B cells and tertiary lymphoid structures promote immunotherapy response.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

Autoimmune antibodies correlate with immune checkpoint therapy-induced toxicities.

Immune checkpoint (IC) therapy provides substantial benefits to cancer patients but can also cause distinctive toxicities termed immune-related adverse events (irAEs). Biomarkers to predict toxicities will be necessary to improve management of patients receiving IC therapy. We relied on serological analysis of recombinant cDNA expression libraries to evaluate plasma samples from patients treated with IC therapy and identified autoantibodies, both in pretreatment and on-treatment samples prior to the development of irAEs, which correlate with the development of immune-related hypophysitis (anti-GNAL and anti-ITM2B autoantibodies) and pneumonitis (anti-CD74 autoantibody). We developed an enzyme-linked immunosorbent assay and tested additional patient samples to confirm our initial findings. Collectively, our data suggest that autoantibodies may correlate with irAEs related to IC therapy, and specific autoantibodies may be detected early for the management of irAEs.

Comparison of immune infiltrates in melanoma and pancreatic cancer highlights VISTA as a potential target in pancreatic cancer.

Immune checkpoint therapy (ICT) has transformed cancer treatment in recent years; however, treatment response is not uniform across tumor types. The tumor immune microenvironment plays a critical role in determining response to ICT; therefore, understanding the differential immune infiltration between ICT-sensitive and ICT-resistant tumor types will help to develop effective treatment strategies. We performed a comprehensive analysis of the immune tumor microenvironment of an ICT-sensitive tumor (melanoma, n = 44) and an ICT-resistant tumor (pancreatic cancer, n = 67). We found that a pancreatic tumor has minimal to moderate infiltration of CD3, CD4, and CD8 T cells; however, the immune infiltrates are predominantly present in the stromal area of the tumor and are excluded from tumoral area compared with melanoma, where the immune infiltrates are primarily present in the tumoral area. Metastatic pancreatic ductal adenocarcinomas (PDACs) had a lower infiltration of total T cells compared with resectable primary PDACs, suggesting that metastatic PDACs have poor immunogenicity. Further, a significantly higher number of CD68+ macrophages and VISTA+ cells (also known as V-domain immunoglobulin suppressor of T cell activation) were found in the pancreatic stromal area compared with melanoma. We identified VISTA as a potent inhibitory checkpoint that is predominantly expressed on CD68+ macrophages on PDACs. These data suggest that VISTA may be a relevant immunotherapy target for effective treatment of patients with pancreatic cancer.

The distribution of T-cell subsets and the expression of immune checkpoint receptors and ligands in patients with newly diagnosed and relapsed acute myeloid leukemia.

BACKGROUND: Phenotypic characterization of immune cells in the bone marrow (BM) of patients with acute myeloid leukemia (AML) is lacking. METHODS: T-cell infiltration was quantified on BM biopsies from 13 patients with AML, and flow cytometry was performed on BM aspirates (BMAs) from 107 patients with AML who received treatment at The University of Texas MD Anderson Cancer Center. The authors evaluated the expression of inhibitory receptors (programmed cell death protein 1 [PD1], cytotoxic T-lymphocyte antigen 4 [CTLA4], lymphocyte-activation gene 3 [LAG3], T-cell immunoglobulin and mucin-domain containing-3 [TIM3]) and stimulatory receptors (glucocorticoid-induced tumor necrosis factor receptor-related protein [GITR], OX40, 41BB [a type 2 transmembrane glycoprotein receptor], inducible T-cell costimulatory [ICOS]) on T-cell subsets and the expression of their ligands (41BBL, B7-1, B7-2, ICOSL, PD-L1, PD-L2, and OX40L) on AML blasts. Expression of these markers was correlated with patient age, karyotype, baseline next-generation sequencing for 28 myeloid-associated genes (including P53), and DNA methylation proteins (DNA methyltransferase 3α, isocitrate dehydrogenase 1[IDH1], IDH2, Tet methylcytosine dioxygenase 2 [TET2], and Fms-related tyrosine kinase 3 [FLT3]). RESULTS: On histochemistry evaluation, the T-cell population in BM appeared to be preserved in patients who had AML compared with healthy donors. The proportion of T-regulatory cells (Tregs) in BMAs was higher in patients with AML than in healthy donors. PD1-positive/OX40-positive T cells were more frequent in AML BMAs, and a higher frequency of PD1-positive/cluster of differentiation 8 (CD8)-positive T cells coexpressed TIM3 or LAG3. PD1-positive/CD8-positive T cells were more frequent in BMAs from patients who had multiply relapsed AML than in BMAs from those who had first relapsed or newly diagnosed AML. Blasts in BMAs from patients who had TP53-mutated AML were more frequently positive for PD-L1. CONCLUSIONS: The preserved T-cell population, the increased frequency of regulatory T cells, and the expression of targetable immune receptors in AML BMAs suggest a role for T-cell-harnessing therapies in AML.

