Acquired small cell lung cancer resistance to Chk1 inhibitors involves Wee1 up-regulation.

Platinum-based chemotherapy has been the cornerstone treatment for small cell lung cancer (SCLC) for decades, but no major progress has been made in the past 20 years with regard to overcoming chemoresistance. As the cell cycle checkpoint kinase 1 (Chk1) plays a key role in DNA damage response to chemotherapeutic drugs, we explored the mechanisms of acquired drug resistance to the Chk1 inhibitor prexasertib in SCLC. We established prexasertib resistance in two SCLC cell lines and found that DNA copy number, messengerRNA (mRNA) and protein levels of the cell cycle regulator Wee1 significantly correlate with the level of acquired resistance. Wee1 small interfering RNA (siRNA) or Wee1 inhibitor reversed prexasertib resistance, whereas Wee1 transfection induced prexasertib resistance in parental cells. Reverse phase protein microarray identified up-regulated proteins in the resistant cell lines that are involved in apoptosis, cell proliferation and cell cycle. Down-regulation of CDK1 and CDC25C kinases promoted acquired resistance in parental cells, whereas down-regulation of p38MAPK reversed the resistance. High Wee1 expression was significantly correlated with better prognosis of resected SCLC patients. Our results indicate that Wee1 overexpression plays an important role in acquired resistance to Chk1 inhibition. We also show that bypass activation of the p38MAPK signaling pathway may contribute to acquired resistance to Chk1 inhibition. The combination of Chk1 and Wee1 inhibitors may provide a new therapeutic strategy for the treatment of SCLC.

Pembrolizumab in Relapsed and Refractory Mycosis Fungoides and Sézary Syndrome: A Multicenter Phase II Study.

PURPOSE: To assess the efficacy of pembrolizumab in patients with advanced relapsed or refractory mycosis fungoides (MF) or Sézary syndrome (SS). PATIENTS AND METHODS: CITN-10 is a single-arm, multicenter phase II trial of 24 patients with advanced MF or SS. Patients were treated with pembrolizumab 2 mg/kg every 3 weeks for up to 24 months. The primary end point was overall response rate by consensus global response criteria. RESULTS: Patients had advanced-stage disease (23 of 24 with stage IIB to IV MF/SS) and were heavily pretreated with a median of four prior systemic therapies. The overall response rate was 38% with two complete responses and seven partial responses. Of the nine responding patients, six had 90% or more improvement in skin disease by modified Severity Weighted Assessment Tool, and eight had ongoing responses at last follow-up. The median duration of response was not reached, with a median response follow-up time of 58 weeks. Immune-related adverse events led to treatment discontinuation in four patients. A transient worsening of erythroderma and pruritus occurred in 53% of patients with SS. This cutaneous flare reaction did not result in treatment discontinuation for any patient. The flare reaction correlated with high PD-1 expression on Sézary cells but did not associate with subsequent clinical responses or lack of response. Treatment responses did not correlate with expression of PD-L1, total mutation burden, or an interferon-γ gene expression signature. CONCLUSION: Pembrolizumab demonstrated significant antitumor activity with durable responses and a favorable safety profile in patients with advanced MF/SS.

Descriptive analysis of genetic aberrations and cell of origin in Richter transformation.

Richter transformation (RT) is a progression from chronic lymphocytic leukemia (CLL) to a more aggressive lymphoma, most often diffuse large B-cell lymphoma (DLBCL). Due to the rarity of the disease, data regarding the molecular profile and cell of origin (COO) of RT is limited. We performed immunohistochemistry analysis for COO determination and next-generation sequencing for gene mutation analysis in 11 RT patients. Seventy-nine percent of our patients were classified as non-GCB phenotype. Of the 57 unique mutations identified, the three most commonly mutated genes were TP53, TET2, and CREBBP. Neither TET2 nor CREBBP has been previously described in RT. Our analysis provides additional information to help guide further investigation of both the diagnosis and treatment of this complex and heterogeneous disease.

Acquired SETD2 mutation and impaired CREB1 activation confer cisplatin resistance in metastatic non-small cell lung cancer.

