Decreased progenitor TCF1 + T-cells correlate with COVID-19 disease severity.

COVID-19, caused by SARS-CoV-2, can lead to a severe inflammatory disease characterized by significant lymphopenia. However, the underlying cause for the depletion of T-cells in COVID-19 patients remains incompletely understood. In this study, we assessed the presence of different T-cell subsets in the progression of COVID-19 from mild to severe disease, with a focus on TCF1 expressing progenitor T-cells that are needed to replenish peripheral T-cells during infection. Our results showed a preferential decline in TCF1+ progenitor CD4 and CD8+ T-cells with disease severity. This decline was seen in various TCF1+ subsets including naive, memory and effector-memory cells, and surprisingly, was accompanied by a loss in cell division as seen by a marked decline in Ki67 expression. In addition, TCF1+ T-cells showed a reduction in pro-survival regulator, BcL2, and the appearance of a new population of TCF1 negative caspase-3 expressing cells in peripheral blood from patients with severe disease. The decline in TCF1+ T-cells was also seen in a subgroup of severe patients with vitamin D deficiency. Lastly, we found that sera from severe patients inhibited TCF1 transcription ex vivo which was attenuated by a blocking antibody against the cytokine, interleukin-12 (IL12). Collectively, our findings underscore the potential significance of TCF1+ progenitor T-cells in accounting for the loss of immunity in severe COVID-19 and outline an array of markers that could be used to identify disease progression.

A next-generation BRAF inhibitor overcomes resistance to BRAF inhibition in patients with BRAF-mutant cancers using pharmacokinetics-informed dose escalation.

RAF inhibitors have transformed treatment for BRAF V600-mutant cancer patients, but clinical benefit is limited by adaptive induction of ERK signaling, genetic alterations that induce BRAF V600 dimerization, and poor brain penetration. Next-generation pan-RAF dimer inhibitors are limited by narrow therapeutic index. PF-07799933 (ARRY-440) is a brain-penetrant, selective, pan-mutant BRAF inhibitor. PF-07799933 inhibited signaling in vitro, disrupted endogenous mutant-BRAF:wild-type-CRAF dimers, and spared wild-type ERK signaling. PF-07799933 ± binimetinib inhibited growth of mouse xenograft tumors driven by mutant BRAF that functions as dimers and by BRAF V600E with acquired resistance to current RAF inhibitors. We treated patients with treatment-refractory BRAF-mutant solid tumors in a first-in-human clinical trial (NCT05355701) that utilized a novel, flexible, pharmacokinetics-informed dose escalation design that allowed rapid achievement of PF-07799933 efficacious concentrations. PF-07799933 ± binimetinib was well-tolerated and resulted in multiple confirmed responses, systemically and in the brain, in BRAF-mutant cancer patients refractory to approved RAF inhibitors.

Improved platform for breast cancer circulating tumor cell enrichment and characterization with next-generation sequencing technology.

Circulating tumor cells (CTCs) represent cells shed from the primary tumor or metastatic sites and can be used to monitor treatment response and tumor recurrence. However, CTCs circulate in extremely low numbers making in-depth analysis beyond simple enumeration challenging when collected from peripheral blood. Furthermore, tumor heterogeneity, a hallmark of many tumors, especially breast cancer, further complicates CTC characterization. To overcome this limitation, we developed a platform based on the large-scale isolation of CTCs by apheresis, allowing us to collect CTCs in large numbers, which were preserved live in liquid nitrogen for further characterization. Flow cytometry followed by cell sorting (FACS) was performed using a combination of antibodies directed against cell surface markers of white blood cells (CD45) and epithelial tumor cells (CK8). Analysis of subpopulations CD45+/- and CK8+/- by bulk RNA sequencing (RNAseq) and the CD45-/CK8 positive population by single-cell RNAseq was performed. The CD45- population was enriched using CD45 magnetic beads separation and examined by IHC for pan-cytokeratin and immunofluorescence (IF) for specific markers, including the elusive circulating cancer stem cells (CSCs). CSC-rich mammospheres were grown in vitro for further analysis and treated to examine their response to chemotherapeutic agents. Finally, mammospheres were transplanted into the mammary fat pad and bone of immunodeficient mice to examine tumor growth in vivo. This platform enables the detection and collection of CTCs in early and late-stage breast cancer patients of every subtype. Markers including CD44/24, ALDH1 and CXCR4 were identified by IF and showed high expression following mammosphere culture, which responded predictably to chemotherapeutic agents. Mammospheres were also transplanted into nude mice and induced tumors in the mammary fat pad and bone following intra-tibial transplantation. Finally, bulk RNA analysis of the FACS isolated CD45+/- and CK8+/- cells showed a clear separation of CD45- away from CD45+ populations. Single-cell RNAseq of the FACS isolated CD45-/CK8+ cells showed the presence of 4-5 clusters, confirming the high degree of heterogeneity of CTCs. Our platform for large-scale isolation of CTCs using apheresis is suitable for an in-depth analysis of the cancer phenotype and may eventually allow evaluation in real-time of the disease process to optimize cancer regimens.