Nivolumab for previously treated unresectable metastatic anal cancer (NCI9673): a multicentre, single-arm, phase 2 study.

BACKGROUND: Squamous cell carcinoma of the anal canal (SCCA) is a rare malignancy associated with infection by human papillomavirus (HPV). No consensus treatment approach exists for the treatment of metastatic disease. Because intratumoral HPV oncoproteins upregulate immune checkpoint proteins such as PD-1 to evade immune-mediated cytotoxicity, we did a trial of the anti-PD-1 antibody nivolumab for patients with metastatic SCCA. METHODS: We did this single-arm, multicentre, phase 2 trial at ten academic centres in the USA. We enrolled patients with treatment-refractory metastatic SCCA, who were given nivolumab every 2 weeks (3 mg/kg). The primary endpoint was response according to Response Evaluation Criteria in Solid Tumors, version 1.1, in the intention-to-treat population. At the time of data cutoff, the study was ongoing, with patients continuing to receive treatment. The study is registered with ClinicalTrials.gov, number NCT02314169. RESULTS: We screened 39 patients, of whom 37 were enrolled and received at least one dose of nivolumab. Among the 37 patients, nine (24% [95% CI 15-33]) had responses. There were two complete responses and seven partial responses. Grade 3 adverse events were anaemia (n=2), fatigue (n=1), rash (n=1), and hypothyroidism (n=1). No serious adverse events were reported. INTERPRETATION: To our knowledge, this is the first completed phase 2 trial of immunotherapy for SCCA. Nivolumab is well tolerated and effective as a monotherapy for patients with metastatic SCCA. Immune checkpoint blockade appears to be a promising approach for patients with this orphan disease. FUNDING: National Cancer Institute/Cancer Therapy Evaluation Program, the HPV and Anal Cancer Foundation, the E B Anal Cancer Fund, The University of Texas MD Anderson Moon Shots Program, and an anonymous philanthropic donor.

Activation of nuclear factor κB (NF-κB) in prostate cancer is mediated by protein kinase C epsilon (PKCepsilon).

Protein kinase C ε (PKCε) has emerged as an oncogenic kinase and plays important roles in cell survival, mitogenesis and invasion. PKCε is up-regulated in most epithelial cancers, including prostate, breast, and lung cancer. Here we report that PKCε is an essential mediator of NF-κB activation in prostate cancer cells. A strong correlation exists between PKCε overexpression and NF-κB activation status in prostate cancer cells. Moreover, transgenic overexpression of PKCε in the mouse prostate causes preneoplastic lesions that display significant NF-κB hyperactivation. PKCε RNAi depletion or inhibition in prostate cancer cells diminishes NF-κB translocation to the nucleus with subsequent impairment of both activation of NF-κB transcription and induction of NF-κB responsive genes in response to the proinflammatory cytokine tumor necrosis factor α (TNFα). On the other hand, PKCε overexpression in normal prostate cells enhances activation of the NF-κB pathway. A mechanistic analysis revealed that TNFα activates PKCε via a C1 domain/diacylglycerol-dependent mechanism that involves phosphatidylcholine-phospholipase C. Moreover, PKCε facilitates the assembly of the TNF receptor-I signaling complex to trigger NF-κB activation. Our studies identified a molecular link between PKCε and NF-κB that controls key responses implicated in prostate cancer progression.

