
Eligible patients with CRC must have received at least 2 prior
cancer therapies and patients with other cancer types must have
received at least 1 prior cancer therapy. Patients with untreated
brain metastases, history of HIV, hepatitis B, hepatitis C,
clinically significant ascites/effusions, or autoimmune disease
were excluded.
[0057] Study Oversight
[0058] Initial drafts of the manuscript were prepared by a subset
of the authors and all authors contributed to the final manuscript.
All the authors made the decision to submit the manuscript for
publication. The principal investigator and study sponsor vouch for
the accuracy and completeness of the data reported as well as
adherence to the protocol.
[0059] HLA Typing
[0060] HLA-A, HLA-B and HLA-C Sequence Based Typing can be divided
into three distinct steps, as described below. A generic, A*02
specific, B generic, B group specific, C generic and C*07 specific
PCR and sequencing mixes were made in the JHU core facility.
Celera's AlleleSEQR HLA-B Sequence Based Typing kit was used for B
generic SBT. The HLA-A typing scheme is composed of two PCR
reactions, A generic and A*02 specific. A generic amplicon
encompasses partial exon 1-partial exon 5. A*02 amplicon
encompasses partial intron 1-partial exon 5. HLA-B typing scheme is
composed of two PCR reactions, B generic and B group specific. The
B generic PCR is a multiplexed reaction containing two PCR
amplicons encompassing exon 2-exon 3 and exon 4-exon 7. B group
specific amplicon encompasses partial intron 1-partial exon 5.
HLA-C typing scheme is composed of two PCR reactions, C generic and
C*07 specific. C generic and C*07 specific amplicons encompasses
exons 1-7.
[0061] The specificity of the HLA-A and B PCR employed AmpliTaq
Gold DNA polymerase. The GeneAmp High Fidelity enzyme is used for
the HLA-C and C*07 PCR mixes. This enzyme is a mix of two
polymerases: AmpliTaq DNA polymerase (non-proofreading polymerase)
and a proofreading polymerase. This enzyme mix is necessary to
produce efficient and robust amplification of the larger full
length HLA-C amplicon.
[0062] PCR product purification was performed using Exonuclease I
and Shrimp Alkaline Phosphatase The A generic and B generic
amplicons were bi-directionally sequenced for exons 2,3,4. The C
generic amplicon was bi-directionally sequenced for exons 2,3 and
sequenced in a single direction for exons 1,4,5,6,7. A*02 specific,
B group specific and C*07 specific amplicons were sequenced in a
single direction for exons 2,3. All sequencing reactions were
performed with Big Dye Terminator V1.1 from Applied Biosystems and
sequenced with an ABI Prism 3500XL Genetic Analyzer. Conexio
Genomic's "Assign SBT" allele assignment software was used to
process the data files.
[0063] Mismatch Repair Status Testing.sup.1,2
[0064] Six slides of tumor and normal (uninvolved lymph node or
margin of resection) were cut (5 microns each), deparaffinized
(xylene), and one stained with hematoxylin and eosin (H+E). A tumor
area containing at least 20% neoplastic cells, designated by a
board-certified Anatomic Pathologist was macrodissected using the
Pinpoint DNA isolation system (Zymo Research, Irvine, Calif.),
digested in proteinase K for 8 hours and DNA was isolated using a
QIAamp DNA Mini Kit (Qiagen, Valencia, Calif.). MSI was assessed
using the MSI Analysis System (Promega, Madison, Wis.), composed of
5 pseudomonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21,
NR-24 and MONO-27) to detect MSI and 2-pentanucleotide repeat loci
(PentaC and PentaD) to confirm identity between normal and tumor
samples, per manufacturer's instructions. Following amplification
of 50-100 ng DNA, the fluorescent PCR products were sized on an
Applied Biosystems 3130x1 capillary electrophoresis instrument
(Invitrogen, Calsbad, Calif.). Pentanucleotide loci confirmed
identity in all cases. Controls included water as a negative
control and a mixture of 80% germline DNA with 20% MSI cancer DNA
as a positive control. The size in bases was determined for each
microsatellite locus and tumors were designated as MSI if two or
more mononucleotide loci varied in length compared to the germline
DNA.
[0065] Sequencing Analysis
[0066] Samples
[0067] Samples provided as FFPE blocks or frozen tissue underwent
pathological review to determine tumor cellularity. Tumors were
macrodissected to remove contaminating normal tissue, resulting in
samples containing >20% neoplastic cells. Matched normal samples
were provided as blood, saliva or normal tissue obtained from
surgery.