Phase II LEAP-004 Study of Lenvatinib Plus Pembrolizumab for Melanoma With Confirmed Progression on a Programmed Cell Death Protein-1 or Programmed Death Ligand 1 Inhibitor Given as Monotherapy or in Combination.

PURPOSE: Effective treatments are needed for melanoma that progresses on inhibitors of programmed cell death protein-1 (PD-1) or its ligand (PD-L1). We conducted the phase II LEAP-004 study to evaluate the combination of the multikinase inhibitor lenvatinib and the PD-1 inhibitor pembrolizumab in this population (ClinicalTrials.gov identifier: NCT03776136). METHODS: Eligible patients with unresectable stage III-IV melanoma with confirmed progressive disease (PD) within 12 weeks of the last dose of a PD-1/L1 inhibitor given alone or with other therapies, including cytotoxic T-cell lymphocyte-associated antigen 4 (CTLA-4) inhibitors, received lenvatinib 20 mg orally once daily plus ≤ 35 doses of pembrolizumab 200 mg intravenously once every 3 weeks until PD or unacceptable toxicity. The primary end point was objective response rate (ORR) per RECIST, version 1.1, by independent central review. RESULTS: A total of 103 patients were enrolled and treated. The median study follow-up was 15.3 months. ORR in the total population was 21.4% (95% CI, 13.9 to 30.5), with three (2.9%) complete responses and 19 (18.4%) partial responses. The median duration of response was 8.3 months (range, 3.2-15.9+). ORR was 33.3% in the 30 patients with PD on prior anti-PD-1 plus anti-CTLA-4 therapy. The median progression-free survival and overall survival in the total population were 4.2 months (95% CI, 3.8 to 7.1) and 14.0 months (95% CI, 10.8 to not reached), respectively. Grade 3-5 treatment-related adverse events occurred in 47 (45.6%) patients, most commonly hypertension (21.4%); one patient died from a treatment-related event (decreased platelet count). CONCLUSION: Lenvatinib plus pembrolizumab provides clinically meaningful, durable responses in patients with advanced melanoma with confirmed PD on prior PD-1/L1 inhibitor-based therapy, including those with PD on anti-PD-1 plus anti-CTLA-4 therapy. The safety profile was as expected. These data support lenvatinib plus pembrolizumab as a potential regimen for this population of high unmet need.

The Mutational Spectrum of Pre- and Post-Neoadjuvant Chemotherapy Triple-Negative Breast Cancers.

The response of triple-negative breast cancer (TNBC) patients to pre-operative (neoadjuvant chemotherapy) is a critical factor of their outcome. To determine the effects of chemotherapy on the tumor genome and to identify mutations associated with chemoresistance and sensitivity, we performed whole exome sequencing on pre/post-chemotherapy tumors and matched lymphocytes from 26 patients. We observed great inter-tumoral heterogeneity with no gene mutated recurrently in more than four tumors besides TP53. Although the degree of response to chemotherapy in residual tumors was associated with more subclonal changes during chemotherapy, there was minimal evolution between pre/post-tumors. Indeed, gene sets enriched for mutations in pre- and post-chemotherapy tumors were very similar and reflected genes involved in the biological process of neurogenesis. Somatically mutated genes present in chemosensitive tumors included COL1A2, PRMD15, APOBEC3B, PALB2 and histone protein encoding genes, while BRCA1, ATR, ARID1A, XRCC3 and genes encoding for tubulin-associated proteins were present in the chemoresistant tumors. We also found that the mutational spectrum of post-chemotherapy tumors was more reflective of matching metastatic tumor biopsies than pre-chemotherapy samples. These findings support a portrait of modest ongoing genomic instability with respect to single-nucleotide variants induced by or selected for by chemotherapy in TNBCs.

