PRAME Immunohistochemistry for Distinguishing Vulvar and Vaginal Melanoma From Benign Melanocytic Nevi.

Vulvovaginal melanoma (VVM) is a rare but deadly disease, accounting for 5% of all vulvar malignancies, with a 5-yr survival rate of only 47% for all stages of the disease. VVM is a distinct subset of melanoma, with a unique genomic profile and underlying pathogenesis unassociated with sun exposure. Distinguishing these rare malignancies from very common pigmented lesions of the vulva and vagina is challenging as histologic features often overlap between entities. PReferentially expressed Antigen in MElanoma (PRAME) is a melanoma-associated protein, and immunohistochemistry (IHC) for PRAME distinguishes cutaneous, oral mucosal, and retinal melanoma from atypical nevi. Given the biological differences between VVM and cutaneous melanoma, the utility of PRAME IHC for the diagnosis of VVM is unknown. We accrued a cohort of 20 VVM and 21 benign vulvar melanocytic nevi. We found that nuclear PRAME IHC staining with 4+ intensity was present in 85% of the VVM and 0% of the nevi. With the assistance of PRAME IHC, we found evidence of close or positive margin involvement in 3 of 10 cases where margins were originally diagnosed as negative for melanoma in situ. Our study is the first to assess PRAME IHC in a cohort of VVM cases and provides confidence for using PRAME IHC to assist with diagnosis and margin assessment in this rare disease.

Biomarkers for Immune Checkpoint Inhibitors in Renal Cell Carcinoma.

Immune checkpoint inhibitor (ICI) therapy has revolutionized renal cell carcinoma treatment. Patients previously thought to be palliative now occasionally achieve complete cures from ICI. However, since immunotherapies stimulate the immune system to induce anti-tumor immunity, they often lead to adverse autoimmunity. Furthermore, some patients receive no benefit from ICI, thereby unnecessarily risking adverse events. In many tumor types, PD-L1 expression levels, immune infiltration, and tumor mutation burden predict the response to ICI and help inform clinical decision making to better target ICI to patients most likely to experience benefits. Unfortunately, renal cell carcinoma is an outlier, as these biomarkers fail to discriminate between positive and negative responses to ICI therapy. Emerging biomarkers such as gene expression profiles and the loss of pro-angiogenic proteins VHL and PBRM-1 show promise for identifying renal cell carcinoma cases likely to respond to ICI. This review provides an overview of the mechanistic underpinnings of different biomarkers and describes the theoretical rationale for their use. We discuss the effectiveness of each biomarker in renal cell carcinoma and other cancer types, and we introduce novel biomarkers that have demonstrated some promise in clinical trials.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Indicators Differentiating Thrombotic Thrombocytopenic Purpura From Other Thrombotic Microangiopathies in a Canadian Apheresis Referral Center.

OBJECTIVES: Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) caused by ADAMTS13 deficiency with mortality of up to 90% in the absence of treatment, typically therapeutic plasma exchange (TPE). TTP presents similarly to other TMAs in which TPE is ineffective and associated with morbidity and additional costs. Thus, we sought to assess clinical and laboratory parameters differentiating TTP from other TMAs in our institution's catchment population. METHODS: We reviewed 8 years of data from a Canadian provincial apheresis center, including 100 patients with suspected TMA who underwent ADAMTS13 testing, 35 of whom were diagnosed with TTP. We assessed clinical and laboratory parameters to identify discriminators of TTP and assigned PLASMIC TTP prediction scores. RESULTS: We observed a higher frequency of neurologic symptoms, more severe thrombocytopenia, and less creatinine elevation in TTP relative to other TMAs. High PLASMIC scores (6-7 points) had 83% sensitivity and 88% specificity for TTP diagnoses; however, ADAMTS13 activity testing was required for correct diagnoses in 14 cases. CONCLUSIONS: Clinical and laboratory parameters including PLASMIC scoring may lead to misdiagnosis in some cases of TMA. ADAMST13 activity testing provides definitive diagnosis of TTP, supporting the role of rapid turnaround ADAMTS13 testing for appropriate treatment of TMAs.

Assessment of long non-coding RNA expression reveals novel mediators of the lung tumour immune response.

