Cancer of the ampulla of Vater: analysis of the whole genome sequence exposes a potential therapeutic vulnerability

BACKGROUND: Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scale clinical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers. METHODS: We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNA from a 63 year-old man who underwent a pancreaticoduodenectomy by whole genome sequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations. RESULTS: We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition. CONCLUSIONS: Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers

Case report: whole exome sequencing of primary cardiac angiosarcoma highlights potential for targeted therapies

Abstract Background Primary cardiac angiosarcomas are rare, but they are the most aggressive type of primary cardiac neoplasms. When patients do present, it is with advanced pulmonary and/or cardiac symptoms. Therefore, many times the correct diagnosis is not made at the time of initial presentation. These patients have metastatic disease and the vast majority of these patients die within a few months after diagnosis. Currently the treatment choices are limited and there are no targeted therapies available. Case presentation A 56-year-old male presented with shortness of breath, night sweats, and productive cough for a month. Workup revealed pericardial effusion and multiple bilateral pulmonary nodules suspicious for metastatic disease. Transthoracic echocardiogram showed a large pericardial effusion and a large mass in the base of the right atrium. Results of biopsy of bilateral lung nodules established a diagnosis of primary cardiac angiosarcoma. Aggressive pulmonary disease caused rapid deterioration; the patient went on hospice and subsequently died. Whole exome sequencing of the patient\u2019s postmortem tumor revealed a novel KDR (G681R) mutation, and focal high-level amplification at chromosome 1q encompassing MDM4 , a negative regulator of TP53. Conclusion Mutations in KDR have been reported previously in angiosarcomas. Previous studies also demonstrated that KDR mutants with constitutive KDR activation could be inhibited with specific KDR inhibitors in vitro. Thus, patients harboring activating KDR mutations could be candidates for treatment with KDR-specific inhibitors

STAT3 Is Activated by JAK2 Independent of Key Oncogenic Driver Mutations in Non-Small Cell Lung Carcinoma

Constitutive activation of STAT3 is a common feature in many solid tumors including non-small cell lung carcinoma (NSCLC). While activation of STAT3 is commonly achieved by somatic mutations to JAK2 in hematologic malignancies, similar mutations are not often found in solid tumors. Previous work has instead suggested that STAT3 activation in solid tumors is more commonly induced by hyperactive growth factor receptors or autocrine cytokine signaling. The interplay between STAT3 activation and other well-characterized oncogenic “driver” mutations in NSCLC has not been fully characterized, though constitutive STAT3 activation has been proposed to play an important role in resistance to various small-molecule therapies that target these oncogenes. In this study we demonstrate that STAT3 is constitutively activated in human NSCLC samples and in a variety of NSCLC lines independent of activating KRAS or tyrosine kinase mutations. We further show that genetic or pharmacologic inhibition of the gp130/JAK2 signaling pathway disrupts activation of STAT3. Interestingly, treatment of NSCLC cells with the JAK1/2 inhibitor ruxolitinib has no effect on cell proliferation and viability in two-dimensional culture, but inhibits growth in soft agar and xenograft assays. These data demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and plays critical roles in tumor cell behavior that may not be effectively inhibited by drugs that selectively target these driver mutations

Paired Tumor and Normal Whole Genome Sequencing of Metastatic Olfactory Neuroblastoma

Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression.Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects.This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations

Tailoring Tyrosine Kinase Inhibitors to Fit the Lung Cancer Genome

Tyrosine kinase inhibitors (TKIs) have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies with defined genetic lesions has proven to be remarkably effective. Recent efforts to characterize the spectrum of genetic lesions found in non-small cell lung carcinoma (NSCLC) have provided important insights into the molecular basis of this disease and have also revealed a wide array of tyrosine kinases that might be effectively targeted for rationally designed therapies. The findings of these studies, however, also provide a cautionary tale about the limitations of single-agent therapies, which fail to account for the genetic heterogeneity and pathway redundancy that characterize advanced NSCLC. Emergence of drug resistance mechanisms to specific TKIs, such as gefitinib and erlotinib, suggests that more sophisticated chemotherapeutic paradigms that target multiple pathways at the same time will be required to effectively treat this disease

DNA methylation in multiple myeloma is weakly associated with gene transcription.