Impact of the 21-Gene Recurrence Score Assay on Treatment Decisions and Cost in Patients with Node-Positive Breast Cancer: A Multicenter Study in Quebec.

BACKGROUND: The 21-gene Breast Recurrence Score (RS) assay, "the assay", has led to a paradigm shift for patients with hormone receptor-positive, node-negative early breast cancer and is emerging as an important tool to assist physician-patient decisions in foregoing chemotherapy in node-positive patients. We wanted to better understand the impact of the RS assay in node-positive patients upon physician treatment decisions and treatment cost in Quebec, Canada. PATIENTS AND METHODS: We conducted a multicenter, prospective observational trial for Estrogen/Progesterone Receptor (ER/PR)- positive, Human Epidermal Growth Factor Receptor 2 (HER2)-negative breast cancer patients with 1-3 positive lymph nodes. Physicians completed a questionnaire indicating treatment choice prior to and post availability of RS results. The primary endpoint was change in the physician's recommendation for chemotherapy prior to and post assay results. Secondary endpoints included change in physician's expressed level of confidence, and changes in estimated cost of recommended treatments prior to and post assay results. RESULTS: For the entire cohort, physician recommendation for chemotherapy was reduced by an absolute 67.1% by knowledge of the RS assay result (P < .0001). Physician recommendation of chemotherapy was decreased by 75.9% for patients RS result <14 (P < .0001); and 67.5% for patients with RS result 14-25 (P < .0001). Changes in treatment recommendations were associated with an overall reduction in cost by 73.7% per patient, and after incorporating the cost of the RS test, a cost benefit of $823 CAN at 6-month follow-up. CONCLUSION: Altogether, we established that the assay led to a two-third reduction in the use of chemotherapy, and was a cost-effective approach for hormone receptor-positive, node-positive breast cancer.

Helicobacter pylori serology is associated with worse overall survival in patients with melanoma treated with immune checkpoint inhibitors.

The microbiome is now regarded as one of the hallmarks of cancer and several strategies to modify the gut microbiota to improve immune checkpoint inhibitor (ICI) activity are being evaluated in clinical trials. Preliminary data regarding the upper gastro-intestinal microbiota indicated that Helicobacter pylori seropositivity was associated with a negative prognosis in patients amenable to ICI. In 97 patients with advanced melanoma treated with ICI, we assessed the impact of H. pylori on outcomes and microbiome composition. We performed H. pylori serology and profiled the fecal microbiome with metagenomics sequencing. Among the 97 patients, 22% were H. pylori positive (Pos). H. pylori Pos patients had a significantly shorter overall survival (p = .02) compared to H. pylori negative (Neg) patients. In addition, objective response rate and progression-free survival were decreased in H. pylori Pos patients. Metagenomics sequencing did not reveal any difference in diversity indexes between the H. pylori groups. At the taxa level, Eubacterium ventriosum, Mediterraneibacter (Ruminococcus) torques, and Dorea formicigenerans were increased in the H. pylori Pos group, while Alistipes finegoldii, Hungatella hathewayi and Blautia producta were over-represented in the H. pylori Neg group. In a second independent cohort of patients with NSCLC, diversity indexes were similar in both groups and Bacteroides xylanisolvens was increased in H. pylori Neg patients. Our results demonstrated that the negative impact of H. pylori on outcomes seem to be independent from the fecal microbiome composition. These findings warrant further validation and development of therapeutic strategies to eradicate H. pylori in immuno-oncology arena.

Parathyroid Hormone-Related Protein Inhibition Blocks Triple-Negative Breast Cancer Expansion in Bone Through Epithelial to Mesenchymal Transition Reversal.