[0068] Sample Preparation and Next-Generation Sequencing.sup.3
[0069] Sample preparation, library construction, exome capture,
next generation sequencing, and bioinformatics analyses of tumor
and normal samples were performed at Personal Genome Diagnostics,
Inc. (Baltimore, Md.). In brief, DNA was extracted from frozen or
formalin-fixed paraffin embedded (FFPE) tissue, along with matched
blood or saliva samples using the Qiagen DNA FFPE tissue kit or
Qiagen DNA blood mini kit (Qiagen, CA). Genomic DNA from tumor and
normal samples were fragmented and used for Rumina TruSeq library
construction (Illumina, San Diego, Calif.) according to the
manufacturer's instructions or as previously described4. Briefly,
50 nanograms (ng)-3 micrograms (.mu.g) of genomic DNA in 100
microliters (.mu.l) of TE was fragmented in a Covaris sonicator
(Covaris, Woburn, Mass.) to a size of 150-450 bp. To remove
fragments smaller than 150 bp, DNA was purified using Agencourt
AMPure XP beads (Beckman Coulter, IN) in a ratio of 1.0 to 0.9 of
PCR product to beads twice and washed using 70% ethanol per the
manufacturer's instructions. Purified, fragmented DNA was mixed
with 36 .mu.l of H2O, 10 .mu.l of End Repair Reaction Buffer, 5
.mu.l of End Repair Enzyme Mix (cat# E6050, NEB, Ipswich, Mass.).
The 100 .mu.l end-repair mixture was incubated at 20.degree. C. for
30 min, and purified using Agencourt AMPure XP beads (Beckman
Coulter, IN) in a ratio of 1.0 to 1.25 of PCR product to beads and
washed using 70% ethanol per the manufacturer's instructions. To
A-tail, 42 .mu.l of end-repaired DNA was mixed with 5 .mu.l of
10.times.dA Tailing Reaction Buffer and 3 .mu.l of Klenow
(exo-)(cat# E6053, NEB, Ipswich, Mass.). The 50 .mu.l mixture was
incubated at 37.degree. C. for 30 min and purified using Agencourt
AMPure XP beads (Beckman Coulter, IN) in a ratio of 1.0 to 1.0 of
PCR product to beads and washed using 70% ethanol per the
manufacturer's instructions. For adaptor ligation, 25 .mu.l of
A-tailed DNA was mixed with 6.7 .mu.l of H2O, 3.3 .mu.l of
PE-adaptor (Illumina), 10 .mu.l of 5.times. Ligation buffer and 5
.mu.l of Quick T4 DNA ligase (cat# E6056, NEB, Ipswich, Mass.). The
ligation mixture was incubated at 20.degree. C. for 15 min and
purified using Agencourt AMPure XP beads (Beckman Coulter, IN) in a
ratio of 1.0 to 0.95 and 1.0 of PCR product to beads twice and
washed using 70% ethanol per the manufacturer's instructions. To
obtain an amplified library, twelve PCRs of 25 .mu.l each were set
up, each including 15.5 .mu.l of H2O, 5 .mu.l of 5.times. Phusion
HF buffer, 0.5 .mu.l of a dNTP mix containing 10 mM of each dNTP,
1.25 .mu.l of DMSO, 0.25 .mu.l of Illumina PE primer #1, 0.25 .mu.l
of Illumina PE primer #2, 0.25 .mu.l of Hotstart Phusion
polymerase, and 2 .mu.l of the DNA. The PCR program used was:
98.degree. C. for 2 minutes; 12 cycles of 98.degree. C. for 15
seconds, 65.degree. C. for 30 seconds, 72.degree. C. for 30
seconds; and 72.degree. C. for 5 min. DNA was purified using
Agencourt AMPure XP beads (Beckman Coulter, IN) in a ratio of 1.0
to 1.0 of PCR product to beads and washed using 70% ethanol per the
manufacturer's instructions. Exonic or targeted regions were
captured in solution using the Agilent SureSelect v.4 kit according
to the manufacturer's instructions (Agilent, Santa Clara, Calif.).
The captured library was then purified with a Qiagen MinElute
column purification kit and eluted in 17 .mu.l of 70.degree. C. EB
to obtain 15 .mu.l of captured DNA library. (5) The captured DNA
library was amplified in the following way: Eight 30 uL PCR
reactions each containing 19 .mu.l of H2O, 6 .mu.l of 5.times.
Phusion HF buffer, 0.6 .mu.l of 10 mM dNTP, 1.5 .mu.l of DMSO, 0.30
.mu.l of Illumina PE primer #1, 0.30 .mu.l of Illumina PE primer
#2, 0.30 .mu.l of Hotstart Phusion polymerase, and 2 .mu.l of
captured exome library were set up. The PCR program used was:
98.degree. C. for 30 seconds; 14 cycles (exome) or 16 cycles
(targeted) of 98.degree. C. for 10 seconds, 65.degree. C. for 30
seconds, 72.degree. C. for 30 seconds; and 72.degree. C. for 5 min.
