Patent Application
20210363592
The presently-disclosed subject matter generally relates to treatment of breast cancer. In particular, certain embodiments of the presently-disclosed subject matter relate to predicting whether a subject who has received neoadjuvant chemotherapy will benefit from proceeding with a surgery and/or other therapy.
Patients with breast cancer are often treated with chemotherapy prior to surgery, which is referred to as neoadjuvant chemotherapy (NAC). NAC is sometimes provided in combination with immunotherapy. For example, anti-PD-L1 immunotherapy in combination with nab-paclitaxel has been approved for metastatic triple-negative breast cancer (TNBC).1 Furthermore, addition of the anti-programmed death-1 (PD-1) monoclonal antibody pembrolizumab to neoadjuvant chemotherapy (NAC) can significantly enhance TNBC pathological complete response (pCR) rates2.
Thus, existing clinical data indicate that chemotherapy combinations with immunotherapy demonstrate enhanced efficacy compared to chemotherapy alone. However, these results suggest a growing need to better understand how chemotherapy modulates the tumor-immune microenvironment (TIME).
High levels of stromal tumor-infiltrating lymphocytes (sTILs) in a pre-treatment biopsy are predictive of pCR in TNBC patients treated with NAC.3 In NAC-treated TNBC patients with residual disease (RD) at surgery or in untreated primary TNBC tumors, higher sTILs in the resected tumor also confer improved prognosis4-7.
However, uncertainty in connection with the immunomodulatory effect of chemotherapy on sTILs in patients, as well as the impact of chemotherapy on TIME, raises questions about the efficacy of sTTILS as a marker for anti-timer immunity in patients who have received NAC or NAC in combination with immunotherapy.
Following NAC, at the time planned for surgery, some patients are identified as having a pathological complete response (pCR). For pCR patients, because no tumor cells remain, one might conclude that surgery is unnecessary. Nonetheless, it is often the practice to perform surgery remove the ‘tumor scar’ from the patient. These pCR patients have a very good outcome (low chance of recurrence).
Following NAC, at the time planned for surgery, some patients are identified as having residual disease (RD). For RD patients, surgery is performed to extract the remaining tumor. However, despite the tumor extraction, the RD patients are more likely to have a recurrence.
Identifying patients at risk of recurrence, particularly in those with RD, is a major challenge. If patients could be included or excluded from having a risk of recurrence or RD, recommendations for further treatment or cessation of treatment could be appropriately provided. Patients who could benefit from surgery and/or further treatment could be identified, patients with a low-risk of recurrence could have additional piece of mind after surgery, and patients identified as having a pCR could ultimately be spared unproductive surgery.
The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.
This Summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
The presently-disclosed subject matter includes methods detecting expression of a combination of genes in a sample from a subject having breast cancer and who has received neoadjuvant chemotherapy (NAC), methods of determining likelihood of residual disease (RD) or pathological complete response (pCR), and methods of providing recommendations for further treatment or cessation of treatment could. The presently-disclosed subject matter provide methods, whereby subjects who could benefit from surgery and/or further treatment could be identified, and subjects identified as having a pCR could ultimately be spared unproductive surgery.
The method as disclosed herein is envisioned for use in connection with a subjecting having breast cancer. In some embodiments, the subject has triple-negative breast cancer (TNBC). The method as disclosed herein is also envisioned for use in connection with a subject who has received neoadjuvant chemotherapy (NAC). In some embodiments, it is possible that the subject will be consider surgery or other additional treatment.
In some embodiments of the presently-disclosed subject matter the method involves obtaining or having obtained a biological sample from the subject; and detecting or having detected expression levels in the sample of genes in one or more signatures.
Some embodiments make use of a first signature, sometimes referred to herein as a cytotoxic signature or a residual disease (RD) signature, includes the following eight genes PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, FGFBP2, and HLA-DRB5. In some embodiments of the method, at least five genes of considered.
Some embodiments of the method include a step of calculating a first signature score by adding the expression level of each of the genes selected for detection from PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, and FGFBP2; and subtracting the expression level of HLA-DRB5, if detected. The expression level can be expressed, for example, transcript count for the gene(s) being detected. In some embodiments, the method further involves identifying the subject as having a likelihood of residual disease (RD) when the first signature score is greater than a standardized control; or identifying the subject as having a likelihood of pathological complete response (pCR) when the first signature score is less than a standardized control. The standardized control can be selected according to methods know to those skilled in the art, for example, by detection of normalization genes.
Some embodiments of the method include a step of identifying the subject as having a likelihood of residual disease (RD) and/or cancer recurrence when there is an elevated level of each of the genes selected for detection from PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, and FGFBP2, and a reduced level of HLA-DRB5, if detected. Some embodiments of the method include a step of identifying the subject as having a likelihood of pathological complete response (pCR) when there is a reduced level of each of the genes selected for detection from PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, and FGFBP2, and an elevated level of HLA-DRB5, if detected.