Parathyroid hormone-related protein (PTHrP) plays a major role in skeletal metastasis but its action mechanism has not been fully defined. We previously demonstrated the crucial importance of PTHrP in promoting mammary tumor initiation, growth, and metastasis in a mouse model with a mammary epithelium-targeted Pthlh gene ablation. We demonstrate here a novel mechanism for bone invasion involving PTHrP induction of epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs) regulation. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated Pthlh gene ablation was used to study EMT markers, phenotype, and invasiveness in two triple-negative breast cancer (TNBC) cell types (established MDA-MB-231 and patient-derived PT-TNBC cells). In vitro, Pthlh ablation in TNBC cells reduced EMT markers, mammosphere-forming ability, and CD44high/CD24low cells ratio. In vivo, cells were injected intratibially into athymic nude mice, and therapeutic treatment with our anti-PTHrP blocking antibody was started 2 weeks after skeletal tumors were established. In vivo, compared to control, lytic bone lesion from Pthlh -ablated cells decreased significantly over 2 weeks by 27% for MDA-MB-231 and by 75% for PT-TNBC-injected mice (p < 0.001). Micro-CT (µCT) analyses also showed that antibody therapy reduced bone lytic volume loss by 52% and 48% for non-ablated MDA-MB-231 and PT-TNBC, respectively (p < 0.05). Antibody therapy reduced skeletal tumor burden by 45% and 87% for non-ablated MDA-MB-231 and PT-TNBC, respectively (p < 0.002) and caused a significant decrease of CSC/EMT markers ALDH1, vimentin, and Slug, and an increase in E-cadherin in bone lesions. We conclude that PTHrP is a targetable EMT molecular driver and suggest that its pharmacological blockade can provide a potential therapeutic approach against established TNBC-derived skeletal lesions. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Spatially mapping the immune landscape of melanoma using imaging mass cytometry.

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8+CD45RO+Ki67+) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses.

Multicenter, double-blind, placebo-controlled trial of seviprotimut-L polyvalent melanoma vaccine in patients with post-resection melanoma at high risk of recurrence.

BACKGROUND: Most patients with advanced melanomas relapse after checkpoint blockade therapy. Thus, immunotherapies are needed that can be applied safely early, in the adjuvant setting. Seviprotimut-L is a vaccine containing human melanoma antigens, plus alum. To assess the efficacy of seviprotimut-L, the Melanoma Antigen Vaccine Immunotherapy Study (MAVIS) was initiated as a three-part multicenter, double-blind, placebo-controlled phase III trial. Results from part B1 are reported here. METHODS: Patients with AJCC V.7 stage IIB-III cutaneous melanoma after resection were randomized 2:1, with stage stratification (IIB/C, IIIA, IIIB/C), to seviprotimut-L 40 mcg or placebo. Recurrence-free survival (RFS) was the primary endpoint. For an hypothesized HR of 0.625, one-sided alpha of 0.10, and power 80%, target enrollment was 325 patients. RESULTS: For randomized patients (n=347), arms were well-balanced, and treatment-emergent adverse events were similar for seviprotimut-L and placebo. For the primary intent-to-treat endpoint of RFS, the estimated HR was 0.881 (95% CI: 0.629 to 1.233), with stratified logrank p=0.46. However, estimated HRs were not uniform over the stage randomized strata, with HRs (95% CIs) for stages IIB/IIC, IIIA, IIIB/IIIC of 0.67 (95% CI: 0.37 to 1.19), 0.72 (95% CI: 0.35 to 1.50), and 1.19 (95% CI: 0.72 to 1.97), respectively. In the stage IIB/IIC stratum, the effect on RFS was greatest for patients <60 years old (HR=0.324 (95% CI: 0.121 to 0.864)) and those with ulcerated primary melanomas (HR=0.493 (95% CI: 0.255 to 0.952)). CONCLUSIONS: Seviprotimut-L is very well tolerated. Exploratory efficacy model estimation supports further study in stage IIB/IIC patients, especially younger patients and those with ulcerated melanomas. TRIAL REGISTRATION NUMBER: NCT01546571.

Gut microbiota signatures are associated with toxicity to combined CTLA-4 and PD-1 blockade.

Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but also a high rate of immune-related adverse events. Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 patients with advanced melanoma treated with CICB, with a high rate of any ≥grade 3 immune-related adverse events (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T-cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1ß in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Prognostic and predictive value of circulating tumor DNA during neoadjuvant chemotherapy for triple negative breast cancer.