Resistance to chemotherapy remains a critical barrier to effective cancer treatment. Although cisplatin is one of the most commonly used chemotherapeutic agents in the treatment of non-small cell lung cancer (NSCLC), mechanisms of resistance to this drug are not fully understood. Here, we report a novel cisplatin-resistance mechanism involving SET Domain Containing 2 (SETD2), a histone H3 lysine 36 (H3K36) trimethyltransferase, and cAMP-responsive element-binding protein 1 (CREB1). A549 cells selected in vivo to give brain metastases exhibited cisplatin resistance and decreased expression of phosphorylated CREB1. Next-generation sequencing (NGS) analysis identified a missense mutation in SETD2 (p.T1171K), and we demonstrated that SETD2-mediated trimethylation of H3K36 (H3K36me3) and CREB1 phosphorylation are critical for cellular sensitivity to cisplatin. Moreover, we showed that suppression of SETD2 or CREB1 and ectopic expression of mutant SETD2 conferred cisplatin resistance through inhibition of H3K36me3 and ERK activation in NSCLC cells. Our results provide evidence that SETD2 and CREB1 contribute to cisplatin cytotoxicity via regulation of the ERK signaling pathway, and their inactivation may lead to cisplatin resistance.

Reprint of: Circulating cell-free DNA mutation patterns in early and late stage colon and pancreatic cancer.

Cancer is a heterogeneous disease harboring diverse subclonal populations that can be discriminated by their DNA mutations. Environmental pressure selects subclones that ultimately drive disease progression and tumor relapse. Circulating cell-free DNA (ccfDNA) can be used to approximate the mutational makeup of cancer lesions and can serve as a marker for monitoring disease progression at the molecular level without the need for invasively acquired samples from primary or metastatic lesions. This potential for molecular analysis makes ccfDNA attractive for the study of clonal evolution and for uncovering emerging therapeutic resistance or sensitivity. We assessed ccfDNA from colon and pancreatic adenocarcinoma patients using next generation sequencing of 56 cancer-associated genes at the time of primary resectable disease and metastatic progression and compared this to the mutational patterns of the primary tumor. 28%-47% of non-synonymous mutations in the primary tumors were also detected in the ccfDNA while 71%-78% mutations found in ccfDNA were not detected in the primary tumors. ccfDNA collected at the time of progression harbored 3-5 new mutations not detected in ccfDNA at the earlier collection time points. We conclude that incorporation of ccfDNA analysis provides crucial insights into the changing molecular makeup of progressive colon and pancreatic cancer.

Prevention of Lipid Peroxidation-derived Cyclic DNA Adduct and Mutation in High-Fat Diet-induced Hepatocarcinogenesis by Theaphenon E.

Obesity is associated with cancer risk and its link with liver cancer is particularly strong. Obesity causes non-alcoholic fatty liver disease (NAFLD) that could progress to hepatocellular carcinoma (HCC). Chronic inflammation likely plays a key role. We carried out a bioassay in the high-fat diet (HFD)-fed C57BL/6J mice to provide insight into the mechanisms of obesity-related HCC by studying γ-OHPdG, a mutagenic DNA adduct derived from lipid peroxidation. In an 80-week bioassay, mice received a low-fat diet (LFD), high-fat diet (HFD), and HFD with 2% Theaphenon E (TE) (HFD+TE). HFD mice developed a 42% incidence of HCC and LFD mice a 16%. Remarkably, TE, a standardized green tea extract formulation, completely blocked HCC in HFD mice with a 0% incidence. γ-OHPdG measured in the hepatic DNA of mice fed HFD and HFD+TE showed its levels increased during the early stages of NAFLD in HFD mice and the increases were significantly suppressed by TE, correlating with the tumor data. Whole-exome sequencing showed an increased mutation load in the liver tumors of HFD mice with G>A and G>T as the predominant mutations, consistent with the report that γ-OHPdG induces G>A and G>T. Furthermore, the mutation loads were significantly reduced in HFD+TE mice, particularly G>T, the most common mutation in human HCC. These results demonstrate in a relevant model of obesity-induced HCC that γ-OHPdG formation during fatty liver disease may be an initiating event for accumulated mutations that leads to HCC and this process can be effectively inhibited by TE. Cancer Prev Res; 11(10); 665-76. ©2018 AACR.

Pembrolizumab in patients with thymic carcinoma: a single-arm, single-centre, phase 2 study.