Some embodiments of the presently-disclosed subject matter involve a second signature, sometimes referred to herein as a IFN/complement signature or pathological complete response (pCR) signature. The second signature includes the following sixty genes: SERPING1, IFIT3, IFI44L, IFI44, LAP3, FCGR1A, EPSTI1, IFIT2, TNFSF10, WARS1, IFITM3, MX1, MT2A, BATF2, IL15, IFIT1, STAT1, GBP4, ISG15, OAS3, JAK2, VAMP5, FGL2, PLSCR1, OASL, SAMD9L, USP18, SECTM1, APOL6, PLA2G4A, UBE2L6, CFB, PSME2, OAS2, STAT2, PARP14, CASP1, IFI35, HLA-DMA, GCH1, CD86, IL15RA, DDX60, LATS2, BST2, NMI, IFIH1, CASP4, EIF2AK2, PARP9, GBP2, TENT5A, OAS1, C1QC, C1QA, C2, KYNU, MMP14, PDP1, and CASP10.
In some embodiments of the method, at least ten genes of the second signature are considered. For example, in some embodiments the following ten genes of the second signature are considered: C1QC, CASP10, JAK2, IL15, TNFSF10, C1QA, IFIT3, EPSTI1, PSME2, and LAP3. In some embodiments of the method, more than 10 or even all sixty of the genes of the second signature are considered.
Some embodiments of the method include a step of calculating a second signature score by adding the expression level of each of the genes selected for detection. The expression level can be expressed, for example, transcript count for the gene(s) being detected. In some embodiments, the method also involves identifying the subject as having a likelihood of residual disease (RD) when the second signature score is less than a standardized control; or identifying the subject as having a likelihood of pathological complete response (pCR) when the second signature score is greater than a standardized control.
In some embodiments, the method includes identifying the subject as having a likelihood of residual disease (RD) and/or cancer recurrence when there is a reduced level of each of the genes in the second signature that are selected for detection. In some embodiments, the method includes identifying the subject as having a likelihood of pathological complete response (pCR) when there is an elevated level of each of the genes in the second signature that are selected for detection.
Some embodiments of the method further include administering or recommending administration of additional chemotherapy prior to surgery and/or administration of additional chemotherapy after surgery when the subject is identified as having a likelihood of RD; or proceeding or recommending proceeding with surgery without administering additional chemotherapy when the subject is identified has having a likelihood of pCR.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:
The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
The presently-disclosed subject matter includes methods detecting expression of a combination of genes in a sample from a subject having breast cancer and who has received neoadjuvant chemotherapy (NAC), methods of determining likelihood of residual disease (RD) or pathological complete response (pCR), and methods of providing recommendations for further treatment or cessation of treatment could. The presently-disclosed subject matter provide methods, whereby subjects who could benefit from surgery and/or further treatment could be identified, and subjects identified as having a pCR could ultimately be spared unproductive surgery.
The method as disclosed herein is envisioned for use in connection with a subjecting having breast cancer. In some embodiments, the subject has triple-negative breast cancer (TNBC). The method as disclosed herein is also envisioned for use in connection with a subject who has received neoadjuvant chemotherapy (NAC). In some embodiments, it is possible that the subject will be consider surgery or other additional treatment.
In some embodiments of the presently-disclosed subject matter the method involves obtaining or having obtained a biological sample from the subject; and detecting or having detected expression levels in the sample of genes in one or more signatures.
In some embodiments of the presently-disclosed subject matter the biological sample is a peripheral blood sample. In some embodiments, the biological sample is a buffy coat fraction of the whole peripheral blood, or purified immune cells from whole peripheral blood. In some embodiments, the biological sample is a sample comprising monocytes. In some embodiments, the biological sample is a tumor sample or a sample obtained from the tumor-immune microenvironment. In some embodiments, the biological sample is from a lymph node.
As noted, methods of the presently-disclosed subject matter involve detecting or having detected expression levels in the sample of genes in one or more signatures. Some embodiments make use of a first signature, sometimes referred to herein as a cytotoxic signature or a residual disease (RD) signature, includes the following eight genes PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, FGFBP2, and HLA-DRB5. In some embodiments of the method, at least five genes of considered. In some embodiments of the method, six, seven, or all eight of the genes of the first signature are considered.
Some embodiments of the method include a step of calculating a first signature score by adding the expression level of each of the genes selected for detection from PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, and FGFBP2; and subtracting the expression level of HLA-DRB5, if detected. The expression level can be expressed, for example, transcript count for the gene(s) being detected. In some embodiments, the method further involves identifying the subject as having a likelihood of residual disease (RD) when the first signature score is greater than a standardized control; or identifying the subject as having a likelihood of pathological complete response (pCR) when the first signature score is less than a standardized control. The standardized control can be selected according to methods know to those skilled in the art, for example, by detection of normalization genes.