The tumour immune microenvironment is a crucial mediator of lung tumourigenesis, and characterizing the immune landscape of patient tumours may guide immunotherapy treatment regimens and uncover novel intervention points. We sought to identify the landscape of tumour-infiltrating immune cells in the context of long non-coding RNA (lncRNAs), known regulators of gene expression. We examined the lncRNA profiles of lung adenocarcinoma (LUAD) tumours by interrogating RNA sequencing data from microdissected and non-microdissected samples (BCCRC and TCGA). Subsequently, analysis of single-cell RNA sequencing data from lung tumours and flow-sorted healthy peripheral blood mononuclear cells identified lncRNAs in immune cells, highlighting their biological and prognostic relevance. We discovered lncRNA expression patterns indicative of regulatory relationships with immune-related protein-coding genes, including the relationship between AC008750.1 and NKG7 in NK cells. Activation of NK cells in vitro was sufficient to induce AC008750.1 expression. Finally, siRNA-mediated knockdown of AC008750.1 significantly impaired both the expression of NKG7 and the anti-tumour capacity of NK cells. We present an atlas of cancer-cell extrinsic immune cell-expressed lncRNAs, in vitro evidence for a functional role of lncRNAs in anti-tumour immune activity, which upon further exploration may reveal novel clinical utility as markers of immune infiltration.

Hyperspectral cell sociology reveals spatial tumor-immune cell interactions associated with lung cancer recurrence.

BACKGROUND: The tumor microenvironment (TME) is a complex mixture of tumor epithelium, stroma and immune cells, and the immune component of the TME is highly prognostic for tumor progression and patient outcome. In lung cancer, anti-PD-1 therapy significantly improves patient survival through activation of T cell cytotoxicity against tumor cells. Direct contact between CD8+ T cells and target cells is necessary for CD8+ T cell activity, indicating that spatial organization of immune cells within the TME reflects a critical process in anti-tumor immunity. Current immunohistochemistry (IHC) imaging techniques identify immune cell numbers and densities, but lack assessment of cell-cell spatial relationships (or "cell sociology"). Immune functionality, however, is often dictated by cell-to-cell contact and cannot be resolved by simple metrics of cell density (for example, number of cells per mm2). To address this issue, we developed a Hyperspectral Cell Sociology technology platform for the analysis of cell-cell interactions in multi-channel IHC-stained tissue. METHODS: Tissue sections of primary tumors from lung adenocarcinoma patients with known clinical outcome were stained using multiplex IHC for CD3, CD8, and CD79a, and hyperspectral image analysis determined the phenotype of all cells. A Voronoi diagram for each cell was used to approximate cell boundaries, and the cell type of all neighboring cells was identified and quantified. Monte Carlo analysis was used to assess whether cell sociology patterns were likely due to random distributions of the cells. RESULTS: High density of intra-tumoral CD8+ T cells was significantly associated with non-recurrence of tumors. A cell sociology pattern of CD8+ T cells surrounded by tumor cells was more significantly associated with non-recurrence compared to CD8+ T cell density alone. CD3+ CD8- T cells surrounded by tumor cells was also associated with non-recurrence, but at a similar significance as cell density alone. Cell sociology metrics improved recurrence classifications of 12 patients. Monte Carlo re-sampling analysis determined that these cell sociology patterns were non-random. CONCLUSION: Hyperspectral Cell Sociology expands our understanding of the complex interplay between tumor cells and immune infiltrate. This technology could improve predictions of responses to immunotherapy and lead to a deeper understanding of anti-tumor immunity.

Somatic mutation-associated T follicular helper cell elevation in lung adenocarcinoma.

T follicular helper cells (Tfh) play crucial roles in the development of humoral immunity. In the B cell-rich germinal center of lymphoid organs, they select for high-affinity B cells and aid in their maturation. While Tfh have known roles in B cell malignancies and have prognostic value in some epithelial cancers, their role in lung tumour initiation and development is unknown. Through immune cell deconvolution, we observed significantly increased Tfh in tumours from two independent cohorts of lung adenocarcinomas and found that this upregulation occurs early in tumour development. A subset of tumours were stained for T and B cells using multicolour immunohistochemistry, which revealed the presence of tumour-adjacent tertiary lymphoid organs in 17/20 cases each with an average of 16 Tfh observed in the germinal center. Importantly, Tfh levels were correlated with tumour mutational load and immunogenic cancer testis antigens, suggesting their involvement in mounting an active immune response against tumour neoantigens.

Mapping the human T cell repertoire to recurrent driver mutations in MYD88 and EZH2 in lymphoma.