Response to neoadjuvant chemotherapy (NAC) in triple negative breast cancer (TNBC) is highly prognostic and determines whether adjuvant chemotherapy is needed if residual tumor is found at surgery. To evaluate the predictive and prognostic values of circulating tumor DNA (ctDNA) in this setting, we analyzed tumor and serial bloods from 26 TNBC patients collected prior, during, and after NAC. Individual digital droplet PCR assays were developed for 121 variants (average 5/patient) identified from tumor sequencing, enabling ctDNA detection in 96% of patients at baseline. Mutant allele frequency at baseline was associated with clinical characteristics. Levels drastically fell after one cycle of NAC, especially in patients whose tumors would go on to have a pathological complete response (pCR), but then rose significantly before surgery in patients with significant residual tumor at surgery (p = 0.0001). The detection of ctDNA early during treatment and also late at the end of NAC before surgery was strongly predictive of residual tumor at surgery, but its absence was less predictive of pCR, especially when only TP53 variants are considered. ctDNA detection at the end of neoadjuvant chemotherapy indicated significantly worse relapse-free survival (HR = 0.29 (95% CI 0.08-0.98), p = 0.046), and overall survival (HR = 0.27 95% CI 0.075-0.96), p = 0.043). Hence, individualized multi-variant ctDNA testing during and after NAC prior to surgery has prognostic and predictive value in early TNBC patients.

Enhanced Anticancer Effect of a Combination of S-adenosylmethionine (SAM) and Immune Checkpoint Inhibitor (ICPi) in a Syngeneic Mouse Model of Advanced Melanoma.

Immune checkpoint inhibitors (ICPi) targeting the PD-1/PD-L1 pathway have shown marked success in patients with advanced melanoma. However, 60-70% of patients fail to respond, warranting a therapeutic intervention that could increase response rates. We and others have shown that S-adenosylmethionine (SAM), a universal methyl donor, has significant anticancer effects in numerous cancers previously; however, its effect on melanoma progression has not been evaluated. Interestingly, SAM was reported to be essential for T cell activation and proliferation and, thus, could potentially cooperate with ICPi and block melanoma progression. In this study, we examined the antitumor effects of SAM and ICPi alone and in combination in a well-established melanoma mouse model wherein syngeneic C57BL/6 mouse were subcutaneously (orthotopic) injected with B16-F1 cells. Treatment of mice with either SAM or anti-PD-1 antibody alone resulted in significant reduction in tumor volumes and weights; effects that were highest in mice treated with a combination of SAM+anti-PD-1. RNA-sequencing analysis of the primary tumors showed numerous differentially expressed genes (DEGs) following treatment with SAM+anti-PD-1, which was shown to downregulate cancer, MAPK, and tyrosine kinase pathways. Indeed, SAM+anti-PD-1 reversed the aberrant expression of some known melanoma genes. Tumor immunophenotyping revealed the SAM+anti-PD-1 combination was significantly more effective than either SAM or anti-PD-1 as the CD8+ T cells had higher activation, proliferation, and cytokine production compared to all other groups. This study shows that the combination of currently approved agents SAM and ICPi can effectively block melanoma via alteration of key genes/pathways implicated in cancer and immune response pathways, providing the rationale for the initiation of clinical trials with SAM and ICPi.

Five-Year Outcomes With Nivolumab in Patients With Wild-Type BRAF Advanced Melanoma.