Antitumor activity of tumor-targeted RNA replicase-based plasmid that expresses interleukin-2 in a murine melanoma model.

Double-stranded RNA (dsRNA) has multiple antitumor mechanisms that may be used to control tumor growth. Previously we have shown that treatment of solid tumors with a plasmid that encodes Sindbis viral RNA replicase complex, pSIN-ß, significantly inhibited the growth of tumors in mice. In the present study, we evaluated the feasibility of further improving the antitumor activity of the pSIN-ß plasmid by incorporating interleukin-2 (IL2) gene into the plasmid. The resultant pSIN-IL2 plasmid was delivered to mouse melanoma cells that overexpress the sigma receptor. Here we report that the pSIN-IL2 plasmid was more effective at controlling the growth of B16 melanoma in mice when complexed with sigma receptor-targeted liposomes than with the untargeted liposomes. Importantly, the pSIN-IL2 plasmid was more effective than pSIN-ß plasmid at controlling the growth of B16 melanoma in mice, and B16 tumor-bearing mice that were treated with pSIN-IL2 had an elevated number of activated CD4(+), CD8(+), and natural killer cells, as compared to those treated with pSIN-ß. The RNA replicase-based, IL2-expressing plasmid may have applications in melanoma gene therapy.

Neoadjuvant Durvalumab Alone or Combined with Novel Immuno-Oncology Agents in Resectable Lung Cancer: The Phase II NeoCOAST Platform Trial.

Neoadjuvant chemoimmunotherapy improves pathologic complete response rate and event-free survival in patients with resectable non-small cell lung cancer (NSCLC) versus chemotherapy alone. NeoCOAST was the first randomized, multidrug platform trial to examine novel neoadjuvant immuno-oncology combinations for patients with resectable NSCLC, using major pathologic response (MPR) rate as the primary endpoint. Eighty-three patients received a single cycle of treatment: 26 received durvalumab (anti-PD-L1) monotherapy, 21 received durvalumab plus oleclumab (anti-CD73), 20 received durvalumab plus monalizumab (anti-NKG2A), and 16 received durvalumab plus danvatirsen (anti-STAT3 antisense oligonucleotide). MPR rates were higher for patients in the combination arms versus durvalumab alone. Safety profiles for the combinations were similar to those of durvalumab alone. Multiplatform immune profiling suggested that improved MPR rates in the durvalumab plus oleclumab and durvalumab plus monalizumab arms were associated with enhanced effector immune infiltration of tumors, interferon responses and markers of tertiary lymphoid structure formation, and systemic functional immune cell activation. SIGNIFICANCE: A neoadjuvant platform trial can rapidly generate clinical and translational data using candidate surrogate endpoints like MPR. In NeoCOAST, patients with resectable NSCLC had improved MPR rates after durvalumab plus oleclumab or monalizumab versus durvalumab alone and tumoral transcriptomic signatures indicative of augmented immune cell activation and function. See related commentary by Cooper and Yu, p. 2306. This article is featured in Selected Articles from This Issue, p. 2293.

Protective role of cathepsin L in mouse skin carcinogenesis.

Lysosomal cysteine protease cathepsin L (CTSL) is believed to play a role in tumor progression and is considered a marker for clinically invasive tumors. Studies from our laboratory using the classical mouse skin carcinogenesis model, with 7,12-dimethyl-benz[a]anthracene (DMBA) for initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) for promotion, showed that expression of CTSL is increased in papillomas and squamous cell carcinomas (SCC). We also carried out carcinogenesis studies using Ctsl-deficient nackt (nkt) mutant mice on three different inbred backgrounds. Unexpectedly, the multiplicity of papillomas was significantly higher in Ctsl-deficient than in wild-type mice on two unrelated backgrounds. Topical applications of TPA or DMBA alone to the skin of nkt/nkt mice did not induce papillomas, and there was no increase in spontaneous tumors in nkt/nkt mice on any of the three inbred backgrounds. Reduced epidermal cell proliferation in Ctsl-deficient nkt/nkt mice after TPA treatment suggested that they are not more sensitive than wild-type mice to TPA promotion. We also showed that deficiency of CTSL delays terminal differentiation of keratinocytes, and we propose that decreased elimination of initiated cells is at least partially responsible for the increased papilloma formation in the nackt model.