Some embodiments of the method include a step of identifying the subject as having a likelihood of residual disease (RD) and/or cancer recurrence when there is an elevated level of each of the genes selected for detection from PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, and FGFBP2, and a reduced level of HLA-DRB5, if detected. Some embodiments of the method include a step of identifying the subject as having a likelihood of pathological complete response (pCR) when there is a reduced level of each of the genes selected for detection from PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, and FGFBP2, and an elevated level of HLA-DRB5, if detected.
Some embodiments of the method further include administering or recommending administration of additional chemotherapy prior to surgery and/or administration of additional chemotherapy after surgery when the subject is identified as having a likelihood of RD; or proceeding or recommending proceeding with surgery without administering additional chemotherapy when the subject is identified has having a likelihood of pCR.
Some embodiments of the presently-disclosed subject matter involve a second signature, sometimes referred to herein as a IFN/complement signature or pathological complete response (pCR) signature. The second signature includes the following sixty genes: SERPING1, IFIT3, IFI44L, IFI44, LAP3, FCGR1A, EPSTI1, IFIT2, TNFSF10, WARS1, IFITM3, MX1, MT2A, BATF2, IL15, IFIT1, STAT1, GBP4, ISG15, OAS3, JAK2, VAMP5, FGL2, PLSCR1, OASL, SAMD9L, USP18, SECTM1, APOL6, PLA2G4A, UBE2L6, CFB, PSME2, OAS2, STAT2, PARP14, CASP1, IFI35, HLA-DMA, GCH1, CD86, IL15RA, DDX60, LATS2, BST2, NMI, IFIH1, CASP4, EIF2AK2, PARP9, GBP2, TENT5A, OAS1, C1QC, C1QA, C2, KYNU, MMP14, PDP1, and CASP10.
In some embodiments of the method, at least ten genes of the second signature are considered. For example, in some embodiments the following ten genes of the second signature are considered: C1QC, CASP10, JAK2, IL15, TNFSF10, C1QA, IFIT3, EPSTI1, PSME2, and LAP3. In some embodiments of the method, more than 10 or even all sixty of the genes of the second signature are considered.
Some embodiments of the method include a step of calculating a second signature score by adding the expression level of each of the genes selected for detection. The expression level can be expressed, for example, transcript count for the gene(s) being detected. In some embodiments, the method also involves identifying the subject as having a likelihood of residual disease (RD) when the second signature score is less than a standardized control; or identifying the subject as having a likelihood of pathological complete response (pCR) when the second signature score is greater than a standardized control.
In some embodiments, the method includes identifying the subject as having a likelihood of residual disease (RD) and/or cancer recurrence when there is a reduced level of each of the genes in the second signature that are selected for detection. In some embodiments, the method includes identifying the subject as having a likelihood of pathological complete response (pCR) when there is an elevated level of each of the genes in the second signature that are selected for detection.
Some embodiments of the method further include administering or recommending administration of additional chemotherapy prior to surgery and/or administration of additional chemotherapy after surgery when the subject is identified as having a likelihood of RD; or proceeding or recommending proceeding with surgery without administering additional chemotherapy when the subject is identified has having a likelihood of pCR.
As will be apparent to the skilled artisan upon studying this document, it some embodiments, a method could make use of both the first signature and the second signature, while in other methods only one of the signatures is employed.
In some embodiment of the presently disclosed subject matter, the method involves extracting mRNA from the biological sample. In this regard, the method can also involve measuring in the extracted mRNA the levels of mRNA of genes from the first signature or genes from the second signature. In some embodiments, method also involves measuring in the extracted mRNA the levels of mRNA of normalization genes to control for the individual sample mRNA content. For example, PTPRC, RPL13a, and/or TBP can be used as normalization genes. As will be recognized by the skilled artisan, measurements of mRNA levels can be made using various technologies known in the art, such as, for example, nanoString mRNA profiling, RNA sequencing, or realtime qPCR.
In some embodiments of the presently-disclosed subject matter, detecting expression levels of genes in a sample can be achieved by using a probe for detecting a gene expression product. In this regard, reference is made to Table 1.
In some embodiments of the presently-disclosed subject matter, expression levels of PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, FGFBP2, and/or HLA-DRB5 in the sample can be measured using a probe that selectively detects sequence selected from: SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, and 22.
In some embodiments of the presently-disclosed subject matter, expression levels of PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, FGFBP2, and/or HLA-DRB5 in the sample can be measured using a probe comprising a sequence selected from the group consisting of SEQ ID NO: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, and 24.
While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.
