Ryan J. Sullivan

BRAF Inhibition Is Associated with Enhanced Melanoma Antigen Expression and a More Favorable Tumor Microenvironment in Patients with Metastatic Melanoma

Purpose: To evaluate the effects of BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma. Experimental Design: Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10 to 14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition (dabrafenib + trametinib) and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T-cell markers, and immunomodulatory cytokines. Results: Treatment with either BRAF inhibitor alone or BRAF + MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8+ T-cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines [interleukin (IL)-6 and IL-8] and an increase in markers of T-cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 was increased on treatment. A decrease in melanoma antigen expression and CD8 T-cell infiltrate was noted at time of progression on BRAF inhibitor alone and was reversed with combined BRAF and MEK inhibition. Conclusions: Together, these data suggest that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T-cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T-cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma. Clin Cancer Res; 19(5); 1225–31. ©2013 AACR.

Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

Single-cell expression profiles of melanoma Tumors harbor multiple cell types that are thought to play a role in the development of resistance to drug treatments. Tirosh et al. used single-cell sequencing to investigate the distribution of these differing genetic profiles within melanomas. Many cells harbored heterogeneous genetic programs that reflected two different states of genetic expression, one of which was linked to resistance development. Following drug treatment, the resistance-linked expression state was found at a much higher level. Furthermore, the environment of the melanoma cells affected their gene expression programs. Science, this issue p. 189 Melanoma cells show transcriptional heterogeneity. To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.

Transforming Fusions of FGFR and TACC Genes in Human Glioblastoma

Oncogenic TACC-tics Human cancers exhibit many types of genomic rearrangements—including some that juxtapose sequences from two unrelated genes—thereby creating fusion proteins with oncogenic activity. Functional analysis of these fusion genes can provide mechanistic insights into tumorigenesis and potentially lead to effective drugs, as famously illustrated by the BCR-ABL gene in chronic myelogenous leukemia. Singh et al. (p. 1231, published online 26 July) identify and characterize a fusion gene present in 3% of human glioblastomas, a deadly brain cancer. In the resultant fusion protein, the tyrosine kinase region of the fibroblast growth factor receptor (FGFR) is joined to a domain from a transforming acidic coiled-coil (TACC) protein. The TACC-FGFR protein is oncogenic, shows unregulated kinase activity, localizes to the mitotic spindle, and disrupts chromosome segregation. In mice, FGFR inhibitors slowed the growth of tumors driven by the TACC-FGFR gene, suggesting that a subset of glioblastoma patients may benefit from these types of drugs. A fusion gene detected in a small subset of human brain tumors encodes a potentially druggable target. The brain tumor glioblastoma multiforme (GBM) is among the most lethal forms of human cancer. Here, we report that a small subset of GBMs (3.1%; 3 of 97 tumors examined) harbors oncogenic chromosomal translocations that fuse in-frame the tyrosine kinase coding domains of fibroblast growth factor receptor (FGFR) genes (FGFR1 or FGFR3) to the transforming acidic coiled-coil (TACC) coding domains of TACC1 or TACC3, respectively. The FGFR-TACC fusion protein displays oncogenic activity when introduced into astrocytes or stereotactically transduced in the mouse brain. The fusion protein, which localizes to mitotic spindle poles, has constitutive kinase activity and induces mitotic and chromosomal segregation defects and triggers aneuploidy. Inhibition of FGFR kinase corrects the aneuploidy, and oral administration of an FGFR inhibitor prolongs survival of mice harboring intracranial FGFR3-TACC3–initiated glioma. FGFR-TACC fusions could potentially identify a subset of GBM patients who would benefit from targeted FGFR kinase inhibition.

MAP Kinase Pathway Alterations in BRAF-Mutant Melanoma Patients with Acquired Resistance to Combined RAF/MEK Inhibition

Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.

Resistance to BRAF-targeted therapy in melanoma

BRAF mutations are identified in 40-50% of patients with melanoma. Treatment of these patients with either of two BRAF inhibitors (vemurafenib, dabrafenib) or the MEK inhibitor trametinib is associated with improved clinical benefit (response rate, progression free survival, and overall survival) compared with treatment with chemotherapy in three phase III trials. Unfortunately, most patients, including those who experience initial, profound tumour regression, have evidence of disease progression within 6-8 months after commencing therapy with one of these agents. The mechanisms of resistance are varied and include activation of alternative signalling pathways as well as reactivating the MAP kinase pathway through alternative means. This review describes relevant aspects of MAP kinase pathway signalling, summarises the clinical data with BRAF and MEK inhibitors, presents the known resistance mechanisms to BRAF inhibitor therapy, and provides some strategies for how resistance may be overcome.