A Randomized Phase II Study of MEDI0680 in Combination with Durvalumab versus Nivolumab Monotherapy in Patients with Advanced or Metastatic Clear-cell Renal Cell Carcinoma.

PURPOSE: MEDI0680 is a humanized anti-programmed cell death-1 (PD-1) antibody, and durvalumab is an anti-PD-L1 antibody. Combining treatment using these antibodies may improve efficacy versus blockade of PD-1 alone. This phase II study evaluated antitumor activity and safety of MEDI0680 plus durvalumab versus nivolumab monotherapy in immunotherapy-naïve patients with advanced clear-cell renal cell carcinoma who received at least one prior line of antiangiogenic therapy. PATIENTS AND METHODS: Patients received either MEDI0680 (20 mg/kg) with durvalumab (750 mg) or nivolumab (240 mg), all intravenous, every 2 weeks. The primary endpoint was investigator-assessed objective response rate (ORR). Secondary endpoints included best overall response, progression-free survival (PFS), safety, overall survival (OS), and immunogenicity. Exploratory endpoints included changes in circulating tumor DNA (ctDNA), baseline tumor mutational burden, and tumor-infiltrated immune cell profiles. RESULTS: Sixty-three patients were randomized (combination, n = 42; nivolumab, n = 21). ORR was 16.7% [7/42; 95% confidence interval (CI), 7.0-31.4] with combination treatment and 23.8% (5/21; 95% CI, 8.2-47.2) with nivolumab. Median PFS was 3.6 months in both arms; median OS was not reached in either arm. Because of adverse events, 23.8% of patients discontinued MEDI0680 and durvalumab and 14.3% of patients discontinued nivolumab. In the combination arm, reduction in ctDNA fraction was associated with longer PFS. ctDNA mutational analysis did not demonstrate an association with response in either arm. Tumor-infiltrated immune profiles showed an association between immune cell activation and response in the combination arm. CONCLUSIONS: MEDI0680 combined with durvalumab was safe and tolerable; however, it did not improve efficacy versus nivolumab monotherapy.

Phase 1b study of pegylated arginine deiminase (ADI-PEG 20) plus Pembrolizumab in advanced solid cancers.

Background: Pegylated arginine deiminase (ADI-PEG 20) is a metabolism-based strategy that depletes arginine, resulting in tumoral stress and cytotoxicity. Preclinically, ADI-PEG 20 modulates T-cell activity and enhances the therapeutic efficacy of programmed death-1 (PD-1) inhibition. Methods: A phase 1b study, including a dose-escalation cohort and an expansion cohort, was undertaken to explore the effects of ADI-PEG 20 in combination with pembrolizumab, an anti-PD-1 antibody, for safety, pharmacodynamics, and response. CD3 levels and programmed death-ligand 1 (PD-L1) expression were assessed in paired biopsies collected prior to and after ADI-PEG 20 treatment but before pembrolizumab. Results: Twenty-five patients, nine in the dose-escalation cohort and sixteen in the expansion cohort, were recruited. Treatment was feasible with adverse events consistent with those known for each agent, except for Grade 3/4 neutropenia which was higher than expected, occurring in 10/25 (40%) patients. Mean arginine levels were suppressed for 1-3 weeks, but increased gradually. CD3+ T cells increased in 10/12 (83.3%) subjects following ADI-PEG 20 treatment, including in three partial responders (p = .02). PD-L1 expression was low and increased in 3/10 (30%) of subjects. Partial responses occurred in 6/25 (24%) heavily pretreated patients, in both argininosuccinate synthetase 1 proficient and deficient subjects. Conclusions: The immunometabolic combination was safe with the caveat that the incidence of neutropenia might be increased compared with either agent alone. ADI-PEG 20 treatment increased T cell infiltration in the low PD-L1 tumor microenvironment. The recommended phase 2 doses are 36 mg/m2 weekly for ADI-PEG 20 and 200 mg every 3 weeks for pembrolizumab.