To purify PCR products, a NucleoSpin Extract II purification kit
(Macherey-Nagel, PA) was used following the manufacturer's
instructions. Paired-end sequencing, resulting in 100 bases from
each end of the fragments for exome libraries and 150 bases from
each end of the fragment for targeted libraries, was performed
using Illumina HiSeq 2000/2500 and Illumina MiSeq instrumentation
(Illumina, San Diego, Calif.).
[0070] Primary Processing of Next-Generation Sequencing Data and
Identification of Putative Somatic Mutations3
[0071] Somatic mutations were identified using VariantDx custom
software (Personal Genome Diagnostics, Baltimore, Md.) for
identifying mutations in matched tumor and normal samples. Prior to
mutation calling, primary processing of sequence data for both
tumor and normal samples were performed using Illumina CASAVA
software (v1.8), including masking of adapter sequences. Sequence
reads were aligned against the human reference genome (version
hg18) using ELAND with additional realignment of select regions
using the Needleman-Wunsch method 5. Candidate somatic mutations,
consisting of point mutations, insertions, and deletions were then
identified using VariantDx across the either the whole exome or
regions of interest. VariantDx examines sequence alignments of
tumor samples against a matched normal while applying filters to
exclude alignment and sequencing artifacts. In brief, an alignment
filter was applied to exclude quality failed reads, unpaired reads,
and poorly mapped reads in the tumor. A base quality filter was
applied to limit inclusion of bases with reported phred quality
score >30 for the tumor and >20 for the normal. A mutation in
the tumor was identified as a candidate somatic mutation only when
(i) distinct paired reads contained the mutation in the tumor; (ii)
the number of distinct paired reads containing a particular
mutation in the tumor was at least 10% of read pairs; (iii) the
mismatched base was not present in >1% of the reads in the
matched normal sample as well as not present in a custom database
of common germline variants derived from dbSNP; and (iv) the
position was covered in both the tumor and normal at
>150.times.. Mutations arising from misplaced genome alignments,
including paralogous sequences, were identified and excluded by
searching the reference genome.
[0072] Candidate somatic mutations were further filtered based on
gene annotation to identify those occurring in protein-coding
regions. Functional consequences were predicted using snpEff and a
custom database of CCDS, RefSeq and Ensembl annotations using the
latest transcript versions available on hg18 from UCSC
(https://genome.ucsc.edu/). Predictions were ordered to prefer
transcripts with canonical start and stop codons and CCDS or Refseq
transcripts over Ensembl when available. Finally mutations were
filtered to exclude intronic and silent changes, while retaining
mutations resulting in missense mutations, nonsense mutations,
frameshifts, or splice site alterations. A manual visual inspection
step was used to further remove artifactual changes.
[0073] Mutant Peptide MHC Binding Prediction
[0074] Somatic frameshift, insertions, deletions, and missense
mutations predicted to result in an amino acid change were analyzed
for potential MHC class I binding based on the individual patient's
HLA haplotype. Our initial analysis focused on HLA-A and HLA-B.
Amino acid mutations were linked to their corresponding CCDS
accession number and in instances where this was unavailable,
either a Refseq or ensemble transcript was used to extract the
protein sequence. To identify 8mer, 9mer, and 10 mer epitopes,
amino acid fragments surrounding each mutation were identified.
These 15, 17, and 19 mutant amino acid fragments were analyzed by
the epitope prediction program NetMHC 3.4.6 Epitopes with a
predicted affinity of <50 nm were considered to be strong
potential binders and epitopes with a predicted affinity of <500
nm were considered to be weak potential binders as suggested by the
NetMHC group6.
[0075] To further refine the total neoantigen burden, we repeated
that same process for the complementary wild-type peptide for each
mutant peptide. We then filtered for mutant peptides that were
strong potential binders when the complementary wild-type peptide
was predicted a weak potential binder. These mutant peptides are
referred to as mutation-associated neoantigens (MANA). In the event
that a patient had a (e.g., cases 1, 17 and 21) single MHC
haplotype not supported by NetMHC 3.4, the individual haplotype was
not included in our analysis.
[0076] Statistical Methods
[0077] Design of the Trial.sup.7
[0078] This trial was conducted using a parallel two-stage design
to simultaneously evaluate the efficacy of MK-3475 and MSI as a
treatment selection marker for anti-PD-1 therapy. It consisted of
two-stage phase 2 studies in parallel in the three cohorts of
patients described in the text. The study agent, MK-3475, was
administered at 10 mg/kg intravenously every 14 days.
