Marisa W. Welch

Assigning clinical meaning to somatic and germ-line whole-exome sequencing data in a prospective cancer precision medicine study

Purpose:Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care.Methods:One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences.Results:At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach.Conclusion:The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017

Real-time Genomic Characterization of Advanced Pancreatic Cancer to Enable Precision Medicine

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC.Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096-111. ©2018 AACR.See related commentary by Collisson, p. 1062This article is highlighted in the In This Issue feature, p. 1047.

Altered exocrine function can drive adipose wasting in early pancreatic cancer

Malignancy is accompanied by changes in the metabolism of both cells and the organism1,2. Pancreatic ductal adenocarcinoma (PDAC) is associated with wasting of peripheral tissues, a metabolic syndrome that lowers quality of life and has been proposed to decrease survival of patients with cancer3,4. Tissue wasting is a multifactorial disease and targeting specific circulating factors to reverse this syndrome has been mostly ineffective in the clinic5,6. Here we show that loss of both adipose and muscle tissue occurs early in the development of pancreatic cancer. Using mouse models of PDAC, we show that tumour growth in the pancreas but not in other sites leads to adipose tissue wasting, suggesting that tumour growth within the pancreatic environment contributes to this wasting phenotype. We find that decreased exocrine pancreatic function is a driver of adipose tissue loss and that replacement of pancreatic enzymes attenuates PDAC-associated wasting of peripheral tissues. Paradoxically, reversal of adipose tissue loss impairs survival in mice with PDAC. When analysing patients with PDAC, we find that depletion of adipose and skeletal muscle tissues at the time of diagnosis is common, but is not associated with worse survival. Taken together, these results provide an explanation for wasting of adipose tissue in early PDAC and suggest that early loss of peripheral tissue associated with pancreatic cancer may not impair survival.Pancreatic ductal adenocarcinoma in mice induces loss of adipose tissue through altered function of the exocrine pancreas, and supplementing pancreatic enzymes attenuates the wasting of peripheral tissues induced by pancreatic cancer.

Association of Alterations in Main Driver Genes With Outcomes of Patients With Resected Pancreatic Ductal Adenocarcinoma

Importance Although patients with resected pancreatic adenocarcinoma are at high risk for disease recurrence, few biomarkers are available to inform patient outcomes. Objective To evaluate the alterations of the 4 main driver genes in pancreatic adenocarcinoma and patient outcomes after cancer resection. Design, Setting, and Participants This study analyzed protein expression and DNA alterations for the KRAS, CDKN2A, SMAD4, and TP53 genes by immunohistochemistry and next-generation sequencing in formalin-fixed, paraffin-embedded tumors in 356 patients with resected pancreatic adenocarcinoma who were treated at the Dana-Farber/Brigham and Women’s Cancer Center (October 26, 2002, to May 21, 2012), University of Rochester Medical Center (March 1, 2006, to November 1, 2013), or Stanford Cancer Institute (September 26, 1995, to May 22, 2013). Associations of driver gene alterations with disease-free survival (DFS) and overall survival (OS) were evaluated using Cox proportional hazards regression with estimation of hazard ratios (HRs) and 95% CIs and adjustment for age, sex, tumor characteristics, institution, and perioperative treatment. Data were collected September 9, 2012, to June 28, 2016, and analyzed December 17, 2016, to March 14, 2017. Main Outcomes and Measures The DFS and OS among patients with resected pancreatic adenocarcinoma. Results Of the 356 patients studied, 191 (53.7%) were men and 165 (46.3%) were women, with a median (interquartile range [IQR]) age of 67 (59.0-73.5) years. Patients with KRAS mutant tumors had worse DFS (median [IQR], 12.3 [6.7 -27.2] months) and OS (20.3 [11.3-38.3] months) compared with patients with KRAS wild-type tumors (DFS, 16.2 [8.9-30.5] months; OS, 38.6 [16.6-63.1] months) and had 5-year OS of 13.0% vs 30.2%. Particularly poor outcomes were identified in patients with KRAS G12D-mutant tumors, who had a median (IQR) OS of 15.3 (9.8-32.7) months. Patients whose tumors lacked CDKN2A expression had worse DFS (median, 11.5 [IQR, 6.2-24.5] months) and OS (19.7 [10.9-37.1] months) compared with patients who had intact CDKN2A (DFS, 14.8 [8.2-30.5] months; OS, 24.6 [14.1-44.6] months). The molecular status of SMAD4 was not associated with DFS or OS, whereas TP53 status was associated only with shorter DFS (HR, 1.33; 95% CI, 1.02-1.75; P = .04). Patients had worse DFS and OS if they had a greater number of altered driver genes. Compared with patients with 0 to 2 altered genes, those with 4 altered genes had worse DFS (HR, 1.79 [95% CI, 1.24-2.59; P = .002]) and OS (HR, 1.38 [95% CI, 0.98-1.94; P = .06]). Five-year OS was 18.4% for patients with 0 to 2 gene alterations, 14.1% for those with 3 alterations, and 8.2% for those with 4 alterations. Conclusions and Relevance Patient outcomes are associated with alterations of the 4 main driver genes in resected pancreatic adenocarcinoma.