PURPOSE: The CheckMate 066 trial investigated nivolumab monotherapy as first-line treatment for patients with previously untreated BRAF wild-type advanced melanoma. Five-year results are presented herein. PATIENTS AND METHODS: In this multicenter, double-blind, phase III study, 418 patients with previously untreated, unresectable, stage III/IV, wild-type BRAF melanoma were randomly assigned 1:1 to receive nivolumab 3 mg/kg every 2 weeks or dacarbazine 1,000 mg/m2 every 3 weeks. The primary end point was overall survival (OS), and secondary end points included progression-free survival (PFS), objective response rate (ORR), and safety. RESULTS: Patients were followed for a minimum of 60 months from the last patient randomly assigned (median follow-up, 32.0 months for nivolumab and 10.9 months for dacarbazine). Five-year OS rates were 39% with nivolumab and 17% with dacarbazine; PFS rates were 28% and 3%, respectively. Five-year OS was 38% in patients randomly assigned to dacarbazine who had subsequent therapy, including nivolumab (n = 37). ORR was 42% with nivolumab and 14% with dacarbazine; among patients alive at 5 years, ORR was 81% and 39%, respectively. Of 42 patients treated with nivolumab who had a complete response (20%), 88% (37 of 42) were alive as of the 5-year analysis. Among 75 nivolumab-treated patients alive and evaluable at the 5-year analysis, 83% had not received subsequent therapy; 23% were still on study treatment, and 60% were treatment free. Safety analyses were similar to the 3-year report. CONCLUSION: Results from this 5-year analysis confirm the significant benefit of nivolumab over dacarbazine for all end points and add to the growing body of evidence supporting long-term survival with nivolumab mono-therapy. Survival is strongly associated with achieving a durable response, which can be maintained after treatment discontinuation, even without subsequent systemic therapies.

A Unique Morphological Phenotype in Chemoresistant Triple-Negative Breast Cancer Reveals Metabolic Reprogramming and PLIN4 Expression as a Molecular Vulnerability.

The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. IMPLICATIONS: These findings underlie the importance of a novel morphologic-metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.

Survival Outcomes in Patients With Previously Untreated BRAF Wild-Type Advanced Melanoma Treated With Nivolumab Therapy: Three-Year Follow-up of a Randomized Phase 3 Trial.

Importance: This analysis provides long-term follow-up in patients with BRAF wild-type advanced melanoma receiving first-line therapy based on anti-programmed cell death 1 receptor inhibitors. Objective: To compare the 3-year survival with nivolumab vs that with dacarbazine in patients with previously untreated BRAF wild-type advanced melanoma. Design, Setting, and Participants: This follow-up of a randomized phase 3 trial analyzed 3-year overall survival data from the randomized, controlled, double-blind CheckMate 066 phase 3 clinical trial. For this ongoing, multicenter academic institution trial, patients were enrolled from January 2013 through February 2014. Eligible patients were 18 years or older with confirmed unresectable previously untreated stage III or IV melanoma and an Eastern Cooperative Oncology Group performance status of 0 or 1 but without a BRAF mutation. Interventions: Patients were treated until progression or unacceptable toxic events with nivolumab (3 mg/kg every 2 weeks plus dacarbazine-matched placebo every 3 weeks) or dacarbazine (1000 mg/m2 every 3 weeks plus nivolumab-matched placebo every 2 weeks). Main Outcome and Measure: Overall survival. Results: At minimum follow-ups of 38.4 months among 210 participants in the nivolumab group (median age, 64 years [range, 18-86 years]; 57.6% male) and 38.5 months among 208 participants in the dacarbazine group (median age, 66 years [range, 25-87 years]; 60.1% male), 3-year overall survival rates were 51.2% (95% CI, 44.1%-57.9%) and 21.6% (95% CI, 16.1%-27.6%), respectively. The median overall survival was 37.5 months (95% CI, 25.5 months-not reached) in the nivolumab group and 11.2 months (95% CI, 9.6-13.0 months) in the dacarbazine group (hazard ratio, 0.46; 95% CI, 0.36-0.59; P < .001). Complete and partial responses, respectively, were reported for 19.0% (40 of 210) and 23.8% (50 of 210) of patients in the nivolumab group compared with 1.4% (3 of 208) and 13.0% (27 of 208) of patients in the dacarbazine group. Additional analyses were performed on outcomes with subsequent therapies. Treatment-related grade 3/4 adverse events occurred in 15.0% (31 of 206) of nivolumab-treated patients and in 17.6% (36 of 205) of dacarbazine-treated patients. There were no deaths due to study drug toxic effects. Conclusions and Relevance: Nivolumab led to improved 3-year overall survival vs dacarbazine in patients with previously untreated BRAF wild-type advanced melanoma. Trial Registration: ClinicalTrials.gov identifier: NCT01721772.

Dual MAPK Inhibition Is an Effective Therapeutic Strategy for a Subset of Class II BRAF Mutant Melanomas.