All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.
Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732).
Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.
In certain instances, nucleotides and polypeptides disclosed herein are included in publicly-available databases, such as GENBANK® and SWISSPROT. Information including sequences and other information related to such nucleotides and polypeptides included in such publicly-available databases are expressly incorporated by reference. Unless otherwise indicated or apparent the references to such publicly-available databases are references to the most recent version of the database as of the filing date of this Application.
The present application can “comprise” (open ended) or “consist essentially of” the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.
As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.
The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.
Three cohorts of patients were combined for the tumor profiling study. All included patients received neoadjuvant therapy and had residual disease and matched pre-treatment tissue was required for inclusion. All but 2 (ER+) patients received cytotoxic chemotherapy as part of their regimen. Four patients (ER+) received courses of hormone therapy as part of their neoadjuvant regimen, two of which were in conjunction with cytotoxic chemotherapy. Five patients (HER2+) received HER2-directed therapy as part of their neoadjuvant regimen.
For the ‘Peru’ cohort, clinical characteristics and molecular analysis of the patients (n=48 with matched pre-treatment tissue) were previously described at the Instituto Nacional de Enfermedades Neoplásicas21. Clinical and pathologic data were retrieved from medical records under an institutionally approved protocol (INEN IRB 10-018). For the ‘VICC’ cohort, which included PBMC and whole blood analyses, clinical and pathologic data were retrieved from medical records under an institutionally approved protocol (VICC IRB 030747). For the DARTMOUTH cohort patient samples were collected under a protocol approved by the Dartmouth College Institutional Review Board and the waiver of the subject consent process was IRB-approved. (IRB 28888). Metadata for the primary cohort of patients was provided. For the peripheral blood study, all blood was collected within 14 days preceding definitive surgery. Metadata for the cohort of patients used for the peripheral blood study was provided.
The expression patterns of immune-related genes was examined before and after NAC in a series of 83 breast tumors, including 44 TNBCs, from patients with RD. Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). T cell receptor sequencing (TCRseq) was performed on a subset (n=15) of tumors. Additionally, in four patients undergoing NAC, PD-1-high and PD-1-negative CD3+CD8+ peripheral blood mononuclear cells (PBMCs) were profiled using single-cell RNA sequencing (scRNAseq) and multiplexed cytokine secretion assays. Finally, a scRNAseq-derived signature of activated cytolytic cells was used to measure immune activation in the peripheral blood of 36 patients after NAC (collected within 2 weeks prior to surgery) and 24 untreated patients. The association of this signature was tested with pCR and post-surgical cancer recurrence.
Stromal tumor-infiltrating lymphocytes were analyzed using full face H&E sections from pre-NAC diagnostic biopsies or post-NAC RD surgical specimens. Samples were scored according to the International TILs Working Group Guidelines22-24. The pre-defined cut point of 30%4 was used for all survival analyses.
Gene expression and gene set analysis on pre- and post-NAC formalin-fixed tissues were performed using the nanoString Pan-Cancer Immunology panel (770 genes) according to the manufacturers' standard protocol. Data were normalized according to positive and negative spike-in controls, then endogenous housekeeper controls, and transcript counts were log transformed for downstream analyses. Normalized linear data was obtained. Gene sets were calculated by summing the log 2-transformed normalized NanoString counts for all genes contained in a given gene set. Samples were simultaneously assayed for PAM50 molecular subtyping.
Briefly, 10 μm sections of diagnostic biopsies or residual tumors were used for RNA preparation (Promega Maxwell 16 RNA FFPE) and 50 ng of total RNA>300 nt (assayed on a Agilent Tapestation 2200 Bioanalyzer) was used for input into nCounter hybridizations for Pan-Cancer Immunology panels or 500-1000 ng RNA for PAM50 analysis. Data were normalized according to positive and negative spike-in controls, then endogenous housekeeper controls, and transcript counts were log transformed for downstream analyses. Subtype prediction was performed in R using the genefu package.
For the 8 gene signature analysis in whole blood, a custom NanoString Elements was constructed to measure the gene expression levels of PDCD1, NKG7, LAG3, GZMH, GZMB, GNLY, FGFBP2, HLA-DRB5, and HLA-G, as well as 3 normalization control genes (PTPRC, RPL13a, TBP). RNA was isolated from whole blood (Promega Maxwell 16 Simply RNA Blood) and 150-250 ng was used for input into the nCounter analysis. Data were normalized as above. Linear normalized data were obtained.
On day 1, cryopreserved PBMCs were thawed and resuspended in complete RPMI media with IL-2 (10 ng/ml) at a density of 1-5×106 cells/ml. Cells were recovered at 37° C., 5% CO2, overnight. Plates were prepared by coating with antihuman CD3 (10 μg/ml in PBS, 200-300 μl/well) in a 96-well flat-bottom plate at 4° C., 0/N. On day 2, non-adherent cells for each sample were collected and viability was confirmed, with dead cell depletion by Ficoll.
