CD155 on Tumor Cells Drives Resistance to Immunotherapy by Inducing the Degradation of the Activating Receptor CD226 in CD8+ T Cells.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8+ TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226hi TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226+CD8+ T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.

Tumor intrinsic and extrinsic immune functions of CD155.

CD155 (PVR/necl5/Tage4), a member of the nectin-like family of adhesion molecules, is highly upregulated on tumor cells across multiple cancer types and has been associated with worse patient outcomes. In addition to well described cell-intrinsic roles promoting tumor progression and metastasis, CD155 has now been implicated in immune regulation. The role of CD155 as a potent immune ligand with diverse cell-extrinsic functions is now being defined. CD155 signaling to immune cells is mediated through interactions with the co-stimulatory immune receptor CD226 (DNAM-1) and the inhibitory checkpoint receptors TIGIT and CD96, which are differentially regulated at the cell surface on T cells and NK cells. The integration of signals from CD155 cognate receptors modifies the activity of tumor-infiltrating lymphocytes in a context-dependent manner, making CD155 an attractive target for immune-oncology. Preclinical studies suggest that targeting this axis can improve immune-mediated tumor control, particularly when combined with existing anti-PD-1 checkpoint therapies. In this review, we discuss the roles of CD155 on host and tumor cells in controlling tumor progression and discuss the possibility of targeting CD155 for cancer therapy.

Nicotinamide for skin cancer chemoprevention: effects of nicotinamide on melanoma in vitro and in vivo.

Nicotinamide (NAM), an amide form of vitamin B3, replenishes cellular energy after ultraviolet radiation (UVR) exposure, thereby enhancing DNA repair and reducing UVR's immunosuppressive effects. NAM reduces actinic keratoses and new keratinocyte cancers in high risk individuals, but its effects on melanoma are unknown. Melanomas arising on NAM or placebo within the ONTRAC skin cancer chemoprevention trial (Oral Nicotinamide To Reduce Actinic Cancer) were examined by immunohistochemistry. The effects of NAM (50 µM, 5 mM and 20 mM) on the viability, proliferation and invasiveness of four human melanoma cell lines and on the viability and proliferation of two human melanocyte lines, with and without UV irradiation were also investigated. 50 µM NAM did not affect viability, proliferation or invasion of melanoma or melanocyte cell lines, whereas concentrations too high to be achievable in vivo reduced viability and proliferation. Nicotinamide did not enhance melanoma viability, proliferation or invasiveness in vitro, providing additional confidence in its safety for use in clinical trials in high risk patients. Peritumoral and tumour infiltrating CD4+ and CD8+ lymphocytes were significantly increased in melanomas arising on NAM compared to those arising on placebo. Given the chemopreventive activity of nicotinamide against keratinocyte cancers, its DNA repair enhancing effects in melanocytes and now its potential enhancement of tumour-infiltrating lymphocytes and lack of adverse effects on melanoma cell growth and proliferation, clinical trials of nicotinamide for melanoma chemoprevention are now indicated.

Inter- and intrapatient heterogeneity of indoleamine 2,3-dioxygenase expression in primary and metastatic melanoma cells and the tumour microenvironment.

AIMS: Indoleamine 2,3-dioxygenase (IDO), an immunomodulatory enzyme, facilitates immune escape by tumours and promotes tumour progression. IDO inhibitors with and without additional anti-PD-1 therapy have been evaluated in recent and ongoing melanoma clinical trials, but IDO expression in melanoma tumours, and therefore its potential role as a predictive biomarker remains unknown. This study sought to evaluate IDO expression in immunotherapy-naive metastatic melanoma patients in order to determine patterns of expression in corresponding primary melanomas, locoregional metastases and distant metastases. METHODS AND RESULTS: Here, we evaluated IDO expression using immunohistochemistry in 99 melanoma tumour samples from 43 immunotherapy-naive patients with metastatic melanoma to determine patterns of expression in primary melanomas (n = 29), locoregional metastases (n = 36) and distant metastases (n = 34). Thirty-seven per cent of patients demonstrated tumour IDO expression in at least one specimen. Twelve of 35 patients (34%) with longitudinal specimens (i.e. two or more separate specimens from different disease stages in the same patient) displayed heterogeneous IDO staining between samples. Tumour IDO expression positively correlated with tumour-infiltrating lymphocyte (TIL) score as well as the number of IDO-expressing mononuclear cells in the primary melanoma (P < 0.0001 and P = 0.0011, respectively) and nodal metastases (P = 0.049 and P = 0.037, respectively), but not in distant metastases. Furthermore, tumour IDO expression correlated positively with PD-L1 expression by melanoma cells among all specimens (P = 0.0073). CONCLUSIONS: Therefore, while assessment of tumour IDO expression warrants evaluation in melanoma patient cohorts treated with IDO inhibitors dosed at levels proven to inhibit the target by pharmacodynamic assessment, its utility as a biomarker may be limited by intertumoral heterogeneity.