[0079] For each of Cohort A and B, the co-primary endpoints were
progression-free-survival (irPFS) at 20 weeks and objective
response (irOR) assessed using immune related criteria. A step-down
gatekeeping procedure was used to preserve the overall type I
error. A two-stage Green-Dahlberg design was used to evaluate
irPFS, with interim and final analysis after 15 and 25 patients,
respectively. At stage 1, .gtoreq.1 of 15 free-of-progression at 20
weeks were required to proceed to the second stage, and .gtoreq.4
of 25 free-of-progression at 20 weeks were then required to proceed
to test for irOR, with .gtoreq.4 of 25 responders (irCR or irPR)
indicating promising efficacy in that cohort. Each cohort could be
terminated for efficacy as soon as .gtoreq.4 free-of-progression at
20 weeks and .gtoreq.4 responses were confirmed, or be terminated
for futility as soon as 0 of 15 in stage 1 were free-of-progression
at 20 weeks or .gtoreq.22 subjects had disease progression by 20
weeks. This design achieves 90% power to detect a 20-week irPFS
rate of 25% and 80% power to detect an irOR rate (irORR) of 21%,
with an overall type I error of 0.05 at the null hypothesis of
20-week irPFS rate of 5% and irORR of 5%.
[0080] For Cohort C, the primary endpoint was irPFS at 20 weeks. A
two-stage Green-Dahlberg two-stage design was used, with an interim
and final analysis after 14 and 21 patients; at stage 1, .gtoreq.1
of 14 free-of-progression at 20 weeks were required to proceed to
the second stage, with .gtoreq.4 of 21 free-of-progression at 20
weeks at the end indicating adequate efficacy in Cohort C. The
cohort could be terminated as soon as .gtoreq.4 free-of-progression
at 20 weeks were confirmed. The design has 81% power to detect a
20-week irPFS rate of 25% with a 5% type I error at the null
hypothesis of 20-week irPFS rate of 5%.
[0081] Statistical Analysis
[0082] Response and progression were evaluated using RECIST v1.1
and the immune-related response criteria (irRC) adopted from
Wolchok et al.8, which uses the sum of the products of
bidimensional tumor measurements and incorporates new lesions into
the sum. Progression-free survival (PFS) rates and irPFS rate at
20-weeks was estimated as the proportion of patients who were
free-of-disease progression and alive at 20 weeks after the
initiation of pembrolizumab. Patients who had disease progression
prior to 20 weeks or were enrolled for >20 weeks at the time the
study data were collated were included in the analysis for
estimating 20-week PFS (irPFS) rate. Patients who dropped out early
due to toxicities or worsening disease and therefore did not have
20-week tumor assessment were considered as having progressive
disease. ORR (irORR) was the proportion of patients who achieved
best overall response of CR or PR (irCR or irPR). Patients who were
in the study long enough to have tumor response evaluations were
included in the analysis for estimating response rates. Among those
who responded (CR or PR), duration of response was the time of
first RECIST response to the time of disease progression, and was
censored at the last evaluable tumor assessment for responders who
had not progressed.
[0083] PFS and irPFS were defined as the time from the date of
initial dose to the date of disease progression or the date of
death due to any cause, whichever occurred first. PFS and irPFS
were censored on the date of the last evaluable tumor assessment
documenting absence of progressive disease for patients who were
alive and progression-free. Overall survival (OS) was defined as
the time from the date of initial dose to death due to any cause.
For patients who were still alive at the time of analysis, the OS
time was censored on the last date the patients were known to be
alive. Survival times were summarized by the Kaplan-Meier method.
As a post hoc analysis, log-rank tests were used to compare Cohort
A and B and hazard ratios were estimated based on Cox models.
[0084] The association of percent CEA decline after 1 cycle with
PFS or OS was assessed using landmark analysis based on Cox
regression models. For correlative studies, non-parametric Wilcoxon
test was used to compare mutational load between MMR-deficient and
MMR-proficient patients. The effects of baseline mutational burden
and immune markers on response and survival times were examined
using logistic regression and Cox regression, respectively.
[0085] Immunohistochemistry & Image Analysis
[0086] The fraction of malignant cells exhibiting a membranous
pattern of B7-H1 expression and the percentage at the invasive
front were quantified by three pathologists (R.A.A., F.B., and
J.M.T.) as previously reported9,10. Image analysis was used to
determine the number of CD8 diaminobenzidine (DAB)-stained cells.
Using the H&E-stained slide for each case, we identified the
following regions: i) tumor, ii) invasive front (the boundary
between malignant and non-malignant tissue), and iii) normal
tissue. The CD8-stained slides were scanned at 20.times. equivalent
magnification (0.49 micrometers per pixel) on an Aperio ScanScope
AT. Regions corresponding to tumor, invasive front and normal
tissue (above, from the H&E) were annotated on separate layers
using Aperio ImageScope v12.1.0.5029.