BACKGROUND: Treatment options are limited for patients with thymic carcinoma. These aggressive tumours are not typically associated with paraneoplastic autoimmune disorders, and strong PD-L1 expression has been reported in thymic epithelial tumours. We aimed to assess the activity of pembrolizumab, a monoclonal antibody that targets PD-1, in patients with advanced thymic carcinoma. METHODS: We completed a single-arm phase 2 study of pembrolizumab in patients with recurrent thymic carcinoma who had progressed after at least one line of chemotherapy. This was a single-centre study performed at Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA. Key inclusion criteria were an Eastern Cooperative Oncology Group performance status of 0-2, no history of autoimmune disease or other malignancy requiring treatment or laboratory abnormality, and adequate organ function. Patients received 200 mg of pembrolizumab every 3 weeks for up to 2 years. The primary objective of the study was the proportion of patients who had achieved a response assessed with Response Evaluation Criteria in Solid Tumors version 1.1. Analysis was per protocol, in all eligible patients. The study is registered with ClinicalTrials.gov, number NCT02364076, and is closed to accrual; we report the final analysis. FINDINGS: 41 patients were enrolled from March 12, 2015, to Dec 16, 2016, of whom 40 were eligible and evaluable and one was excluded because of elevated liver enzymes at screening. The median follow-up was 20 months (IQR 14-26). The proportion of patients who achieved a response was 22·5% (95% CI 10·8-38·5); one (3%) patient achieved a complete response, eight (20%) patients achieved partial responses, and 21 (53%) patients achieved stable disease. The most common grade 3 or 4 adverse events were increased aspartate aminotransferase and alanine aminotransferase (five [13%] patients each). Six (15%) patients developed severe autoimmune toxicity, including two (5%) patients with myocarditis. There were 17 deaths at the time of analysis, but no deaths due to toxicity. INTERPRETATION: Pembrolizumab is a promising treatment option in patients with thymic carcinoma. Because severe autoimmune disorders are more frequent in thymic carcinoma than in other tumour types, careful monitoring is essential. FUNDING: Merck & Co.

An endogenous DNA adduct as a prognostic biomarker for hepatocarcinogenesis and its prevention by Theaphenon E in mice.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, mainly because of its poor prognosis. A valid mechanism-based prognostic biomarker is urgently needed. γ-hydroxy-1,N2 -propanodeoxyguanosine (γ-OHPdG) is an endogenously formed mutagenic DNA adduct derived from lipid peroxidation. We examined the relationship of γ-OHPdG with hepatocarcinogenesis in two animal models and its potential role as a prognostic biomarker for recurrence in HCC patients. Bioassays were conducted in xeroderma pigmentosum group A knockout mice and diethylnitrosamine-injected mice, both prone to HCC development. γ-OHPdG levels in the livers of these animals were determined. The effects of antioxidant treatments on γ-OHPdG and hepatocarcinogenesis were examined. Using two independent sets of HCC specimens from patients, we examined the relationship between γ-OHPdG and survival or recurrence-free survival. γ-OHPdG levels in liver DNA showed an age-dependent increase and consistently correlated with HCC development in all three animal models. Theaphenon E treatment significantly decreased γ-OHPdG levels in the liver DNA of xeroderma pigmentosum group A knockout mice and remarkably reduced HCC incidence in these mice to 14% from 100% in the controls. It also effectively inhibited HCC development in the diethylnitrosamine-injected mice. Using clinical samples from two groups of patients, our study revealed that higher levels of γ-OHPdG are strongly associated with low survival (P < 0.0001) and low recurrence-free survival (P = 0.007). CONCLUSION: These results support γ-OHPdG as a mechanism-based, biologically relevant biomarker for predicting the risk of HCC and its recurrence. (Hepatology 2018;67:159-170).

Combined Small Cell Carcinoma of the Lung: Is It a Single Entity?