PTEN deficiency is fully penetrant for prostate adenocarcinoma in C57BL/6 mice via mTOR-dependent growth.

The tumor suppressor phosphatase and tensin homolog (PTEN) is frequently involved in human prostate carcinoma. PTEN is therefore an attractive target for the development of preclinical animal models. Prostate intraepithelial neoplasia lesions develop in mice with Pten heterozygosity, but disease progression has been reported only in combination with either other tumor suppressor gene alterations or the conditional inactivation of both Pten alleles in prostate epithelial cells. We report that on a C57BL/6 background, in contrast to previous studies on mixed 129 genetic backgrounds, Pten locus heterozygosity is fully penetrant for the development of prostate adenocarcinoma. Grossly observable tumors were detected at 6 months of age, and, by 10 to 12 months, 100% of examined mice developed adenocarcinoma of the anterior prostate. Furthermore, double heterozygotes carrying both Pten and Tsc2-null alleles showed no increase relative to Pten(+/-) heterozygotes in either lesion development or progression. Lesions in both Pten(+/-); Tsc2(+/-), and Pten(+/-) mice exhibited loss of PTEN expression and activation of PI3K signaling. PI3K activation occurred early in prostate intraepithelial neoplasia lesion formation in these animals, consistent with loss of PTEN function, and contributed to the etiology of tumors that developed in Pten(+/-) mice. Furthermore, prostate lesion growth in Pten(+/-) mice was dependent on mTOR, as evidenced by a reduction in both phospho-S6 levels and proliferative index after rapamycin treatment.

ARID1A mutation plus CXCL13 expression act as combinatorial biomarkers to predict responses to immune checkpoint therapy in mUCC.

Immune checkpoint therapy (ICT) can produce durable antitumor responses in metastatic urothelial carcinoma (mUCC); however, the responses are not universal. Despite multiple approvals of ICT in mUCC, we lack predictive biomarkers to guide patient selection. The identification of biomarkers may require interrogation of both the tumor mutational status and the immune microenvironment. Through multi-platform immuno-genomic analyses of baseline tumor tissues, we identified the mutation of AT-rich interactive domain-containing protein 1A (ARID1A) in tumor cells and expression of immune cytokine CXCL13 in the baseline tumor tissues as two predictors of clinical responses in a discovery cohort (n = 31). Further, reverse translational studies revealed that CXCL13-/- tumor-bearing mice were resistant to ICT, whereas ARID1A knockdown enhanced sensitivity to ICT in a murine model of bladder cancer. Next, we tested the clinical relevance of ARID1A mutation and baseline CXCL13 expression in two independent confirmatory cohorts (CheckMate275 and IMvigor210). We found that ARID1A mutation and expression of CXCL13 in the baseline tumor tissues correlated with improved overall survival (OS) in both confirmatory cohorts (CheckMate275, CXCL13 data, n = 217; ARID1A data, n = 139, and IMvigor210, CXCL13 data, n = 348; ARID1A data, n = 275). We then interrogated CXCL13 expression plus ARID1A mutation as a combination biomarker in predicting response to ICT in CheckMate275 and IMvigor210. Combination of the two biomarkers in baseline tumor tissues suggested improved OS compared to either single biomarker. Cumulatively, this study revealed that the combination of CXCL13 plus ARID1A may improve prediction capability for patients receiving ICT.

Neoantigen responses, immune correlates, and favorable outcomes after ipilimumab treatment of patients with prostate cancer.