Germline cancer susceptibility gene variants, somatic second hits, and survival outcomes in patients with resected pancreatic cancer

PurposeGermline variants in double-strand DNA damage repair (dsDDR) genes (e.g., BRCA1/2) predispose to pancreatic adenocarcinoma (PDAC) and may predict sensitivity to platinum-based chemotherapy and poly(ADP) ribose polymerase (PARP) inhibitors. We sought to determine the prevalence and significance of germline cancer susceptibility gene variants in PDAC with paired somatic and survival analyses.MethodsUsing a customized next-generation sequencing panel, germline/somatic DNA was analyzed from 289 patients with resected PDAC ascertained without preselection for high-risk features (e.g., young age, personal/family history). All identified variants were assessed for pathogenicity. Outcomes were analyzed using multivariable-adjusted Cox proportional hazards regression.ResultsWe found that 28/289 (9.7%; 95% confidence interval [CI] 6.5–13.7%) patients carried pathogenic/likely pathogenic germline variants, including 21 (7.3%) dsDDR gene variants (3 BRCA1, 4 BRCA2, 14 other dsDDR genes [ATM, BRIP1, CHEK2, NBN, PALB2, RAD50, RAD51C]), 3 Lynch syndrome, and 4 other genes (APC p.I1307K, CDKN2A, TP53). Somatic sequencing and immunohistochemistry identified second hits in the tumor in 12/27 (44.4%) patients with germline variants (1 failed sequencing). Compared with noncarriers, patients with germline dsDDR gene variants had superior overall survival (hazard ratio [HR] 0.54; 95% CI 0.30–0.99; P = 0.05).ConclusionNearly 10% of PDAC patients harbor germline variants, although the majority lack somatic second hits, the therapeutic significance of which warrants further study.

Plasma inflammatory cytokines and survival of pancreatic cancer patients

Objectives: Inflammation and inflammatory conditions have been associated with pancreatic cancer risk and progression in a number of clinical, epidemiological, and animal model studies. The goal of the present study is to identify plasma markers of inflammation associated with survival of pancreatic cancer patients, and assess their joint contribution to patient outcome. Methods: We measured circulating levels of four established markers of inflammation (C‐reactive protein (CRP), interleukin‐6 (IL‐6), soluble tumor necrosis factor receptor type II (sTNF‐RII), and macrophage inhibitory cytokine‐1 (MIC‐1)) in 446 patients enrolled in an ongoing prospective clinic‐based study. Hazard ratios (HRs) and 95% confidence intervals (CI) for death were estimated using multivariate Cox proportional hazards models. Results: Overall mortality was significantly increased in patients in the top quartile of CRP (HR = 2.52, 95% CI: 1.82–3.49), IL‐6 (HR = 2.78, 95% CI: 2.03–3.81), sTNF‐RII (HR = 2.00, 95% CI: 1.46–2.72), and MIC‐1 (HR = 2.53, 95% CI: 1.83–3.50), compared to those in the bottom quartile (P‐trend <0.0001 for all four comparisons). Furthermore, patients with higher circulating concentrations of all four cytokines had a median survival of 3.7 months; whereas, those with lower levels had a median survival of 19.2 months (HR = 4.55, 95% CI: 2.87–7.20, P‐trend <0.0001). Conclusion: Individual elevated plasma inflammatory cytokines are associated with significant and dramatic reductions in pancreatic cancer patient survival. Furthermore, we observed an independent combined effect of those cytokines on patient survival, suggesting that multiple inflammatory pathways are likely involved in PDAC progression. Future research efforts to target the inflammatory state using combination strategies in pancreatic cancer patients are warranted.