Oncogenic "driver" mutations are theoretically attractive targets for the immunotherapy of lymphoid cancers, yet the proportion that can be recognized by T cells remains poorly defined. To address this issue without any confounding effects of the patient's immune system, we assessed T cells from 19 healthy donors for recognition of three common driver mutations in lymphoma: MYD88L265P, EZH2Y641F , and EZH2Y641N . Donors collectively expressed the 10 most prevalent HLA class I alleles, including HLA-A*02:01. Peripheral blood T cells were primed with peptide-loaded dendritic cells (DC), and reactive T cells were assessed for recognition of naturally processed mutant versus wild type full-length proteins. After screening three driver mutations across 17-26 HLA class I alleles and 3 × 106-3 × 107 T cells per donor, we identified CD4+ T cells against EFISENCGEII from EZH2Y641N (presented by HLA-DRB1*13:02) and CD8+ T cells against RPIPIKYKA from MYD88L265P (presented by HLA-B*07:02). We failed to detect RPIPIKYKA-specific T cells in seven other HLA-B*07:02-positive donors, including two lymphoma patients. Thus, healthy donors harbor T cells specific for common driver mutations in lymphoma. However, such responses appear to be rare due to the combined limitations of antigen processing, HLA restriction, and T cell repertoire size, highlighting the need for highly individualized approaches for selecting targets.

A library-based screening method identifies neoantigen-reactive T cells in peripheral blood prior to relapse of ovarian cancer.

Mutated cancer antigens, or neoantigens, represent compelling immunological targets and appear to underlie the success of several forms of immunotherapy. While there are anecdotal reports of neoantigen-specific T cells being present in the peripheral blood and/or tumors of cancer patients, effective adoptive cell therapy (ACT) against neoantigens will require reliable methods to isolate and expand rare, neoantigen-specific T cells from clinically available biospecimens, ideally prior to clinical relapse. Here, we addressed this need using "mini-lines", large libraries of parallel T cell cultures, each originating from only 2,000 T cells. Using small quantities of peripheral blood from multiple time points in an ovarian cancer patient, we screened over 3.3 × 106 CD8+ T cells by ELISPOT for recognition of peptides corresponding to the full complement of somatic mutations (n = 37) from the patient's tumor. We identified ten T cell lines which collectively recognized peptides encoding five distinct mutations. Six of the ten T cell lines recognized a previously described neoantigen from this patient (HSDL1L25V), whereas the remaining four lines recognized peptides corresponding to four other mutations. Only the HSDL1L25V-specific T cell lines recognized autologous tumor. HSDL1L25V-specific T cells comprised at least three distinct clonotypes and could be identified and expanded from peripheral blood 3-9 months prior to the first tumor recurrence. These T cells became undetectable at later time points, underscoring the dynamic nature of the response. Thus, neoantigen-specific T cells can be expanded from small volumes of blood during tumor remission, making pre-emptive ACT a plausible clinical strategy.

Low Mutation Burden in Ovarian Cancer May Limit the Utility of Neoantigen-Targeted Vaccines.

Due to advances in sequencing technology, somatically mutated cancer antigens, or neoantigens, are now readily identifiable and have become compelling targets for immunotherapy. In particular, neoantigen-targeted vaccines have shown promise in several pre-clinical and clinical studies. However, to date, neoantigen-targeted vaccine studies have involved tumors with exceptionally high mutation burdens. It remains unclear whether neoantigen-targeted vaccines will be broadly applicable to cancers with intermediate to low mutation burdens, such as ovarian cancer. To address this, we assessed whether a derivative of the murine ovarian tumor model ID8 could be targeted with neoantigen vaccines. We performed whole exome and transcriptome sequencing on ID8-G7 cells. We identified 92 somatic mutations, 39 of which were transcribed, missense mutations. For the 17 top predicted MHC class I binding mutations, we immunized mice subcutaneously with synthetic long peptide vaccines encoding the relevant mutation. Seven of 17 vaccines induced robust mutation-specific CD4 and/or CD8 T cell responses. However, none of the vaccines prolonged survival of tumor-bearing mice in either the prophylactic or therapeutic setting. Moreover, none of the neoantigen-specific T cell lines recognized ID8-G7 tumor cells in vitro, indicating that the corresponding mutations did not give rise to bonafide MHC-presented epitopes. Additionally, bioinformatic analysis of The Cancer Genome Atlas data revealed that only 12% (26/220) of HGSC cases had a ≥90% likelihood of harboring at least one authentic, naturally processed and presented neoantigen versus 51% (80/158) of lung cancers. Our findings highlight the limitations of applying neoantigen-targeted vaccines to tumor types with intermediate/low mutation burdens.

Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival.

Somatic missense mutations can initiate tumorogenesis and, conversely, anti-tumor cytotoxic T cell (CTL) responses. Tumor genome analysis has revealed extreme heterogeneity among tumor missense mutation profiles, but their relevance to tumor immunology and patient outcomes has awaited comprehensive evaluation. Here, for 515 patients from six tumor sites, we used RNA-seq data from The Cancer Genome Atlas to identify mutations that are predicted to be immunogenic in that they yielded mutational epitopes presented by the MHC proteins encoded by each patient's autologous HLA-A alleles. Mutational epitopes were associated with increased patient survival. Moreover, the corresponding tumors had higher CTL content, inferred from CD8A gene expression, and elevated expression of the CTL exhaustion markers PDCD1 and CTLA4. Mutational epitopes were very scarce in tumors without evidence of CTL infiltration. These findings suggest that the abundance of predicted immunogenic mutations may be useful for identifying patients likely to benefit from checkpoint blockade and related immunotherapies.

Surveillance of the tumor mutanome by T cells during progression from primary to recurrent ovarian cancer.

PURPOSE: Cancers accumulate mutations over time, each of which brings the potential for recognition by the immune system. We evaluated T-cell recognition of the tumor mutanome in patients with ovarian cancer undergoing standard treatment. EXPERIMENTAL DESIGN: Tumor-associated T cells from 3 patients with ovarian cancer were assessed by ELISPOT for recognition of nonsynonymous mutations identified by whole exome sequencing of autologous tumor. The relative levels of mutations and responding T cells were monitored in serial tumor samples collected at primary surgery and first and second recurrence. RESULTS: The vast majority of mutations (78/79) were not recognized by tumor-associated T cells; however, a highly specific CD8(+) T-cell response to the mutation hydroxysteroid dehydrogenase-like protein 1 (HSDL1)(L25V) was detected in one patient. In the primary tumor, the HSDL1(L25V) mutation had low prevalence and expression, and a corresponding T-cell response was undetectable. At first recurrence, there was a striking increase in the abundance of the mutation and corresponding MHC class I epitope, and this was accompanied by the emergence of the HSDL1(L25V)-specific CD8(+) T-cell response. At second recurrence, the HSDL1(L25V) mutation and epitope continued to be expressed; however, the corresponding T-cell response was no longer detectable. CONCLUSION: The immune system can respond to the evolving ovarian cancer genome. However, the T-cell response detected here was rare, was transient, and ultimately failed to prevent disease progression. These findings reveal the limitations of spontaneous tumor immunity in the setting of standard treatments and suggest a high degree of ignorance of tumor mutations that could potentially be reversed by immunotherapy.

Profound CD8+ T cell immunity elicited by sequential daily immunization with exogenous antigen plus the TLR3 agonist poly(I:C).

The development of vaccines that elicit robust CD8(+) T cell immunity has long been a subject of intense investigation. Although whole exogenous protein has not historically been considered as useful for eliciting CD8(+) T cell immunity, we report herein that whole, protein antigen is capable of eliciting profound levels of CD8(+) T cell immunity if it is administered via repeated, daily subcutaneous immunization in combination with the TLR3 agonist poly(I:C). Mice immunized for four consecutive days with 100 µg of either whole exogenous OVA or whole HPV16 E7 protein combined with 10 µg of poly(I:C) mounted remarkable antigen-specific CD8(+) T cell responses as measured by tetramer staining and ELISPOT analysis of splenocytes and peripheral blood, with up to 30% of peripheral CD8(+) T cells being antigen specific within 7-8 days of vaccination. CD8(+) T cell immunity elicited using this vaccination approach was critically dependent upon cross presentation, as either whole protein or long synthetic peptides were highly effective immunogens whereas minimal peptide epitopes were not. Vaccine-induced CD8(+) T cells were also able to regress large, established tumors in vivo. Together these data suggest that 'cluster' vaccination with exogenous antigen combined with TLR3 agonist may constitute a profoundly important advancement in therapeutic vaccine design.