For each sample, where sufficient, volume was split in half for each of the following negative isolations: with one half of cells from each sample, CD4 T cells were isolated with CD4+ negative isolation kit following Miltenyi protocol (130-096-533); with the other half of cells from each sample, CD8 T cells were isolated with CD8+ negative isolation kit following Miltenyi protocol (130-096-495). The PD-1+ and PD-1−subsets were from isolated CD4 or CD8 T cells by staining with PE-conjugated anti-PD-1 antibody using the manufacturer's protocol (Miltenyi, 130-096-164) as follows: 1) stain each subset with 10 ul stain:100 ul Robosep buffer for every 1×10{circumflex over ( )}7 total cells; 2) incubate at 4° C. for 10 mins; 3) rinse cells by adding 1-2 mL of Robosep and C/F at 300×g for 10 mins; 4) aspirate supernatant and resuspend cells pellets in 80 ul buffer per 1×107 total cells. PD-1+ cells were then isolated with anti-PE microbeads following the manufacturer's protocol (Miltenyi, 130-097-054).
Cells were resuspended in complete RPMI media at a density of 1×106/ml and seeded into wells of the CD3-coated 96-well flat-bottom plate with soluble anti-human CD28 (5 ug/ml). Plates were incubated at 37° C., 5% CO2 for 24 hrs. On day 3, supernatants (100 ul per well) were collected from all wells and stored at −80° C. for population assays. T cells were collected and stained with Brilliant Violet cell membrane stain and AlexaFluor-647-conjugated anti-CD8 at RT for 20 min. Cells were resuspended in complete RPMI media for single-cell Isoplexis assay (human T cell panel), performed according to the manufacturer's standard protocol. Data were collected and analyzed 24 hours later (day 4).
TP53 gene sequencing was performed using either the Foundation Medicine assay as previously reported21 or using the SureMASTR TP53 sequencing assay (Agilent). For the later, purified DNA from FFPE breast tumor sections were amplified and sequenced according to the manufacturer's standard protocol. Samples were sequenced to a depth of −10,000 and mutations were called using the SureCall software (Agilent). Mutation allele frequency was set at 5% and only likely functional (early stops, frameshift deletions and known recurrent hotspot single-nucleotide variation mutations) were selected for sample annotation.
TCR sequencing and clonality quantification was assessed in FFPE samples of breast cancer specimens or PBMCs. For FFPE tissue, DNA or RNA was extracted from 10 μm sections using the Promega Maxwell 16 FFPE DNA or FFPE RNA kits and the manufacturer's protocol. For PBMCs, PD-1HI and PD-1NEG CD8+ T cells sorted by fluorescence-activated cell sorting from samples isolated from EDTA collection tubes and processed using a Ficoll gradient. At least 100K cells were collected, centrifuged, and utilized for RNA purification. TCRs were sequenced using survey level immunoSEQ™ (DNA; Adaptive Biotechnologies) and the Immunoverse assay (RNA; ArcherDX), as previously described39,40. Sequencing results were evaluated using the immunoSEQ analyzer version 3.0 or Archer Immunoverse analyzer. CDR3 sequences and frequency tables were extracted from the manufacturers' analysis platforms and imported into R for analysis using the Immunarch package (immunarch.com)25 in R. Shannon entropy, a measure of sample diversity, was calculated on the clonal abundance of all productive TCR sequences in the data set. Shannon entropy was normalized by dividing Shannon entropy by the logarithm of the number of unique productive TCR sequences. This normalized entropy value was then inverted (1—normalized entropy) to produce the ‘clonality’ metric. TCR6 clonotypes and metadata based on the primary cohort (
PD-1HI and PD-1NEG CD8+ T cells were sorted by fluorescence-activated cell sorting from peripheral blood mononuclear cells isolated from EDTA collection tubes and processed using a Ficoll gradient. Each sample (targeting 5,000 cells/sample) was processed for single cell 5′ RNA sequencing utilizing the 10× Chromium system. Libraries were prepared using P/N 1000006, 1000080, and 1000020 following the manufacturer's protocol. The libraries were sequenced using the NovaSeq 6000 with 150 bp paired end reads. RTA (version 2.4.11; Illumina) was used for base calling and analysis was completed using 10× Genomics Cell Ranger software v2.1.1. Data were analyzed in R using the filtered h5 gene matrices in the Seurat26,27 package (R). Briefly, samples were merged, and all cells were scored for mitochondrial gene expression (a marker of dying cells) and cell cycle genes to determine phase. Data were transformed using SCTransform, regressing against mitochondrial gene expression and cell cycle phase. Dimensional reduction was performed using Harmony28.