BACKGROUND: SCLC accounts for 15% and 20% of all lung cancers, with combined SCLC (CSCLC) comprising 2% to 5%. Little is known about the clinical characteristics and molecular changes associated with the various histologic components. METHODS: A total of 205 SCLC cases were resected between 2005 and 2015. Clinical and pathologic features were analyzed. All CSCLC cases were confirmed by histologic examination and immunohistochemistry. The individual components were microdissected using a novel automated dissection system, and DNA was extracted and subjected to targeted exome sequencing. RESULTS: A total of 10 cases of CSCLC were identified out of 170 cases with adequate histologic material; squamous cell carcinoma comprised the second component in half of these (n = 5). There were no significant differences between CSCLC and pure SCLC with respect to clinical features. The median follow-up time was 36 months. The median survival times of patients with pure SCLC and CSCLC were 58 months and 26 months, respectively (p = 0.030). The different components of three cases of CSCLC were deemed adequate for microdissection and sequencing. Approximately 75% of the identified somatic mutations were present in both components. There were also 15 gene mutations or six amplifications unique to only one of the components. CONCLUSIONS: We identified no significant clinical or pathologic differences between pure SCLC and CSCLC; CSCLC was associated with decreased overall survival compared with pure SCLC. The histologic components of CSCLC had high genetic concordance but also showed divergent genotypes. These findings may suggest a common precursor with subsequent acquisition of oncogenic changes in CSCLC.

Circulating cell-free DNA mutation patterns in early and late stage colon and pancreatic cancer.

Cancer is a heterogeneous disease harboring diverse subclonal populations that can be discriminated by their DNA mutations. Environmental pressure selects subclones that ultimately drive disease progression and tumor relapse. Circulating cell-free DNA (ccfDNA) can be used to approximate the mutational makeup of cancer lesions and can serve as a marker for monitoring disease progression at the molecular level without the need for invasively acquired samples from primary or metastatic lesions. This potential for molecular analysis makes ccfDNA attractive for the study of clonal evolution and for uncovering emerging therapeutic resistance or sensitivity. We assessed ccfDNA from colon and pancreatic adenocarcinoma patients using next generation sequencing of 56 cancer-associated genes at the time of primary resectable disease and metastatic progression and compared this to the mutational patterns of the primary tumor. 28%-47% of non-synonymous mutations in the primary tumors were also detected in the ccfDNA while 71%-78% mutations found in ccfDNA were not detected in the primary tumors. ccfDNA collected at the time of progression harbored 3-5 new mutations not detected in ccfDNA at the earlier collection time points. We conclude that incorporation of ccfDNA analysis provides crucial insights into the changing molecular makeup of progressive colon and pancreatic cancer.

Genomic comparison of early-passage conditionally reprogrammed breast cancer cells to their corresponding primary tumors.

Conditionally reprogrammed cells (CRCs) are epithelial cells that are directly isolated from patients' specimens and propagated in vitro with feeder cells and a Rho kinase inhibitor. A number of these cells have been generated from biopsies of breast cancer patients, including ductal carcinoma in situ and invasive carcinomas. The characterization of their genomic signatures is essential to determine their ability to reflect the natural biology of their tumors of origin. In this study, we performed the genomic characterization of six newly established invasive breast cancer CRC cultures in comparison to the original patients' primary breast tumors (PBT) from which they derived. The CRCs and corresponding PBTs were simultaneously profiled by genome-wide array-CGH, targeted next generation sequencing and global miRNA expression to determine their molecular similarities in the patterns of copy number alterations (CNAs), gene mutations and miRNA expression levels, respectively. The CRCs' epithelial cells content and ploidy levels were also evaluated by flow cytometry. A similar level of CNAs was observed in the pairs of CRCs/PBTs analyzed by array-CGH, with >95% of overlap for the most frequently affected cytobands. Consistently, targeted next generation sequencing analysis showed the retention of specific somatic variants in the CRCs as present in their original PBTs. Global miRNA profiling closely clustered the CRCs with their PBTs (Pearson Correlation, ANOVA paired test, P<0.05), indicating also similarity at the miRNA expression level; the retention of tumor-specific alterations in a subset of miRNAs in the CRCs was further confirmed by qRT-PCR. These data demonstrated that the human breast cancer CRCs of this study maintained at early passages the overall copy number, gene mutations and miRNA expression patterns of their original tumors. The further characterization of these cells by other molecular and cellular phenotypes at late cell passages, are required to further expand their use as a unique and representative ex-vivo tumor model for basic science and translational breast cancer studies.

Therapeutic Effects of XPO1 Inhibition in Thymic Epithelial Tumors.

