Zachary A. Kohutek

Limited heterogeneity of known driver gene mutations among the metastases of individual patients with pancreatic cancer

The extent of heterogeneity among driver gene mutations present in naturally occurring metastases—that is, treatment-naive metastatic disease—is largely unknown. To address this issue, we carried out 60× whole-genome sequencing of 26 metastases from four patients with pancreatic cancer. We found that identical mutations in known driver genes were present in every metastatic lesion for each patient studied. Passenger gene mutations, which do not have known or predicted functional consequences, accounted for all intratumoral heterogeneity. Even with respect to these passenger mutations, our analysis suggests that the genetic similarity among the founding cells of metastases was higher than that expected for any two cells randomly taken from a normal tissue. The uniformity of known driver gene mutations among metastases in the same patient has critical and encouraging implications for the success of future targeted therapies in advanced-stage disease.

Retrotransposon insertions in the clonal evolution of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed after the disease has metastasized; it is among the most lethal forms of cancer. We recently described aberrant expression of an open reading frame 1 protein, ORF1p, encoded by long interspersed element-1 (LINE-1; L1) retrotransposon, in PDAC. To test whether LINE-1 expression leads to somatic insertions of this mobile DNA, we used a targeted method to sequence LINE-1 insertion sites in matched PDAC and normal samples. We found evidence of 465 somatic LINE-1 insertions in 20 PDAC genomes, which were absent from corresponding normal samples. In cases in which matched normal tissue, primary PDAC and metastatic disease sites were available, insertions were found in primary and metastatic tissues in differing proportions. Two adenocarcinomas secondarily involving the pancreas, but originating in the stomach and duodenum, acquired insertions with a similar discordance between primary and metastatic sites. Together, our findings show that LINE-1 contributes to the genetic evolution of PDAC and suggest that somatic insertions are acquired discontinuously in gastrointestinal neoplasms.

FDG-PET maximum standardized uptake value is prognostic for recurrence and survival after stereotactic body radiotherapy for non-small cell lung cancer

OBJECTIVES Glucose metabolic activity measured by [(18)F]-fluoro-2-deoxy-glucose positron emission tomography (FDG-PET) has shown prognostic value in multiple malignancies, but results are often confounded by the inclusion of patients with various disease stages and undergoing various therapies. This study was designed to evaluate the prognostic value of tumor FDG uptake quantified by maximum standardized uptake value (SUVmax) in a large group of early-stage non-small cell lung cancer (NSCLC) patients treated with stereotactic body radiotherapy (SBRT) using consistent treatment techniques. MATERIALS AND METHODS Two hundred nineteen lesions in 211 patients treated with definitive SBRT for stage I NSCLC were analyzed after a median follow-up of 25.2 months. Cox regression was used to determine associations between SUVmax and overall survival (OS), disease-specific survival (DSS), and freedom from local recurrence (FFLR) or distant metastasis (FFDM). RESULTS SUVmax >3.0 was associated with worse OS (p<0.001), FFLR (p=0.003) and FFDM (p=0.003). On multivariate analysis, OS was associated with SUVmax (HR 1.89, p=0.03), gross tumor volume (GTV) (HR 1.94, p=0.005) and Karnofsky performance status (KPS) (HR 0.51, p=0.008). DSS was associated only with SUVmax (HR 2.58, p=0.04). Both LR (HR 11.47, p=0.02) and DM (HR 3.75, p=0.006) were also associated with higher SUVmax. CONCLUSION In a large patient population, SUVmax >3.0 was associated with worse survival and a greater propensity for local recurrence and distant metastasis after SBRT for NSCLC.

Transcriptional Mechanisms of Resistance to Anti–PD-1 Therapy

Purpose: To explore factors associated with response and resistance to anti–PD-1 therapy, we analyzed multiple disease sites at autopsy in a patient with widely metastatic melanoma who had a heterogeneous response. Materials and Methods: Twenty-six melanoma specimens (four premortem, 22 postmortem) were subjected to whole exome sequencing. Candidate immunologic markers and gene expression were assessed in 10 cutaneous metastases showing response or progression during therapy. Results: The melanoma was driven by biallelic inactivation of NF1. All lesions had highly concordant mutational profiles and copy number alterations, indicating linear clonal evolution. Expression of candidate immunologic markers was similar in responding and progressing lesions. However, progressing cutaneous metastases were associated with overexpression of genes associated with extracellular matrix and neutrophil function. Conclusions: Although mutational and immunologic differences have been proposed as the primary determinants of heterogeneous response/resistance to targeted therapies and immunotherapies, respectively, differential lesional gene expression profiles may also dictate anti–PD-1 outcomes. Clin Cancer Res; 23(12); 3168–80. ©2017 AACR. See related commentary by Wilmott et al., p. 2921