Lymph node metastases in resected pancreatic ductal adenocarcinoma: predictors of disease recurrence and survival.

Background:Few studies have simultaneously assessed the prognostic value of the multiple classification systems for lymph node (LN) metastases in resected pancreatic ductal adenocarcinoma (PDAC).Methods:In 600 patients with resected PDAC, we examined the association of LN parameters (AJCC 7th and 8th editions, LN ratio (LNR), and log odds of metastatic LN (LODDS)) with pattern of recurrence and patient survival using logistic regression and Cox proportional hazards regression, respectively. Regression models adjusted for age, sex, margin status, tumour grade, and perioperative therapy.Results:Lymph node metastases classified by AJCC 7th and 8th editions, LNR, and LODDS were associated with worse disease free-survival (DFS) and overall survival (OS) (all Ptrend<0.01). American Joint Committee on Cancer 8th edition effectively predicted DFS and OS, while minimising model complexity. Lymph node metastases had weaker prognostic value in patients with positive margins and distal resections (both Pinteraction<0.03). Lymph node metastases by AJCC 7th and 8th editions did not predict the likelihood of local disease as the first site of recurrence.Conclusions:American Joint Committee on Cancer 8th edition LN classification is an effective and practical tool to predict outcomes in patients with resected PDAC. However, the prognostic value of LN metastases is attenuated in patients with positive resection margins and distal pancreatectomies.

Bead Enhancement of EV Analysis

Extracellular vesicles (EVs) are recognized cancer biomarkers, however, clinical analysis has been difficult due to a lack of simple and sensitive assays. Here, we describe a bead-enhanced flow cytometry method, BEAD flow, using biotinylated EVs captured on streptavidin particles. With this method, we show analysis of patient-derived EVs using a panel of pancreatic cancer biomarkers. BEAD flow is easily translatable to any biomarker or cancer type and can be run with conventional flow cytometers, making it highly flexible and adaptable to diverse research and clinical needs.

Specific detection of cell-free DNA derived from intestinal epithelial cells using methylation patterns

Epithelial cells of the intestine undergo rapid turnover and are thought to be cleared via stool. Disruption of tissue architecture, as occurs in colorectal cancer (CRC), results in the release of material from dying intestinal epithelial cells to blood. This phenomenon could be utilized for diagnosis and monitoring of intestinal diseases, if circulating cell-free DNA (cfDNA) derived from intestinal cells could be specifically identified. Here we describe two genomic loci that are unmethylated specifically in intestinal epithelial cells, allowing for sensitive and specific detection of DNA derived from such cells. As expected, intestinal DNA is found in stool, but not in plasma, of healthy individuals. Patients with inflammatory bowel disease (IBD) have minimal amounts of intestinal cfDNA in the plasma, whereas patients with advanced CRC show a strong signal. The intestinal markers are not elevated in plasma samples from patients with pancreatic ductal adenocarcinoma (PDAC), and a combination of intestine- and pancreas-specific markers allowed for robust differentiation between plasma cfDNA derived from CRC and PDAC patients. Intestinal DNA markers provide a mutation-independent tool for monitoring intestinal dynamics in health and disease.