[0087] CD8-positive lymphocyte density was calculated in each of
the above regions using a custom algorithm implemented in PIP11.
Results were converted to Deepzoom images using the VIPS library12
and visualized using the OpenSeadragon viewer
(http://openseadragon.github.io).
REFERENCES FOR EXAMPLE 3 ONLY
[0088] 1. Bacher J W, Flanagan L A, Smalley R L, et al. Development
of a fluorescent multiplex assay for detection of MSI-High tumors.
Disease markers 2004; 20:237-50. [0089] 2. Murphy K M, Zhang S,
Geiger T, et al. Comparison of the microsatellite instability
analysis system and the Bethesda panel for the determination of
microsatellite instability in colorectal cancers. The Journal of
molecular diagnostics: JMD 2006; 8:305-11. [0090] 3. Jones S,
Anagnostou V, Lytle K, et al. Personalized genomic analyses for
cancer mutation discovery and interpretation. Science translational
medicine 2015; 7:283ra53. [0091] 4. Sausen M, Leary R J, Jones S,
et al. Integrated genomic analyses identify ARID1A and ARID1B
alterations in the childhood cancer neuroblastoma. Nature genetics
2013; 45:12-7. [0092] 5. Needleman S B, Wunsch C D. A general
method applicable to the search for similarities in the amino acid
sequence of two proteins. Journal of molecular biology 1970;
48:443-53. [0093] 6. Lundegaard C, Lamberth K, Harndahl M, Buus S,
Lund O, Nielsen M. NetMHC-3.0: accurate web accessible predictions
of human, mouse and monkey MHC class I affinities for peptides of
length 8-11. Nucleic acids research 2008; 36:W509-12. [0094] 7.
Buyse M, Michiels S, Sargent D J, Grothey A, Matheson A, de Gramont
A. Integrating biomarkers in clinical trials. Expert review of
molecular diagnostics 2011; 11:171-82. [0095] 8. Wolchok J D, Hoos
A, O'Day S, et al. Guidelines for the evaluation of immune therapy
activity in solid tumors: immune-related response criteria.
Clinical cancer research: an official journal of the American
Association for Cancer Research 2009; 15:7412-20. [0096] 9. Llosa N
J, Cruise M, Tam A, et al. The vigorous immune microenvironment of
microsatellite instable colon cancer is balanced by multiple
counter-inhibitory checkpoints. Cancer Discov 2015:43-51. [0097]
10. Taube J M, Anders R A, Young G D, et al. Colocalization of
Inflammatory Response with B7-H1 Expression in Human Melanocytic
Lesions Supports an Adaptive Resistance Mechanism of Immune Escape.
Science Translational Medicine 2012; 4:127ra37. [0098] 11. Cuka N,
Hempel H, Sfanos K, De Marzo A, Cornish T. PIP: An Open Source
Framework for Multithreaded Image Analysis of Whole Slide Images.
LABORATORY INVESTIGATION 2014; 94:398A-A. [0099] 12. Cupitt J,
Martinez K. VIPS: an image processing system for large images.
Electronic Imaging: Science & Technology; 1996: International
Society for Optics and Photonics. p. 19-28.
Example 4
[0100] Patients
[0101] 41 consecutive patients were enrolled and treated between
September 2013 and January 2015. (Table 1). Recruitment included
patients in pursuit of a clinical trial option who were known to
have tumors with mismatch repair, or who had tumors of unknown
status who were then tested. One patient in the MMR-deficient CRC
cohort was enrolled under an IRB eligibility waiver allowing a
grade 3 bilirubin level. A total of 32 CRC patients were enrolled
into Cohorts A and B. All CRC patients received .gtoreq.2 prior
chemotherapy regimens (median=4) except for one MMR-proficient
patient who had received one chemotherapeutic and one
(non-PD1-based) immunotherapeutic regimen.
[0102] Nine subjects diagnosed with MMR-deficient solid tumors
other than CRC were enrolled onto Cohort C. All Cohort C patients
received .gtoreq.1 prior cancer treatments (median=2).
Example 5
[0103] Primary Endpoint Evaluation
[0104] The irORR and irPFS at 20 weeks (FIG. 11 (Table S2)) for
Cohort A were 40% (4 of 10 patients; 95% CI, 12 to 74%) and 78% (7
of 9 patients; 95% CI, 40 to 97%) and for Cohort C were 71% (5 of 7
patients; 95% CI, 29 to 96%) and 67% (4 of 6 patients; 95% CI, 22
to 96%). In Cohort B, comprised of patients with MMR-proficient
CRCs, irORR and 20-week irPFS were 0% (95% CI, 0 to 20%) and 11% (2
of 18 patients; 95% CI, 1 to 35%). Both the MMR-deficient cohorts A
and C reached their predefined early stopping rule for efficacy
when four subjects were free-of-disease progression at 20 weeks and
four objective responses were observed based on immune-related
response criteria (FIG. 11 (Table S2); available on line at New
England Journal of Medicine; incorporated by reference herein; and
supplementary methods, above).