Immune-related gene expression patterns were examined before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 Triple-Negative Breast Cancers (TNBC), from patients with residual disease (RD). Changes in gene expression patterns in the tumor-immune microenvironment (TIME) were tested for association with recurrence-free (RFS) and overall survival (OS). Additionally, the systemic effects of NAC were characterized through single cell analysis (RNAseq and cytokine secretion) of PD-1HI CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood.
Matched archived pre-treatment (diagnostic biopsy) and post-treatment (residual disease surgical specimen) tumor specimens were procured from the series of 83 patients, including the 44 TNBC patients.
Importantly, the study was refined to include only patients who had residual disease at surgery for analysis, thereby excluding patients who achieved pCR. This was a purposeful selection strategy, as patients with pCR usually experience good outcomes, and the focus was instead on patients with RD for whom additional treatment strategies could eventually replace “watchful waiting” approaches. Metadata for the patients, including treating institution, molecular subtype (PAM50), recurrence-free and overall survival (RFS and OS, respectively), TP53 mutation status, and other molecular and clinical data were available.
As sTILs have been described and rigorously validated as both a prognostic factor (in surgical specimens for post-surgical outcomes), and a predictive factor (in diagnostic biopsies for benefit from NAC) in TNBC, but not in other breast cancer subtypes, an initial inquiry was whether these findings were consistent with the study cohort. Using the published cutoff (30%) of sTILs4, higher abundance of sTILs in the post-NAC residual disease in TNBC patients (n=44) was found to be significantly prognostic for both RFS (log-rank p=0.019) and OS (p=0.05;
Thus, in the cohort, abundance of sTILs has the strongest prognostic effect for the post-NAC surgical resection specimen in TNBC tumors with an incomplete response to NAC. These findings, consistent with both retrospective studies and analyses from randomized controlled trials, prompted more detailed molecular studies aimed at understanding how NAC influences the TIME.
To measure transcriptional changes occurring in the tumor-immune microenvironment (TIME) induced by NAC, gene expression profiling was performed for a series of 770 immune-related genes using nanoString (Pan-Cancer Immune Panel), before and after NAC in the entire cohort (n=83). Transcriptional patterns and hierarchical clustering for all data primarily segregated tumors based on receptor status (ER/PR/HER2) and/or molecular subtype, with most luminal/hormone receptor-positive tumors appearing in the first cluster, most HER2-positive tumors in the second cluster, and most basal-like/TNBC tumors in the third cluster (
While the TIME change in sTIL abundance did not prognosticate outcome in TNBC patients, changes in individual immune-related genes were examined for association with outcome. Iterative Cox proportional hazards models were performed, using the delta (A) of each gene (post-NAC minus pre-NAC) in an independent univariate analysis, for both RFS and OS. All analyses are reported using a nominal p-value as well as a false discovery rate (FDR; Benjamini-Hochberg method) q-value for association with RFS or OS. After correction for FDR (q<0.10), upregulation of 11 genes were associated with improved RFS, while upregulation of only one gene was significantly associated with worse RFS (CDH1, which encodes e-cadherin) in the TNBC cohort. Interestingly, e-cadherin is known to interact with killer cell lectin-like receptor G1 (KLRG1), an inhibitory receptor expressed by memory T cells and NK cells9. In contrast, upregulation of a larger number of genes was associated with improved OS (n=189) or reduced OS (n=15) at FDR q<0.10 (
Dimensional reduction through collapsing individual genes into pathways or defined functions can improve interpretation of high-dimensional data. Thus, the gene expression data was collapsed into bioinformatically-categorized immune signatures (sum-scores, defined as the summation of the log 2 expression values for all genes in a category). Organization of the data in this manner and testing the signatures (n=100) for association with RFS and OS yielded a surprising finding—nearly all significant (q<0.10) gene sets (n=37 for RFS and n=77 for OS) identified in this analysis were associated with good outcome (
A robust T cell response is characterized by oligoclonal expansion of antigen-specific T cells. Therefore, it was determined whether clonality of T cells in the TIME was altered during NAC. In a subset of samples (n=15; 8 TNBC, 7 non-TNBC), T cell receptor (TCR) 6 chain sequencing was performed using the ImmunoSeq assay to estimate the number of unique T cell clones (diversity), and the presence of expanded T cell clones in the TIME before and after NAC. Given the breadth of sTILs fractions observed among breast tumors as well as caveats associated with comparison of samples derived from diagnostic core needle biopsies vs. surgical resections, it was first verified that the number of productive T cells was associated with estimation of sTILs determined on adjacent sections. A strong association was detected between these parameters (R2=0.6; p<0.0001;