HDAC inhibitors restore BRAF-inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma.

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.

Programmed death ligand 1 expression in triple-negative breast cancer is associated with tumour-infiltrating lymphocytes and improved outcome.

AIMS: Triple-negative breast cancer (TNBC) patients generally have a poor outcome; there is a pressing need to identify more effective therapeutic strategies. Clinical trials targeting programmed death 1/programmed death ligand 1 (PD1/PDL1) in melanoma and non-small-cell lung cancer have reported high response rates, and tumoral PDL1 expression has been suggested as a potential biomarker to enrich for patient response to these treatments. There are only very limited data to date reporting the expression of PDL1 in TNBC. METHODS AND RESULTS: PDL1 immunohistochemistry was performed on 161 primary TNBCs and assessed in the tumour as well as immune cells in the stromal compartment. PDL1 expression was very common in TNBC, expressed in the tumour cell membrane (64%), cytoplasm (80%) and stromal (93%) cellular compartments. Cytoplasmic tumoral expression of PDL1 was associated with a lower risk of breast cancer-specific death [hazard ratio (HR) 0.45, P = 0.035] while stromal PDL1 expression was associated with a lower rate of deaths from all causes (HR 0.305, P = 0.0042). Membranous expression of PDL1 was not associated with outcome. While both PDL1 expression and tumour-infiltrating lymphocytes were associated with a better outcome, only lymphovascular invasion and high tumour-infiltrating lymphocytes were independently prognostic for breast cancer-specific death. CONCLUSION: While PDL1 expression is frequent in TNBC, it was not independently prognostic. There were differences in outcome depending on the cellular compartment of PDL1 expression. These data provide further impetus for investigating the utility of immune checkpoint therapies in TNBC, given the clinical significance of tumour-infiltrating lymphocytes (TILs) and PDL1 expression in this cohort.

H3K27 demethylation by JMJD3 at a poised enhancer of anti-apoptotic gene BCL2 determines ERα ligand dependency.

Chromatin represents a repressive barrier to the process of ligand-dependent transcriptional activity of nuclear receptors. Here, we show that H3K27 methylation imposes ligand-dependent regulation of the oestrogen receptor α (ERα)-dependent apoptotic response via Bcl-2 in breast cancer cells. The activation of BCL2 transcription is dependent on the simultaneous inactivation of the H3K27 methyltransferase, EZH2, and the demethylation of H3K27 at a poised enhancer by the ERα-dependent recruitment of JMJD3 in hormone-dependent breast cancer cells. We also provide evidence that this pathway is modified in cells resistant to anti-oestrogen (AE), which constitutively express BCL2. We show that the lack of H3K27 methylation at BCL2 regulatory elements due to the inactivation of EZH2 by the HER2 pathway leads to this constitutive activation of BCL2 in these AE-resistant cells. Our results describe a mechanism in which the epigenetic state of chromatin affects ligand dependency during ERα-regulated gene expression.

Detailed pathological examination of completion node dissection specimens and outcome in melanoma patients with minimal (<0.1 mm) sentinel lymph node metastases.