[0105] The median time of follow-up for patients was 32 weeks
(range, 5-51 weeks) for patients with MMR-deficient CRC (Cohort A),
12 weeks (range, 2-56 weeks) for patients with MMR-proficient CRC
(Cohort B) and 12 weeks (range, 4-42 weeks) for patients with
MMR-deficient non-CRC tumors (Cohort C). All patients evaluable for
20-week irPFS were followed for at least 20 weeks.
Example 6
[0106] Radiographic Evaluation
[0107] Of the ten evaluable MMR-deficient CRC patients in Cohort A,
four (40%; 95% CI, 12-74%) achieved objective responses by RECIST
criteria (Table 2, FIG. 1 and FIG. 3 (S2)). Patients were
considered not evaluable unless they underwent a 12-week scan. The
disease control rate was defined as the fraction of patients who
achieved an objective response or whose disease was stable, and was
90% in Cohort A (9 of 10 patients; 95% CI, 55-100%).
[0108] Of the seven evaluable patients with MMR-deficient cancer
types other than CRC enrolled in Cohort C, five (71%; 95% CI,
29-96%) achieved objective responses (Table 2, FIG. 3 (S2) and FIG.
1) using RECIST criteria and the disease control rate was 71% (5 of
7 patients; 95% CI, 29-96%).
[0109] Patients in Cohort C responded faster than patients in
Cohort A (median time to response by RECIST of 12 vs. 28 weeks,
p=0.03). Furthermore, all six MMR-deficient tumors that were not
associated with Lynch syndrome (100%) achieved an objective
response, whereas only three of eleven tumors (27%) associated with
Lynch Syndrome responded (Table S3; p=0.009; available on-line at
New England Journal of Medicine; and incorporated by reference
herein). No other baseline characteristics showed statistically
significant association with objective responses.
[0110] Of the 18 patients with MMR-proficient CRCs in Cohort B, no
objective responses were observed (Table 2, FIG. 3 (S2) and FIG. 1)
using RECIST criteria and the disease control rate was 11% (2 of 18
patients; 95% CI, 1 to 35%).
[0111] All patients who achieved a response by RECIST criteria
(FIG. 11 (Table 2)) also achieved a response by immune-related
response criteria (FIG. 11 (Table S2)).
Example 7
[0112] Survival
[0113] In Cohort A, the patients with MMR-deficient CRC, median
progression-free survival (PFS) and median overall survival (OS)
were not reached (FIG. 2). In contrast, the patients with
MMR-proficient cancers in Cohort B achieved a PFS of only 2.2
months (95% CI, 1.4-2.8) and a median OS of 5.0 months (95% CI, 3.0
to not estimable). In Cohort C (MMR-deficient non-CRC), the median
PFS was 5.4 months (95% CI, 3 to not estimable) and the median OS
was not reached.
[0114] A post hoc (FIG. 2) comparison of the MMR-deficient and
proficient CRC cohorts showed hazard ratios (HR) for disease
progression (HR=0.10; 95% CI, 0.03-0.37; p<0.001) and overall
survival (HR=0.22; 95% CI, 0.05-1.00; p=0.05), favoring patients
with MMR-deficient CRC.
[0115] To evaluate whether the difference in survival might be due
to prognostic differences, we measured the duration of time
patients had been diagnosed with metastatic disease and the
clinical performance of patients on their previous regimen prior to
enrollment. We found that there was no significant difference
between MMR-deficient vs. MMR-proficient CRC patients with respect
to their duration of metastatic disease (p=0.77; Log-rank test) or
median PFS (p=0.60, Log-rank test) on their prior regimens (FIG. 4
(S3)). We also performed an additional multivariate analysis of PFS
and OS to examine the difference in outcomes between MMR-deficient
CRC and MMR-proficient tumors adjusting for elapsed time since
initial diagnosis. The magnitude of the hazard ratios for PFS (HR
0.04, 95% CI 0.01-0.21, P<0.001) and OS (HR 0.18, 95% CI
0.03-1.01, P=0.05), representing the different effect of
pembrolizumab between MMR-deficient and MMR-proficient tumors, was
maintained after adjusting for this potential difference.
Example 8
[0116] Safety Assessment
[0117] Adverse events occurring in >5% of patients are listed in
Table 3. Select adverse events included rash/pruritus (24%),
thyroiditis/hypothyroidism/hypophysitis (10%), and asymptomatic
pancreatitis (15%). While the numbers were small, thyroid function
abnormalities were limited to the MMR-deficient cohorts (Table
3).