To further explore changes in T cell clonality and function in response to chemotherapy, PBMCs were prospectively collected from four breast cancer patients (including two TNBCs) before and after NAC (
PD-1HICD8+ and PD-1NEG CD8+ T cells from pre-NAC and post-NAC blood (except patient 4, for whom a sufficient pre-NAC sample was not available) were also analyzed by TCR sequencing. While the number of detected T cells was consistent among all samples, the clonotypes detected (unique TCRs) were considerably lower in PD-1HI CD8+ T cells (
Next, scRNAseq was used to describe the post-NAC peripheral PD-1HICD8+ T cell populations at the time of surgery in the blood of two TNBC patients: one with residual disease (Pt. 1) and one with matrix-producing metaplastic TNBC who experienced pCR (Pt. 4). Uniform Manifold Approximation and Projection (UMAP) analysis was performed on Harmony-normalized samples to adjust for inter-sample technical variation, and cells were stratified based on 5 clusters identified through the Louvain algorithm. Although the composition of the cells was largely similar, one cluster (cluster 0′) was identified which was enriched in Pt. 4 (
These data led to two competing hypotheses: 1) Cluster 0 genes, reflective of cytolytic CD8+ T cells, are a positive prognostic factor reflective of robust anti-tumor immunity as evidenced by their enrichment in the metaplastic TNBC patient with pCR; or 2) Cluster 0 genes are reflective of ongoing disease including the micrometastatic component that cannot be sampled from the primary tumor. The second hypothesis is supported by the observations that pCR is less prognostic of RFS and OS in metaplastic disease11,12 and that rates of recurrence following chemotherapy are higher for metaplastic disease than non-metaplastic TNBC11,13,14. Interestingly, matrix-producing metaplastic breast cancer (Pt. 4) has been shown to be associated with pCR to NAC, but often can still recur despite pCR11,15. Follow-up for this individual patient was immature at the time of reporting, and thus recurrence, and therefore presence of micrometastatic disease at the time of sampling, cannot be ruled out.
To determine whether cytolytic signatures representative of cluster 0 genes were associated with disease outcome, archived whole blood was evaluated from a series of 60 breast cancer patients. All samples were collected within 14 days preceding surgical resection for primary breast cancer, with 36 samples having received NAC, in addition to 24 samples from untreated patients. A series of eight genes enriched in cluster 0 (PDCD1, NKG7, LAGS, GZMB, GNLY, FGFBP2, HLA-DRB5), one gene enriched in Pt 1 (RCB-II) over Pt 4 (pCR; HLA-G) (
Nearly all tested genes demonstrated a pattern supporting the hypothesis that gene expression in whole blood is associated with ongoing disease, being highest (or lowest in the case of HLA-G) in untreated patients (who have ongoing tumor burden by virtue of not having received therapy prior to surgery) and those with RD compared to those with pCR. Furthermore, among patients with RD, higher expression (or lower in the case of HLA-G) tended to be observed in patients who had early recurrences in the first 3 years following surgery (and thus may have had micrometstatic disease at the time of surgery). Several of these genes (FGFBP2, GNLY, PDCD1, LAGS, and NKG7) were also significantly differentially expressed or approached statistical significance across the outcome groups (Kruskal-Wallis test). Comparisons were particularly striking between the group of patients with RD who experienced early disease recurrence and the group with pCR following NAC (post-hoc Dunn test;
In these Examples, the prognostic nature of sTILs in the RD of TNBC patients was confirmed, which is not evident in non-TNBCs. Extending this knowledge, enhancement of immunologic activity in the TIME was found to be evident in only a subset of NAC-treated TNBC patients, but this activity correlates with improved RFS and OS. This activation is broad and does not appear confined to particular immunologic functions, likely representing the complexity involved in capturing immunologic activity at a single time point. However, the induction of cytolytic effector cell signatures in the TIME was particularly prognostic.
Interestingly, no immune-specific gene sets were significantly associated with poor outcome in TNBC patients after NAC. Immunologic activation in the TIME was also not accompanied by enhancement of TCR clonality. To determine if immunologic activation could be observed peripherally in patients treated with NAC, a series of functional and immunogenomic experiments were performed on CD8+ T cells isolated from PBMCs of patients undergoing NAC. The focus was specifically on PD-1HI CD8+ T cells as these have been shown to be enriched for tumor-specific T cells8.
This population was confirmed to be more active and clonal by functional single-cell cytokine assays and TCR sequencing. In particular, a significant increase in cytolytic and inflammatory cytokines secreted by PD-1HI CD8+ T cells were detected in two TNBC patients after chemotherapy, but not in two non-TNBC patients. A further characterization of PD-1HI CD8+ cells by scRNAseq identified a population of cytolytic gene (e.g., GNLY, FGFBP2, GZMB)-expressing and checkpoint (e.g., LAG3, FCLR6 and substantially higher PDCD1)-expressing cells present in blood.