BACKGROUND: Nonsentinel lymph nodes (NSLNs) are rarely involved in patients with minimal volume melanoma metastases in sentinel lymph nodes (SLNs). Therefore, it has been suggested that completion lymph node dissection (CLND) is not required. However, the lack of routine immunohistochemical staining and multiple sectioning may have led to failure to identify additional positive nodes. The present study sought to more reliably determine the tumor status of NSLNs in patients with minimally involved SLNs and their clinical outcome. METHODS: A total of 21 tumor-negative CLND specimens from 20 patients with SLN metastases of <0.1 mm in diameter treated between 1991 and 2013 were examined with a more detailed pathologic protocol (five new sections stained with/for H&E, S-100, HMB45, Melan-A, and H&E). Clinical follow-up data were also obtained. RESULTS: Of the 343 examined NSLNs, 1 was found to harbor a 0.18-mm subcapsular sinus metastasis. No metastases were identified in the other NSLNs. Median follow-up was 48 months (range 17-130 months). Six patients (30 %) developed a recurrence. At the end of follow-up, 15 patients (75 %) were alive without sign of melanoma recurrence and 5 patients (25 %) had died of melanoma. Estimated 5-year melanoma-specific survival was 64 %. The patient with the additional positive NSLN remains without recurrence after 130 months follow-up. CONCLUSIONS: Although the risk of additional nodal involvement is low, detailed pathologic examination may identify NSLN metastases not identified using routine protocols. Therefore, nodal clearance appears to be the safest option for these patients, pending the results of prospective trials.

Role of Pirh2 in mediating the regulation of p53 and c-Myc.

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2(-/-) mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor.

Hormone-receptor expression and ovarian cancer survival: an Ovarian Tumor Tissue Analysis consortium study.

BACKGROUND: Few biomarkers of ovarian cancer prognosis have been established, partly because subtype-specific associations might be obscured in studies combining all histopathological subtypes. We examined whether tumour expression of the progesterone receptor (PR) and oestrogen receptor (ER) was associated with subtype-specific survival. METHODS: 12 studies participating in the Ovarian Tumor Tissue Analysis consortium contributed tissue microarray sections and clinical data to our study. Participants included in our analysis had been diagnosed with invasive serous, mucinous, endometrioid, or clear-cell carcinomas of the ovary. For a patient to be eligible, tissue microarrays, clinical follow-up data, age at diagnosis, and tumour grade and stage had to be available. Clinical data were obtained from medical records, cancer registries, death certificates, pathology reports, and review of histological slides. PR and ER statuses were assessed by central immunohistochemistry analysis done by masked pathologists. PR and ER staining was defined as negative (<1% tumour cell nuclei), weak (1 to <50%), or strong (≥50%). Associations with disease-specific survival were assessed. FINDINGS: 2933 women with invasive epithelial ovarian cancer were included: 1742 with high-grade serous carcinoma, 110 with low-grade serous carcinoma, 207 with mucinous carcinoma, 484 with endometrioid carcinoma, and 390 with clear-cell carcinoma. PR expression was associated with improved disease-specific survival in endometrioid carcinoma (log-rank p<0·0001) and high-grade serous carcinoma (log-rank p=0·0006), and ER expression was associated with improved disease-specific survival in endometrioid carcinoma (log-rank p<0·0001). We recorded no significant associations for mucinous, clear-cell, or low-grade serous carcinoma. Positive hormone-receptor expression (weak or strong staining for PR or ER, or both) was associated with significantly improved disease-specific survival in endometrioid carcinoma compared with negative hormone-receptor expression, independent of study site, age, stage, and grade (hazard ratio 0·33, 95% CI 0·21-0·51; p<0·0001). Strong PR expression was independently associated with improved disease-specific survival in high-grade serous carcinoma (0·71, 0·55-0·91; p=0·0080), but weak PR expression was not (1·02, 0·89-1·18; p=0·74). INTERPRETATION: PR and ER are prognostic biomarkers for endometrioid and high-grade serous ovarian cancers. Clinical trials, stratified by subtype and biomarker status, are needed to establish whether hormone-receptor status predicts response to endocrine treatment, and whether it could guide personalised treatment for ovarian cancer. FUNDING: Carraresi Foundation and others.

ARID1A mutations in endometriosis-associated ovarian carcinomas.

BACKGROUND: Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described. METHODS: We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI­SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas. RESULTS: ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions. CONCLUSIONS: These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital­University of British Columbia Hospital Foundation.).

Low expression of the X-linked ribosomal protein S4 in human serous epithelial ovarian cancer is associated with a poor prognosis.