Previous studies have now demonstrated that both genic and global hypomethylation characterizes the multiple myeloma (MM) epigenome. Whether these methylation changes are associated with global and corresponding increases (or decreases) in transcriptional activity are poorly understood. The purpose of our current study was to correlate DNA methylation levels in MM to gene expression. We analyzed matching datasets generated by the GoldenGate methylation BeadArray and Affymetrix gene expression platforms in 193 MM samples. We subsequently utilized two independent statistical approaches to identify methylation-expression correlations. In the first approach, we used a linear correlation parameter by computing a Pearson correlation coefficient. In the second approach, we discretized samples into low and high methylation groups and then compared the gene expression differences between the groups. Only methylation of 2.1% and 25.3% of CpG sites on the methylation array correlated to gene expression by Pearson correlation or the discretization method, respectively. Among the genes with methylation-expression correlations were IGF1R, DLC1, p16, and IL17RB. In conclusion, DNA methylation may directly regulate relatively few genes and suggests that additional studies are needed to determine the effects of genome-wide methylation changes in MM

STAT3 phosphorylation is mediated by the tyrosine kinase JAK2 in NSCLC.

<p><b>A</b>, The indicated NSCLC lines were plated at a fixed density and allowed to adhere overnight. Cells were then treated for 16 hours with DMSO (con) or a 2-fold escalating dose of the JAK1/2 inhibitor ruxolitinib ranging from 0.25 to 4.0 µM (left-right). Protein lysates were harvested and the phosphorylation status of STAT3 (pSTAT3^Y705), AKT (pAKT^S473), ERK (pERK^T202/Y204), and S6 (pS6^S235/S236) was evaluated by immunoblot. Equal amounts of protein (20 µg) were added for each sample. <b>B</b>, The same NSCLC lines were plated as in A, but were treated with a fixed dose of ruxolitinib (1 µM) for the indicated time. Phospho-protein status was evaluated in 20 µg of protein per sample as in A. <b>C</b>, NCI-H1703 cells were infected with lentiviral vectors containing a control shRNA (NT, non-targeting) or one of two shRNAs targeted to human JAK2 (sh1 or sh2). Protein lysates were harvested and evaluated for JAK2 expression and STAT3 phosphorylation by immunoblot. Blots for total STAT3 and tubulin were included to demonstrate equal loading. <b>D</b>, Representative images of immunohistochemical stains for total JAK2, total STAT3 and phosphorylated STAT3 (pSTAT3^Y705) performed on a tissue microarray containing 245 pathologically verified human NSCLC samples. Images were captured from staining performed serial sections of the same tumor samples. <b>E</b>, Summary of staining results for JAK2, STAT3 and pSTAT3^Y705 on the NSCLC tissue microarray (<i>n</i> = 245). Note that all but one of the samples with positive nuclear pSTAT3^Y705 staining (52/53, 98.1%) were also positive for JAK2.</p

STAT3 phosphorylation is mediated by IL-6 family cytokines in NSCLC cell lines.

<p><b>A</b>, The indicated NSCLC lines were plated at fixed densities and allowed to adhere overnight in serum-containing media. Cells were then treated for 24 hours in serum-free media containing anti-gp130 neutralizing antibody or a control mouse IgG (2.0 µg/mL each). Protein lysates were harvested and the phosphorylation status of STAT3 (pSTAT3^Y705), AKT (pAKT^S473), ERK (pERK^T202/Y204), and S6 (pS6^S235/S236) was evaluated by immunoblot. Equal amounts of protein (30 µg) were added for each sample. <b>B</b>, NSCLC lines were plated at fixed densities and allowed to adhere overnight in serum-containing media. Cells were then serum-starved, and conditioned media was collected at 48 hours. Secreted interleukin-6 (IL-6) levels were measured from the indicated cell lines using a bead-based immunoassay platform. Values shown are averages of duplicate measurements for each sample.</p