Example 9
[0118] Tumor Markers
[0119] In the two CRC cohorts, baseline CEA levels were evaluable
and above the upper limit of normal (3 mg/dl), in 29 of 32 patients
prior to enrollment. Major CEA declines occurred in seven of the
ten patients with MMR-deficient CRC and in none of the 19 patients
with MMR-proficient CRCin which CEA was evaluable (FIG. 1 and FIG.
5 (S4)). In non-CRC MMR-deficient patients, tumor marker levels
(CEA, CA19-9 or CA-125) were elevated above the upper limit of
normal in four patients. CA19-9 or CA-125 declines of >70%
occurred in three of these four patients. Tumor marker kinetics of
all 3 cohorts are shown in FIG. 1. The level of CEA decline after 1
dose (between days 14 and 28) of pembrolizumab was predictive of
both progression-free (p=0.01) and overall survival outcomes
(p=0.02). The CEA response occurred well in advance of radiographic
confirmation of disease control (range, 10 to 35 weeks). In
contrast, patients who progressed showed rapid biomarker elevation
within 30 days of initiating therapy. Thus, changes in CEA levels
significantly preceded and correlated with ultimate radiographic
changes.
Example 10
[0120] Genomic Analysis
[0121] Analysis of whole-exome sequences showed an average of 1,782
somatic mutations per tumor in MMR-deficient patients (n=9)
compared with 73 mutations per tumor in MMR-proficient patients
(n=6) (non-parametric Wilcoxon test, p=0.007) (FIGS. 6A-6B (S5);
see also Table S3 which is available on-line at New England Journal
of Medicine; incorporated by reference herein). Most (63%) of these
mutations are predicted to alter amino acids.
[0122] These mutations were then assessed for their immunogenic
potential in the context of each patient's individual MHC
haplotype. We thereby identified an average of 578 and 21 potential
mutation-associated neoantigens from the tumors of MMR-deficient
and MMR-proficient patients, respectively (Table S3; which is
available on-line at New England Journal of Medicine; incorporated
by reference herein). The fraction of potential mutation-associated
neoantigens among all somatic mutations was similar in both cohorts
(averaging 32% and 29% in MMR-deficient and -proficient patients,
respectively). High numbers of somatic mutations and potential
mutation-associated neoantigens were associated with improved
progression-free survival and with a trend in favor of objective
response (FIG. 13 (S5) and FIG. 12 (Table S4); also available on
line at New England Journal of Medicine; incorporated by reference
herein).
Example 11
[0123] Immunohistochemistry
[0124] Expression of CD8 and PD-L1 were evaluated by
immunohistochemistry within the tumor and at the invasive fronts of
the tumor in the 30 cases in which tumor tissue was available (FIG.
7 (S6); also available on line at New England Journal of Medicine;
incorporated by reference herein). Tumors from patients in Cohorts
A and C contained a greater density of CD8-positive lymphoid cells
than did tumors from Cohort B patients (FIG. 8 (S7); p=0.10) and
CD8-labeling was associated with a trend favoring objective
response and stable disease (FIG. 9 (S8) and FIG. 13 (Table S5);
also available on line at New England Journal of Medicine;
incorporated by reference herein). This CD8-positive lymphoid
infiltrate was especially prominent at the invasive fronts of the
tumors (FIG. 8 (S7); p=0.04). Significant membranous PD-L1
expression only occurred in MMR-deficient patients and was
prominent on tumor infiltrating lymphocytes (TILs) and
tumor-associated macrophages located at the tumors' invasive fronts
(FIG. 8 (S7); p=0.04). Expression of CD8 and PD-L1 were not
statistically associated with PFS or OS (FIG. 13 (Table S5)).