BACKGROUND: The X-linked ribosomal protein S4 (RPS4X), which is involved in cellular translation and proliferation, has previously been identified as a partner of the overexpressed multifunctional protein YB-1 in several breast cancer cells. Depletion of RPS4X results in consistent resistance to cisplatin in such cell lines. METHODS: As platinum-based chemotherapy is a standard first line therapy used to treat patients with ovarian cancer, we evaluated the prognostic value of RPS4X and YB-1 at the protein level in specimen from 192 high-grade serous epithelial ovarian cancer patients. RESULTS: Immunohistochemistry studies indicated that high expression of RPS4X was associated with a lower risk of death and later disease progression (HR = 0.713, P = 0.001 and HR = 0.761, P = 0.001, respectively) as compared to low expression of RPS4X. In contrast, YB-1 was not significantly associated with either recurrence or survival time in this cohort. Finally, the depletion of RPS4X with different siRNAs in two different ovarian cancer cell lines reduced their proliferative growth rate but more importantly increased their resistance to cisplatin. CONCLUSION: Altogether, these results suggest that the levels of RPS4X could be a good indicator for resistance to platinum-based therapy and a prognostic marker for ovarian cancer. Our study also showed that RPS4X is an independent prognostic factor in patients with serous epithelial ovarian cancer.

Inhibition of CD39 unleashes macrophage antibody-dependent cellular phagocytosis against B-cell lymphoma.

Redirection of tumor-associated macrophages to eliminate tumor cells holds great promise for overcoming therapeutic resistance to rituximab and other antibody drugs. Here, we determined the expression of ectonucleotidases CD39 and CD73 in diffuse large B-cell lymphoma (DLBCL), and examined the impact of extracellular ATP (eATP) metabolism on macrophage-mediated anti-lymphoma immunity. Immunostaining of tissue microarray samples showed that CD39 (the ecto-enzyme for eATP hydrolysis) was highly expressed in tumors with the non-germinal center B-cell-like (non-GCB) subtype, and to a lesser extent tumors with the GCB subtype. By contrast, the expression of CD73 (the ecto-enzyme for adenosine generation) was undetectable in tumor cells. Pharmacological blockade of CD39 prevented eATP degradation and enhanced engulfment of antibody-coated lymphoma cells by macrophages in a P2X7 receptor-dependent manner, indicating that eATP fueled antibody-dependent cellular phagocytosis (ADCP) activity. Importantly, inhibition of CD39 augmented in vivo anti-lymphoma effects by therapeutic antibodies including rituximab and daratumumab. Furthermore, the addition of a CD39 inhibitor to anti-CD20 and anti-CD47 combination therapy significantly improved survival in a disseminated model of aggressive B-cell lymphoma, supporting the benefit of dual targeting CD39-mediated eATP hydrolysis and CD47-mediated "don't eat me" signal. Together, preventing eATP degradation may be a potential approach to unleash macrophage-mediated anti-lymphoma immunity.

Crk adaptor proteins act as key signaling integrators for breast tumorigenesis.

INTRODUCTION: CT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo. METHODS: The clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer. RESULTS: A gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was associated with diminished cell proliferation and was rescued by expression of non-shRNA targeted CrkI/II. Perturbations in tumor progression correlated with altered integrin signaling, including decreased cell spreading, diminished p130Cas phosphorylation, and Cdc42 activation. CONCLUSIONS: These data highlight the physiological importance of Crk proteins in regulating growth of aggressive basal breast cancer cells and identify Crk-dependent signaling networks as promising therapeutic targets.

Mutation of FOXL2 in granulosa-cell tumors of the ovary.

BACKGROUND: Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery. METHODS: We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays. RESULTS: All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors. CONCLUSIONS: Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Characterization of the molecular differences between ovarian endometrioid carcinoma and ovarian serous carcinoma.

The histopathological diagnosis of high-grade endometrioid and serous carcinoma of the ovary is poorly reproducible under the current morphology based classification system, especially for anaplastic, high-grade tumours. The transcription factor Wilms' tumour-1 (WT1) is differentially expressed among the gynaecological epithelia from which epithelial ovarian cancers (EOCs) are believed to originate. In EOCs, WT1 protein is observed in the majority of serous carcinomas and in up to 30% of endometrioid carcinomas. It is unclear whether the latter is a reflection of the actual incidence of WT1 protein expression in endometrioid carcinomas, or whether a significant number of high-grade serous carcinomas have been misclassified as endometrioid carcinoma. Several genetic aberrations are reported to occur in EOCs. These include mutation of the TP53 gene, aberrant activation of beta-catenin signalling and loss of PTEN protein expression, among others. It is unclear whether these aberrations are histotype-specific. The aim of this study was to better define the molecular characteristics of serous and endometrioid carcinomas in an attempt to address the problems with the current histopathological classification methods. Gene expression profiles were analysed to identify reproducible gene expression phenotypes for endometrioid and serous carcinomas. Tissue microarrays (TMA) were used to assess the incidence of TP53, beta-catenin and PTEN aberrations in order to correlate their occurrence with WT1 as an immunohistochemistry based biomarker of serous histotype. It was found that nuclear WT1 protein expression can identify misclassified high-grade endometrioid carcinomas and these tumours should be reassigned to serous histotype. Although low-grade endometrioid carcinomas rarely progress to high-grade carcinomas, a combined WT1-negative, TP53-positive immunophenotype may identify an uncommon high-grade subtype of ovarian endometrioid carcinoma. GEO database: array data accession number GSE6008.