PURPOSE: Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to dMAPKi. EXPERIMENTAL DESIGN: Tumors from patients with BRAF wild-type (WT), V600E (class I), and L597S (class II) metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and compared these with WT or V600E/K BRAF mutant cells. Cell lines and PDXs were treated with BRAFi (vemurafenib, dabrafenib, encorafenib, and LY3009120), MEKi (cobimetinib, trametinib, and binimetinib), or the combination. We identified 2 patients with BRAF L597S metastatic melanoma who were treated with dMAPKi. RESULTS: BRAFi impaired MAPK signaling and cell growth in class I and II BRAF mutant cells. dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class II BRAF mutations and prolonged survival of mice with class II BRAF mutant melanoma brain metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi. CONCLUSIONS: Class II BRAF mutant melanoma is growth inhibited by dMAPKi. Responses to dMAPKi have been observed in 2 patients with class II BRAF mutant melanoma. These data provide rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma.See related commentary by Johnson and Dahlman, p. 6107.

DNA methylation signatures of breast cancer in peripheral T-cells.

BACKGROUND: Immune surveillance acts as a defense mechanism in cancer, and its disruption is involved in cancer progression. DNA methylation reflects the phenotypic identity of cells and recent data suggested that DNA methylation profiles of T cells and peripheral blood mononuclear cells (PBMC) are altered in cancer progression. METHODS: We enrolled 19 females with stage 1 and 2, nine with stage 3 and 4 and 9 age matched healthy women. T cells were isolated from peripheral blood and extracted DNA was subjected to Illumina 450 K DNA methylation array analysis. Raw data was analyzed by BMIQ, ChAMP and ComBat followed by validation of identified genes by pyrosequencing. RESULTS: Analysis of data revealed ~ 10,000 sites that correlated with breast cancer progression and established a list of 89 CG sites that were highly correlated (p < 0.01, r > 0.7, r < - 0.7) with breast cancer progression. The vast majority of these sites were hypomethylated and enriched in genes with functions in the immune system. CONCLUSIONS: The study points to the possibility of using DNA methylation signatures as a noninvasive method for early detection of breast cancer and its progression which need to be tested in clinical studies.

Abscopal Effect in a Stage IV Melanoma Patient who Progressed on Pembrolizumab.

In this case report, we present the clinical course of a woman with locally advanced mucosal melanoma of the oral cavity. She was initially treated with surgery with adjuvant local radiation of 50 Gy in 20 fractions. She quickly relapsed with an aggressive regional recurrence of the disease on the neck and with numerous pulmonary metastases. Immunotherapy with pembrolizumab was started, with initial good response and reduction in the size of the lesion in the neck. The regression, however, was short-lived, as the mass quickly grew at a remarkable rate and the lung lesions progressed significantly. Palliative local radiation of 24 Gy in three fractions delivered at days zero, seven, and 21 to the neck mass was eventually given with the goal to alleviate symptoms. An immediate tumor regression was observed after the first fraction of radiotherapy. Remarkably, the lung lesions had also started regressing following radiation. We believe this to be a case of abscopal effect witnessed during the delivery of radiotherapy. A review of the recent literature is also presented here.

Multifaceted Role of the Urokinase-Type Plasminogen Activator (uPA) and Its Receptor (uPAR): Diagnostic, Prognostic, and Therapeutic Applications.

The plasminogen activator (PA) system is an extracellular proteolytic enzyme system associated with various physiological and pathophysiological processes. A large body of evidence support that among the various components of the PA system, urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 and -2 (PAI-1 and PAI-2) play a major role in tumor progression and metastasis. The binding of uPA with uPAR is instrumental for the activation of plasminogen to plasmin, which in turn initiates a series of proteolytic cascade to degrade the components of the extracellular matrix, and thereby, cause tumor cell migration from the primary site of origin to a distant secondary organ. The components of the PA system show altered expression patterns in several common malignancies, which have identified them as ideal diagnostic, prognostic, and therapeutic targets to reduce cancer-associated morbidity and mortality. This review summarizes the various components of the PA system and focuses on the role of uPA-uPAR in different biological processes especially in the context of malignancy. We also discuss the current state of knowledge of uPA-uPAR-targeted diagnostic and therapeutic strategies for various malignancies.