Exportin 1 (XPO1) mediates nuclear export of many cellular factors known to play critical roles in malignant processes, and selinexor (KPT-330) is the first XPO1-selective inhibitor of nuclear export compound in advanced clinical development phase for cancer treatment. We demonstrated here that inhibition of XPO1 drives nuclear accumulation of important cargo tumor suppressor proteins, including transcription factor FOXO3a and p53 in thymic epithelial tumor (TET) cells, and induces p53-dependent and -independent antitumor activity in vitro Selinexor suppressed the growth of TET xenograft tumors in athymic nude mice via inhibition of cell proliferation and induction of apoptosis. Loss of p53 activity or amplification of XPO1 may contribute to resistance to XPO1 inhibitor in TET. Using mass spectrometry-based proteomics analysis, we identified a number of proteins whose abundances in the nucleus and cytoplasm shifted significantly following selinexor treatment in the TET cells. Furthermore, we found that XPO1 was highly expressed in aggressive histotypes and advanced stages of human TET, and high XPO1 expression was associated with poorer patient survival. These results underscore an important role of XPO1 in the pathogenesis of TET and support clinical development of the XPO1 inhibitor for the treatment of patients with this type of tumors. Cancer Res; 77(20); 5614-27. ©2017 AACR.

PI3K as a Potential Therapeutic Target in Thymic Epithelial Tumors.

INTRODUCTION: Thymic epithelial tumors (TETs) are rare tumors originating from the epithelium of the thymus with limited therapeutic options beyond surgery. The pathogenesis of TETs is poorly understood, and the scarcity of model systems for these rare tumors makes the study of their biology very challenging. METHODS: A new cell line (MP57) was established from a thymic carcinoma specimen and characterized using standard biomarker analysis, as well as next-generation sequencing (NGS) and functional assays. Sanger sequencing was used to confirm the mutations identified by NGS. RESULTS: MP57 possesses all the tested thymic epithelial markers and is deemed a bona fide thymic carcinoma cell line. NGS analysis of MP57 identified a mutation in the gene PIK3R2, which encodes a regulatory subunit of PI3K. Further analysis identified different mutations in multiple PI3K subunit genes in another cell line and several primary thymic carcinoma samples, including two catalytic subunits (PIK3CA and PIK3CG) and another regulatory subunit (PIK3R4). Inhibiting PI3K with GDC-0941 resulted in in vitro antitumor activity in TET cells carrying mutant PI3K subunits. CONCLUSIONS: Alterations of PI3K due to mutations in its catalytic or regulatory subunits are observed in a subgroup of TETs, in particular, thymic carcinomas. Targeting PI3K may be an effective strategy to treat these tumors.

Next-Generation Sequencing: Targeting Targeted Therapies.

Next-generation sequencing (NGS) has given new perspective in oncology. With the ongoing development of targeted therapies, NGS is evolving molecular diagnostics by providing comprehensive interrogation of clinically actionable genomic aberrations in tumors. Having this assay as the primary testing method produces clinically beneficial results. See related article by Drilon et al., p. 3631.

Germline variation in cancer-susceptibility genes in a healthy, ancestrally diverse cohort: implications for individual genome sequencing.

Technological advances coupled with decreasing costs are bringing whole genome and whole exome sequencing closer to routine clinical use. One of the hurdles to clinical implementation is the high number of variants of unknown significance. For cancer-susceptibility genes, the difficulty in interpreting the clinical relevance of the genomic variants is compounded by the fact that most of what is known about these variants comes from the study of highly selected populations, such as cancer patients or individuals with a family history of cancer. The genetic variation in known cancer-susceptibility genes in the general population has not been well characterized to date. To address this gap, we profiled the nonsynonymous genomic variation in 158 genes causally implicated in carcinogenesis using high-quality whole genome sequences from an ancestrally diverse cohort of 681 healthy individuals. We found that all individuals carry multiple variants that may impact cancer susceptibility, with an average of 68 variants per individual. Of the 2,688 allelic variants identified within the cohort, most are very rare, with 75% found in only 1 or 2 individuals in our population. Allele frequencies vary between ancestral groups, and there are 21 variants for which the minor allele in one population is the major allele in another. Detailed analysis of a selected subset of 5 clinically important cancer genes, BRCA1, BRCA2, KRAS, TP53, and PTEN, highlights differences between germline variants and reported somatic mutations. The dataset can serve a resource of genetic variation in cancer-susceptibility genes in 6 ancestry groups, an important foundation for the interpretation of cancer risk from personal genome sequences.