Tumor CD155 Expression Is Associated with Resistance to Anti-PD1 Immunotherapy in Metastatic Melanoma.

PURPOSE: Resistance to anti-PD1-based immune checkpoint blockade (ICB) remains a problem for the treatment of metastatic melanoma. Tumor cells as well as host myeloid cells can express the immune checkpoint ligand CD155 to regulate immune cell function. However, the effect of tumor CD155 on the immune context of human melanoma has not been well described. This observational study characterizes tumor CD155 ligand expression by metastatic melanoma tumors and correlates results with differences in immune cell features and response to ICB. EXPERIMENTAL DESIGN: Pretreatment tumor specimens, from 155 patients with metastatic melanoma treated with ICB and from 50 patients treated with BRAF/MEK-directed targeted therapy, were assessed for CD155 expression by IHC. Intratumor T-cell features were analyzed using multiplex-immunohistofluorescence for CD8, PD1, and SOX10. Correlations were made between CD155 tumor level and bulk tumor RNA sequencing results, as well as clinical RECIST response and progression-free survival. RESULTS: High pretreatment CD155 tumor levels correlated with high parenchymal PD1+CD8+/CD8+ T-cell ratios (PD1tR) and poor response to anti-PD1 therapy. In PDL1 negative tumors, high CD155 tumor expression was associated with patients who had poor response to combination anti-PD1/CTLA4 therapy. CONCLUSIONS: Our findings are the first to suggest that tumor CD155 supports an increase in the fraction of PD1+CD8+ T cells in anti-PD1 refractory melanoma tumors and, further, that targeting the CD155 pathway might improve response to anti-PD1 therapy for patients with metastatic melanoma.

Timing of neoadjuvant immunotherapy in relation to surgery is crucial for outcome.

Adjuvant immunotherapies targeting CTLA4 or PD-1 recently demonstrated efficacy in the treatment of earlier stages of human cancer. We previously demonstrated using mouse spontaneous metastasis models that neoadjuvant immunotherapy and surgery was superior, compared to surgery and adjuvant immunotherapy, in eradicating the lethal metastatic disease. However, the optimal scheduling between neoadjuvant immunotherapy and surgery and how it impacts on efficacy and development of immune-related adverse events (irAEs) remains undefined. Using orthotopic 4T1.2 and E0771 mouse models of spontaneously metastatic mammary cancer, we varied the schedule and duration of neoadjuvant immunotherapies and surgery and examined how it impacted on long-term survival. In two tumor models, we demonstrated that a short duration (4-5 days) between first administration of neoadjuvant immunotherapy and resection of the primary tumor was necessary for optimal efficacy, while extending this duration (10 days) abrogated immunotherapy efficacy. However, efficacy was also lost if neoadjuvant immunotherapy was given too close to surgery (2 days). Interestingly, an additional 4 adjuvant doses of treatment following a standard 2 doses of neoadjuvant immunotherapy, did not significantly improve overall tumor-free survival regardless of the combination treatment (anti-PD-1+anti-CD137 or anti-CTLA4+anti-PD-1). Furthermore, biochemical immune-related adverse events (irAEs) increased in tumor-bearing mice that received the additional adjuvant immunotherapy. Overall, our data suggest that shorter doses of neoadjuvant immunotherapy scheduled close to the time of surgery may optimize effective anti-tumor immunity and reduce severe irAEs.

Integrated molecular and immunophenotypic analysis of NK cells in anti-PD-1 treated metastatic melanoma patients.

Purpose: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma. Experimental Design: Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line). Results: Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (p < 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells' cytotoxicity. Conclusions: A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.