TABLE-US-00001 TABLE 1 Demographic and Baseline Characteristics of
Patients MMR- MRC- MMR- deficient proficient deficient CRC CRC P
non-CRC Characteristic n = 11 n = 21 values.sup.1 n = 9 Age-years
median 46 61 0.02 57 range (24-65) (32-79) (34-92) Sex-no. (%)
Female 5(45) 8(38) 0.72 4(44) Male 6(55) 13(62) 5(56) Race-no. (%)
white 8(73) 17(81) 0.66 8(89) black 1(9) 3(14) 0(0) other 2(18)
1(5) 1(11) ECOG Performance Status-no. (%).sup.2 0 0(0) 6(29) 0.07
2(22) 1 11(100) 15(71) 7(78) Diagnosis-no. (%) Colon 9(82) 18(86)
>0.99 0(0) Rectal 2(18) 3(14) 0(0) Ampullary/ 0(0) N/A 4(44)
Cholangiocarcinoma Endometrial 0(0) N/A 2(22) Small bowel 0(0) N/A
2(22) Gastric 0(0) N/A 1(11) Histology-no. (%) Well/moderately
7(64) 18(86) 0.20 4(44) differentiated Poorly differentiated 4(36)
3(14) 3(33) Other 0(0) 0(0) 2(22) Stage IV-no. (%) (11)100 21(100)
>0.99 9(100) Liver metastases-no. (%) 6(55) 11(52) >0.99
6(67) Time since first diagnosis-months median 31 58 0.07 23 range
6-95 27-192 2-105 Prior systemic therapies-no. (%) 0 0(0) 0(0) 0.89
1(11) 2 3(27) 4(19) 5(56) 3 3(27) 5(24) 1(11) >4 5(45) 12(57)
2(22) Detected germline mutation or known Lynch-no. (%) Yes 9(82)
0(0) <0.001 4(44) No 2(18) 21(100) 4(44) Unknown 0(0) 0(0) 1(11)
BRAF wild type-no. (%) Yes 8(73) 11(52) 0.64 4(44) No 0(0) 1(5)
0(0) Unknown 3(27) 9(43) 5(56) KRAS wild type-no. (%) Yes 6(55)
13(62) 0.72 4(44) No 5(45) 8(38) 1(11) Unknown 0(0) 0(0) 4(44) MMR,
mismatch repair; CRC, colorectal cancer .sup.1MMR-deficient CRC
versus MMR-proficient CRC .sup.2ECOG, Eastern Cooperative Oncology
Group
TABLE-US-00002 TABLE 2 Objective RECIST responses MMR- MRC- MMR-
deficient proficient deficient CRC CRC non-CRC Type of Response-no.
(%) n = 10 n = 18 n = 7 Complete Response 0(0) 0(0) 1(14).sup.1
Partial Response 4(40) 0(0) 4(57).sup.2 Stable Disease (Week 12)
5(50) 2(11) 0(0) Progressive Disease 1(10) 11(61) 2(29) Not
Evaluable.sup.3 0(0) 5(28) 0(0) Objective Response Rate (%) 40 0 71
95% CI 12-74 0-19 29-96 Disease Control Rate (%).sup.4 90 11 71 95%
CI 55-100 1-35 29-96 Duration of Response- Not N/A.sup.5 Not median
weeks reached reached Time to Response, median 28(13-35) N/A.sup.5
11(10-13) weeks (range) .sup.1Originally PR at 12 weeks that was
converted to CR at 20 weeks .sup.2One PR at 12 weeks .sup.3Patients
were considered not evaluable if they did not undergo a 12 week
scan due to clinical progression. .sup.4The rate of disease control
was defined as the percentage of patients who had a complete
response, partial response or stable disease for 12 weeks or more.
.sup.5No responses recorded for MMR-proficient CRC patients
TABLE-US-00003 TABLE 3 Drug-Related Adverse Events All Grades Grade
3 or 4 Event-no (%).sup.1 N = 41 N = 41 Any 40(98) 17(41) Blood and
Lymphatic Anemia 8(20) 7(17) Lymphopenia 8(20) 8(20) Cardiac Sinus
tachycardia 4(10) 0 Dermatologic Dry skin 5(12) 0 Rash/pruritis
10(24) 0 Endocrine Disorders
Thyroiditis/Hypothyroidism/Hypophysitis 4(10) 0 Gastrointestinal
Abdominal Pain 10(24) 0 Anorexia 4(10) 0 Constipation 8(20) 0
Diarrhea 10(24) 2(5) Dry mouth 5(12) 0 Nausea 5(12) 0 Bowel
Obstruction 3(7) 3(7) Hepatobiliary ALT, elevated 3(7) 2(5)
Pancreatitis.sup.2 6(15) 0 Metabolism and Nutrition Hypoalbuminemia
4(10) 4(10) Hyponatremia 3(7) 3(7) Musculoskeletal Arthralgia 7(17)
0 Myalgia 6(15) 0 Nervous System Dizziness 4(10) 0 Headache 7(17) 0
Psychiatric Insomnia 3(7) 0 Respiratory.sup.3 Allergic Rhinitis
12(29) 0 Cough 4(10) 0 Dyspnea 6(15) 0 Upper Respiratory Infection
3(7) 0 Other Cold intolerance 6(15) 0 Edema 4(10) 0 Fatigue 13(32)
0 Fever 5(12) 0 Pain 14(34) 0 .sup.1Adverse Events occurring in
greater than 5% of patients .sup.2All cases of pancreatitis were
asymptomatic .sup.3One incidence of pneumonitis (2%)
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