EGFR Mutations and ALK Rearrangements Are Associated with Low Response Rates to PD-1 Pathway Blockade in Non–Small Cell Lung Cancer: A Retrospective Analysis

Purpose: PD-1 inhibitors are established agents in the management of non–small cell lung cancer (NSCLC); however, only a subset of patients derives clinical benefit. To determine the activity of PD-1/PD-L1 inhibitors within clinically relevant molecular subgroups, we retrospectively evaluated response patterns among EGFR-mutant, anaplastic lymphoma kinase (ALK)-positive, and EGFR wild-type/ALK-negative patients. Experimental Design: We identified 58 patients treated with PD-1/PD-L1 inhibitors. Objective response rates (ORR) were assessed using RECIST v1.1. PD-L1 expression and CD8+ tumor-infiltrating lymphocytes (TIL) were evaluated by IHC. Results: Objective responses were observed in 1 of 28 (3.6%) EGFR-mutant or ALK-positive patients versus 7 of 30 (23.3%) EGFR wild-type and ALK-negative/unknown patients (P = 0.053). The ORR among never- or light- (≤10 pack years) smokers was 4.2% versus 20.6% among heavy smokers (P = 0.123). In an independent cohort of advanced EGFR-mutant (N = 68) and ALK-positive (N = 27) patients, PD-L1 expression was observed in 24%/16%/11% and 63%/47%/26% of pre–tyrosine kinase inhibitor (TKI) biopsies using cutoffs of ≥1%, ≥5%, and ≥50% tumor cell staining, respectively. Among EGFR-mutant patients with paired, pre- and post-TKI–resistant biopsies (N = 57), PD-L1 expression levels changed after resistance in 16 (28%) patients. Concurrent PD-L1 expression (≥5%) and high levels of CD8+ TILs (grade ≥2) were observed in only 1 pretreatment (2.1%) and 5 resistant (11.6%) EGFR-mutant specimens and was not observed in any ALK-positive, pre- or post-TKI specimens. Conclusions: NSCLCs harboring EGFR mutations or ALK rearrangements are associated with low ORRs to PD-1/PD-L1 inhibitors. Low rates of concurrent PD-L1 expression and CD8+ TILs within the tumor microenvironment may underlie these clinical observations. Clin Cancer Res; 22(18); 4585–93. ©2016 AACR. See related commentary by Gettinger and Politi, p. 4539

Ipilimumab Therapy in Patients With Advanced Melanoma and Preexisting Autoimmune Disorders

IMPORTANCE Ipilimumab and other immune therapies are effective treatment options for patients with advanced melanoma but cause frequent immune-related toxic effects. Autoimmune diseases are common, and the safety and efficacy of ipilimumab therapy in patients with preexisting autoimmune disorders is not known. OBJECTIVE To determine the safety and efficacy of ipilimumab therapy in patients with advanced melanoma with preexisting autoimmune disorders. DESIGN, SETTING, AND PARTICIPANTS Retrospective review of patients with advanced melanoma and preexisting autoimmune disorders who received ipilimumab at 9 academic tertiary referral centers from January 1, 2012, through August 1, 2015. The data analysis was performed on August 24, 2015. EXPOSURE Ipilimumab therapy. MAIN OUTCOMES AND MEASURES Safety, in terms of frequency of autoimmune flares and conventional immune-related adverse events (irAEs), and efficacy, in terms of response rates and overall survival, were evaluated descriptively. RESULTS Of the 30 patients who received ipilimumab (17 [57%] male; median [range] age, 59.5 [30-80] y), 6 had rheumatoid arthritis, 5 had psoriasis, 6 had inflammatory bowel disease, 2 had systemic lupus erythematosus, 2 had multiple sclerosis, 2 had autoimmune thyroiditis, and 7 had other conditions. Thirteen patients (43%) were receiving immunosuppressive therapy at the time of initiation of ipilimumab therapy, most commonly low-dose prednisone or hydroxychloroquine. With ipilimumab treatment, 8 patients (27%) experienced exacerbations of their autoimmune condition necessitating systemic treatment; all were managed with corticosteroids. Conventional grade 3 to 5 irAEs occurred in 10 patients (33%) and were reversible with corticosteroids or with infliximab therapy in 2 cases. One patient with baseline psoriasis died of presumed immune-related colitis after a 1-week delay prior to reporting symptoms. Fifteen patients (50%) had neither autoimmune disease flares nor irAEs. Six patients experienced an objective response (20%), including 1 with a durable complete response. CONCLUSIONS AND RELEVANCE To our knowledge, this is the largest series of patients with preexisting autoimmune disease treated with immune checkpoint inhibitors. Ipilimumab was clinically active and was associated with exacerbations of autoimmune disease and conventional ipilimumab-induced irAEs that were readily manageable with standard therapies when started in a timely fashion. Ipilimumab therapy may be considered in this setting with vigilant clinical monitoring.

Critically ill children during the 2009-2010 influenza pandemic in the United States.