Mutant p53 Together with TGFβ Signaling Influence Organ-Specific Hematogenous Colonization Patterns of Pancreatic Cancer

Purpose: TP53 and the TGFβ pathway are major mediators of pancreatic cancer metastasis. The mechanisms by which they cause hematogenous metastasis have not been fully explored. Experimental Design: KPC (LSL-KRASG12D/+;LSL-Trp53R172H/+; Ptf1aCre/+) mice were generated, and the frequency and morphology of organ-specific hematogenous metastases compared with that seen in KPTC and KTC littermates (Tgfbr2+/−). Key findings were validated in primary cells from each genotype and samples of human pancreatic cancer liver metastases. Results: The frequency of hematogenous metastasis in KPTC mice was significantly lower than for KPC mice (41% vs. 68%, P < 0.05), largely due to a reduction in liver metastases. No differences were found between KPC and KPTC lung metastases, whereas liver metastases in KPTC mice showed a profound extravasation deficiency characterized by sinusoidal growth and lack of desmoplastic stroma. Analogous findings were confirmed in liver samples from patients indicating their clinical relevance. Portal vein colonization as a direct mode of access to the liver was observed in both mice and humans. Secretome analyses of KPC cells revealed an abundance of secreted prometastatic mediators including Col6A1 and Lcn2 that promoted early steps of metastatic colonization. These mediators were overexpressed in primary tumors but not metastases, suggesting that the ability to colonize is, in part, developed within the primary site, a phenomenon we refer to as the “Cinderella effect.” Conclusions: These findings establish a novel paradigm for understanding pancreatic cancer metastasis and the observed clinical latencies of liver versus lung metastases specifically. Clin Cancer Res; 23(6); 1607–20. ©2016 AACR.

An Unusual Genomic Variant of Pancreatic Ductal Adenocarcinoma with an Indolent Clinical Course

We describe an 85-yr-old male of Ashkenazi Jewish descent with biopsy-proven locally advanced pancreatic ductal adenocarcinoma (PDA). The patient underwent a modified course of gemcitabine and stereotactic body radiation therapy and survived for 42 mo with a stable pancreatic head mass and no evidence of metastatic disease before death due to complications from a stroke. Whole-exome sequencing of his tumor revealed a simple genome landscape with no evidence of mutations, copy-number changes, or structural alterations in genes most commonly associated with PDA (i.e., KRAS, CDKN2A, TP53, or SMAD4). An analysis of his germline DNA revealed no pathogenic variants of significance. Whole-exome and whole-genome sequencing identified a somatic mutation of RNF213 and an inversion/deletion of CTNNA2 as the genetic basis of his PDA. Although PDA is classically characterized by a predictable set of mutations, these data suggest that alternate genetic paths to PDA may exist, which can be associated with a more indolent clinical course.

Minimal functional driver gene heterogeneity among untreated metastases

Metastatic drivers same as primary Treatment decisions for cancer patients are increasingly guided by analysis of the gene mutations that drive primary tumor growth. Relatively little is known about driver gene mutations in metastases, which cause most cancer-related deaths. Reiter et al. explored whether the growth of different metastatic lesions within an individual patient is fueled by the same or distinct gene mutations. In a study of 76 untreated metastases from 20 patients with different types of cancer, all metastases within a patient shared the same functional driver gene mutations. Thus, analysis of a single biopsy could help oncologists select the optimal therapy for patients with widespread metastatic disease. Science, this issue p. 1033 The growth of different metastatic lesions within an individual cancer patient is fueled by the same genetic mutations. Metastases are responsible for the majority of cancer-related deaths. Although genomic heterogeneity within primary tumors is associated with relapse, heterogeneity among treatment-naïve metastases has not been comprehensively assessed. We analyzed sequencing data for 76 untreated metastases from 20 patients and inferred cancer phylogenies for breast, colorectal, endometrial, gastric, lung, melanoma, pancreatic, and prostate cancers. We found that within individual patients, a large majority of driver gene mutations are common to all metastases. Further analysis revealed that the driver gene mutations that were not shared by all metastases are unlikely to have functional consequences. A mathematical model of tumor evolution and metastasis formation provides an explanation for the observed driver gene homogeneity. Thus, single biopsies capture most of the functionally important mutations in metastases and therefore provide essential information for therapeutic decision-making.

Oral Cavity Cancer

The oral cavity is the most anterior head and neck site. It is separated from the oropharynx by an imaginary line extending from the anterior tonsillar pillars to the junction of hard and soft palate to the circumvallate papillae.The oral cavity is divided into several subsites including the oral tongue, floor of the mouth, retromolar trigone, alveolar ridge, hard palate, buccal mucosa, gingiva and lips (Fig. 1).