It was contemplated that these activated T cells in blood may be reflective of ongoing anti-tumor immunity, which could signify 1) the potential for ongoing tumor immunologic control and thus better outcome, or 2) persistent disease in the breast or micrometastatic compartment that ultimately leads to recurrence. Intriguingly, higher expression of a gene signature derived from these cytolytic cells in whole blood at the time of surgery was associated with higher disease burden (i.e. in those patients who did not receive NAC and those with RD who experienced disease recurrence within three years).
This finding was similar between TNBC and non-TNBC patients. Thus, peripheral cytotoxic activity, guarded by immune checkpoints, reflect ongoing micrometastatic and primary disease burden, and are useful for predicting disease recurrence and possibly immune checkpoint inhibitor benefit.
In studies presented in these examples, a molecular analysis of the TIME in response to NAC in 83 breast cancer patients is presented, specifically focusing on patients lacking a pCR, as these patients have worse outcomes. Like sTILs, changes in tumor immunity seem to be most prevalent in TNBC, often resulting in decreases in expression of immune-related genes. However, an upregulation of immune-related gene expression in tumors following NAC was associated with a strikingly improved outcome after surgery, specifically in TNBC. Of these genes, those involved in cytotoxic effector cells were among the most robustly associated with outcome. Furthermore, cytokines expressed by PD-1HI CD8+ T cells in the peripheral blood were found to be increased dramatically in TNBC patients following NAC.
Analysis of TCR clonotypes infiltrating into tumors suggested that chemotherapy may preferentially increase the recruitment of new T cell clones into the tumor, rather than expanding the T cells already present. This effect was consistent with that observed in the peripheral blood, where PD-1HI CD8+ T cells, while highly clonal compared to PD-1NEG cells, did not substantially change in clonality during NAC; these observations reflect a lack of clonal expansion in response to NAC, as found in the TIME.
Assessment of peripheral blood represents a unique opportunity to monitor anti-tumor immunity through minimally-invasive means. Using scRNAseq, a population of cytolytic effector T cells were identified in blood that expressed elevated levels of exhaustion/checkpoint genes. A gene expression signature derived from this population was used to test the hypothesis that these highly cytolytic but potentially exhausted cells may be reflective of an ongoing disease process, and therefore a peripheral approximation of disease burden. This hypothesis was confirmed in a validation set of 60 patients and serves as a proof-of-principle for the use of this signature as a possible biomarker of outcome.
Importantly, there has been a paucity of studies looking at the effect of chemotherapy on peripheral blood20, with little data on disease outcomes. These studies study provides a unique assessment and framework for an improved understanding of how chemotherapy alters anti-tumor immunity both in the TIME and the peripheral compartment. These data represent a unique opportunity to better understand patient populations most likely to benefit from the addition of immunotherapy to chemotherapy, particularly in the neoadjuvant setting.
Furthermore, the findings demonstrating the association of expression of key cytolytic and immune-activation genes in the peripheral blood with presence of residual disease and recurrence represent a possible biomarker platform.
The peripheral gene expression signature may identify high-risk populations with potentially exhausted T cells and either primary or micrometastatic disease who are likely to benefit from additional immunotherapeutic strategies.
Peripheral blood in breast cancer patients was examined. With reference to
Using the GSEA findings, the leading edge genes from each of the three upregulated gene sets were taken. Leading edge genes are those most strongly enriched in the pCR patients vs the RD patients for each gene set. Sixty (60) unique genes were identified in the leading edge for the three enriched gene sets (genes and expression shown in heatmap in
The 8 gene cytotoxic signature is defined as PDCD1+NKG7+LAG3+GZMH+GZMB+GNLY+FGFBP2−HLA-G. With reference to
Turning now to
The cytotoxic score is primarily expressed in peripheral blood CD8+ T cells and natural killer (NK) cells, while the IFN/Complement score is primarily expressed in monocytes. Single cell RNA sequencing was done on peripheral blood mononuclear cells (PBMCs) from two breast cancer patients, collected following NAC and prior to surgery). With reference to
CIBERSORTx was used to deconvolute cell type abundance from bulk gene expression data. The heatmap in
For the same cohort of patients on whom peripheral blood gene expression profiling was conducted, clinically measured relative monocyte values were identified in the electronic medical record. With reference to
The synthetic derivative, a de-identified medical record system, was used to identify additional breast cancer patients treated with NAC. With reference to
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:
It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.
This application claims priority from U.S. Provisional Application Ser. No. 62/988,316 filed Mar. 11, 2020, the entire disclosure of which is incorporated herein by this reference.
This invention was made with government support under grant number W81XWH1810149/BC170037 awarded by the Department of Defense. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62988316 | Mar 2020 | US |