BACKGROUND: The 2009 pandemic influenza A (H1N1) (pH1N1) virus continues to circulate worldwide. Determining the roles of chronic conditions and bacterial coinfection in mortality is difficult because of the limited data for children with pH1N1-related critical illness. METHODS: We identified children (<21 years old) with confirmed or probable pH1N1 admitted to 35 US PICUs from April 15, 2009, through April 15, 2010. We collected data on demographics, baseline health, laboratory results, treatments, and outcomes. RESULTS: Of 838 children with pH1N1 admitted to a PICU, the median age was 6 years, 58% were male, 70% had ≥1 chronic health condition, and 88.2% received oseltamivir (5.8% started before PICU admission). Most patients had respiratory failure with 564 (67.3%) receiving mechanical ventilation; 162 (19.3%) received vasopressors, and 75 (8.9%) died. Overall, 71 (8.5%) of the patients had a presumed diagnosis of early (within 72 hours after PICU admission) Staphylococcus aureus coinfection of the lung with 48% methicillin-resistant S aureus (MRSA). In multivariable analyses, preexisting neurologic conditions or immunosuppression, encephalitis (1.7% of cases), myocarditis (1.4% of cases), early presumed MRSA lung coinfection, and female gender were mortality risk factors. Among 251 previously healthy children, only early presumed MRSA coinfection of the lung (relative risk: 8 [95% confidence interval: 3.1–20.6]; P < .0001) remained a mortality risk factor. CONCLUSIONS: Children with preexisting neurologic conditions and immune compromise were at increased risk of pH1N1-associated death after PICU admission. Secondary complications of pH1N1, including myocarditis, encephalitis, and clinical diagnosis of early presumed MRSA coinfection of the lung, were mortality risk factors.

Anti-PD-1 therapy in patients with advanced melanoma and preexisting autoimmune disorders or major toxicity with ipilimumab

Background Anti-PD-1 antibodies (anti-PD-1) have clinical activity in a number of malignancies. All clinical trials have excluded patients with significant preexisting autoimmune disorders (ADs) and only one has included patients with immune-related adverse events (irAEs) with ipilimumab. We sought to explore the safety and efficacy of anti-PD-1 in such patients. Patients and methods Patients with advanced melanoma and preexisting ADs and/or major immune-related adverse events (irAEs) with ipilimumab (requiring systemic immunosuppression) that were treated with anti-PD-1 between 1 July 2012 and 30 September 2015 were retrospectively identified. Results One hundred and nineteen patients from 13 academic tertiary referral centers were treated with anti-PD-1. In patients with preexisting AD (N = 52), the response rate was 33%. 20 (38%) patients had a flare of AD requiring immunosuppression, including 7/13 with rheumatoid arthritis, 3/3 with polymyalgia rheumatica, 2/2 with Sjogrens syndrome, 2/2 with immune thrombocytopaenic purpura and 3/8 with psoriasis. No patients with gastrointestinal (N = 6) or neurological disorders (N = 5) flared. Only 2 (4%) patients discontinued treatment due to flare, but 15 (29%) developed other irAEs and 4 (8%) discontinued treatment. In patients with prior ipilimumab irAEs requiring immunosuppression (N = 67) the response rate was 40%. Two (3%) patients had a recurrence of the same ipilimumab irAEs, but 23 (34%) developed new irAEs (14, 21% grade 3-4) and 8 (12%) discontinued treatment. There were no treatment-related deaths. Conclusions In melanoma patients with preexisting ADs or major irAEs with ipilimumab, anti-PD-1 induced relatively frequent immune toxicities, but these were often mild, easily managed and did not necessitate discontinuation of therapy, and a significant proportion of patients achieved clinical responses. The results support that anti-PD-1 can be administered safely and can achieve clinical benefit in patients with preexisting ADs or prior major irAEs with ipilimumab.

Induction gemcitabine and stereotactic body radiotherapy for locally advanced nonmetastatic pancreas cancer.

PURPOSE Stereotactic body radiotherapy (SBRT) has been used successfully to treat patients with locally advanced pancreas cancer. However, many patients develop metastatic disease soon after diagnosis and may receive little benefit from such therapy. We therefore retrospectively analyzed a planned strategy of initial chemotherapy with restaging and then treatment for those patients with no evidence of metastatic progression with SBRT. METHODS AND MATERIALS Forty-seven patients received gemcitabine (1,000 mg/m(2) per week for 3 weeks then 1 week off) until tolerance, at least six cycles, or progression. Patients without metastases after two cycles were treated with SBRT (tolerance-based dose of 24-36 Gy in 3 fractions) between the third and fourth cycles without interrupting the chemotherapy cycles. RESULTS Eight of the 47 patients (17%) were found to have metastatic disease after two cycles of gemcitabine; the remaining 39 patients received SBRT. The median follow-up for survivors was 21 months (range, 6-36 months). The median overall survival for all patients who received SBRT was 20 months, and the median progression-free survival was 15 months. The local control rate was 85% (33 of 39 patients); and 54% of patients (21 of 39) developed metastases. Late Grade III toxicities such as GI bleeding and obstruction were observed in 9% (3/39) of patients. CONCLUSION For patients with locally advanced pancreas cancer, this strategy uses local therapy for those who are most likely to benefit from it and spares those patients with early metastatic progression from treatment. SBRT delivers such local therapy safely with minimal interruption to systemic chemotherapy, thereby potentially improving the outcome in these patients.