Tumors with high mutational burden (TMB) tend to be responsive to immune checkpoint blockade (ICB) because there are neoantigens available for targeting by reinvigorated T cells, whereas those with low TMB demonstrate limited clinical responses. To determine whether antigen-specific T cell responses can be elicited after treatment with ICB in cancers that have a low TMB, we conducted a clinical trial with ipilimumab in 30 patients with metastatic castration-resistant prostate cancer. We identified two distinct cohorts by survival and progression times: "favorable" (n = 9) and "unfavorable" (n = 10). Patients in the favorable cohort had high intratumoral CD8 T cell density and IFN-γ response gene signature and/or antigen-specific T cell responses. Two patients with a relatively low TMB had T cell responses against unique neoantigens. Moreover, six of nine patients in the favorable group are still alive at the time of analysis, with survival ranging from 33 to 54 months after treatment. All 10 patients in the unfavorable cohort have succumbed to their disease and had survival ranging from 0.6 to 10.3 months. Collectively, our data indicate that immunological correlates associated with effector T cell responses are observed in patients with metastatic prostate cancer who benefit from ICB.

Immune profiling of human tumors identifies CD73 as a combinatorial target in glioblastoma.

Immune checkpoint therapy with anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of many solid tumors. However, the clinical efficacy of immune checkpoint therapy is limited to a subset of patients with specific tumor types1,2. Multiple clinical trials with combinatorial immune checkpoint strategies are ongoing; however, the mechanistic rationale for tumor-specific targeting of immune checkpoints is elusive. To garner an insight into tumor-specific immunomodulatory targets, we analyzed 94 patients representing five different cancer types, including those that respond relatively well to immune checkpoint therapy and those that do not, such as glioblastoma multiforme, prostate cancer and colorectal cancer. Through mass cytometry and single-cell RNA sequencing, we identified a unique population of CD73hi macrophages in glioblastoma multiforme that persists after anti-PD-1 treatment. To test if targeting CD73 would be important for a successful combination strategy in glioblastoma multiforme, we performed reverse translational studies using CD73-/- mice. We found that the absence of CD73 improved survival in a murine model of glioblastoma multiforme treated with anti-CTLA-4 and anti-PD-1. Our data identified CD73 as a specific immunotherapeutic target to improve antitumor immune responses to immune checkpoint therapy in glioblastoma multiforme and demonstrate that comprehensive human and reverse translational studies can be used for rational design of combinatorial immune checkpoint strategies.

Neoadjuvant PD-L1 plus CTLA-4 blockade in patients with cisplatin-ineligible operable high-risk urothelial carcinoma.

Immune checkpoint therapy is being tested in the neoadjuvant setting for patients with localized urothelial carcinoma1,2, with one study reporting data in cisplatin-ineligible patients who received anti-PD-L1 monotherapy2. The study reported that patients with bulky tumors, a known high-risk feature defined as greater than clinical T2 disease, had fewer responses, with pathological complete response rate of 17%2. Here we report on the first pilot combination neoadjuvant trial ( NCT02812420 ) with anti-PD-L1 (durvalumab) plus anti-CTLA-4 (tremelimumab) in cisplatin-ineligible patients, with all tumors identified as having high-risk features (n = 28). High-risk features were defined by bulky tumors, variant histology, lymphovascular invasion, hydronephrosis and/or high-grade upper tract disease3-5. The primary endpoint was safety and we observed 6 of 28 patients (21%) with grade ≥3 immune-related adverse events, consisting of asymptomatic laboratory abnormalities (n = 4), hepatitis and colitis (n = 2). We also observed pathological complete response of 37.5% and downstaging to pT1 or less in 58% of patients who completed surgery (n = 24). In summary, we provide initial safety, efficacy and biomarker data with neoadjuvant combination anti-PD-L1 plus anti-CTLA-4, which warrants further development for patients with localized urothelial carcinoma, especially cisplatin-ineligible patients with high-risk features who do not currently have an established standard-of-care neoadjuvant treatment.

Window-of-opportunity clinical trial of pembrolizumab in patients with recurrent glioblastoma reveals predominance of immune-suppressive macrophages.

BACKGROUND: We sought to ascertain the immune effector function of pembrolizumab within the glioblastoma (GBM) microenvironment during the therapeutic window. METHODS: In an open-label, single-center, single-arm phase II "window-of-opportunity" trial in 15 patients with recurrent (operable) GBM receiving up to 2 pembrolizumab doses before surgery and every 3 weeks afterward until disease progression or unacceptable toxicities occurred, immune responses were evaluated within the tumor. RESULTS: No treatment-related deaths occurred. Overall median follow-up time was 50 months. Of 14 patients monitored, 10 had progressive disease, 3 had a partial response, and 1 had stable disease. Median progression-free survival (PFS) was 4.5 months (95% CI: 2.27, 6.83), and the 6-month PFS rate was 40%. Median overall survival (OS) was 20 months, with an estimated 1-year OS rate of 63%. GBM patients' recurrent tumors contained few T cells that demonstrated a paucity of immune activation markers, but the tumor microenvironment was markedly enriched for CD68+ macrophages. CONCLUSIONS: Immune analyses indicated that pembrolizumab anti-programmed cell death 1 (PD-1) monotherapy alone can't induce effector immunologic response in most GBM patients, probably owing to a scarcity of T cells within the tumor microenvironment and a CD68+ macrophage preponderance.

Comprehensive Molecular Characterization Identifies Distinct Genomic and Immune Hallmarks of Renal Medullary Carcinoma.

Renal medullary carcinoma (RMC) is a highly lethal malignancy that mainly afflicts young individuals of African descent and is resistant to all targeted agents used to treat other renal cell carcinomas. Comprehensive genomic and transcriptomic profiling of untreated primary RMC tissues was performed to elucidate the molecular landscape of these tumors. We found that RMC was characterized by high replication stress and an abundance of focal copy-number alterations associated with activation of the stimulator of the cyclic GMP-AMP synthase interferon genes (cGAS-STING) innate immune pathway. Replication stress conferred a therapeutic vulnerability to drugs targeting DNA-damage repair pathways. Elucidation of these previously unknown RMC hallmarks paves the way to new clinical trials for this rare but highly lethal malignancy.

Enhanced skin carcinogenesis and lack of thymus hyperplasia in transgenic mice expressing human cyclin D1b (CCND1b).

Cyclin D1b is an alternative transcript of the cyclin D1 gene (CCND1) expressed in human tumors. Its abundance is regulated by a single base pair polymorphism at the exon 4/intron 4 boundary (nucleotide 870). Epidemiological studies have shown a correlation between the presence of the G870A allele (that favors the splicing for cyclin D1b) with increased risk and less favorable outcome in several forms of cancer. More recently, it has been shown that, unlike cyclin D1a, the alternative transcript D1b by itself has the capacity to transform fibroblasts in vitro. In order to study the oncogenic potential of cyclin D1b, we developed transgenic mice expressing human cyclin D1b under the control of the bovine K5 promoter (K5D1b mice). Seven founders were obtained and none of them presented any significant phenotype or developed spontaneous tumors. Interestingly, K5D1b mice do not develop the fatal thymic hyperplasia, which is characteristic of the cyclin D1a transgenic mice (K5D1a). Susceptibility to skin carcinogenesis was tested in K5D1b mice using two-stage carcinogenesis protocols. In two independent experiments, K5D1b mice developed higher papilloma multiplicity as compared with wild-type littermates. However, when K5D1b mice were crossed with cyclin D1KO mice, the expression of cyclin D1b was unable to rescue the carcinogenesis-resistant phenotype of the cyclin D1 KO mice. To further explore the role of cyclin D1b in mouse models of carcinogenesis we carried out in silico analysis and in vitro experiments to evaluate the existence of a mouse homologous of the human cyclin D1b transcript. We were unable to find any evidence of an alternatively spliced transcript in mouse Ccnd1. These results show that human cyclin D1b has different biological functions than cyclin D1a and confirm its oncogenic properties.