Person:

Giannakis, Marios

Background: The diversity of clinical tumor profiling approaches (small panels to whole exomes with matched or unmatched germline analysis) may engender uncertainty about their benefits and liabilities, particularly in light of reported germline false positives in tumor-only profiling and use of global mutational and/or neoantigen data. The goal of this study was to determine the impact of genomic analysis strategies on error rates and data interpretation across contexts and ancestries. Methods: We modeled common tumor profiling modalities—large (n = 300 genes), medium (n = 48 genes), and small (n = 15 genes) panels—using clinical whole exomes (WES) from 157 patients with lung or colon adenocarcinoma. We created a tumor-only analysis algorithm to assess germline false positive rates, the impact of patient ancestry on tumor-only results, and neoantigen detection. Results: After optimizing a germline filtering strategy, the germline false positive rate with tumor-only large panel sequencing was 14 % (144/1012 variants). For patients whose tumor-only results underwent molecular pathologist review (n = 91), 50/54 (93 %) false positives were correctly interpreted as uncertain variants. Increased germline false positives were observed in tumor-only sequencing of non-European compared with European ancestry patients (p < 0.001; Fisher’s exact) when basic germline filtering approaches were used; however, the ExAC database (60,706 germline exomes) mitigated this disparity (p = 0.53). Matched and unmatched large panel mutational load correlated with WES mutational load (r2 = 0.99 and 0.93, respectively; p < 0.001). Neoantigen load also correlated (r2 = 0.80; p < 0.001), though WES identified a broader spectrum of neoantigens. Small panels did not predict mutational or neoantigen load. Conclusions: Large tumor-only targeted panels are sufficient for most somatic variant identification and mutational load prediction if paired with expanded germline analysis strategies and molecular pathologist review. Paired germline sequencing reduced overall false positive mutation calls and WES provided the most neoantigens. Without patient-matched germline data, large germline databases are needed to minimize false positive mutation calling and mitigate ethnic disparities. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0333-9) contains supplementary material, which is available to authorized users.

PD-1 immune checkpoint blockade occasionally results in durable clinical responses in advanced metastatic cancers. However, mechanism-based predictors of response to this immunotherapy remain incompletely characterized. We performed comprehensive genomic profiling on a tumor and germline sample from a patient with refractory lung adenocarcinoma who achieved marked long-term clinical benefit from anti-PD-L1 therapy. We discovered activating somatic and germline amino acid variants in JAK3 that promoted PD-L1 induction in lung cancer cells and in the tumor immune microenvironment. These findings suggest that genomic alterations that deregulate cytokine receptor signal transduction could contribute to PD-L1 activation and engagement of the PD-1 immune checkpoint in lung cancer.

Summary Large-scale genomic characterization of tumors from prospective cohort studies may yield new insights into cancer pathogenesis. We performed whole-exome sequencing of 619 incident colorectal cancers (CRCs) and integrated the results with tumor immunity, pathology, and survival data. We identified recurrently mutated genes in CRC, such as BCL9L, RBM10, CTCF, and KLF5, that were not previously appreciated in this disease. Furthermore, we investigated the genomic correlates of immune-cell infiltration and found that higher neoantigen load was positively associated with overall lymphocytic infiltration, tumor-infiltrating lymphocytes (TILs), memory T cells, and CRC-specific survival. The association with TILs was evident even within microsatellite-stable tumors. We also found positive selection of mutations in HLA genes and other components of the antigen-processing machinery in TIL-rich tumors. These results may inform immunotherapeutic approaches in CRC. More generally, this study demonstrates a framework for future integrative molecular epidemiology research in colorectal and other malignancies.

Recent studies have detailed the genomic landscape of primary endometrial cancers, but their evolution into metastases has not been characterized. We performed whole-exome sequencing of 98 tumor biopsies including complex atypical hyperplasias, primary tumors, and paired abdominopelvic metastases to survey the evolutionary landscape of endometrial cancer. We expanded and reanalyzed TCGA-data, identifying novel recurrent alterations in primary tumors, including mutations in the estrogen receptor cofactor NRIP1 in 12% of patients. We found that likely driver events tended to be shared by primary and metastatic tissue-samples, with notable exceptions such as ARID1A mutations. Phylogenetic analyses indicated that the sampled metastases typically arose from a common ancestral subclone that was not detected in the primary tumor biopsy. These data demonstrate extensive genetic heterogeneity within endometrial cancers and relative homogeneity across metastatic sites.

Precision medicine has a goal of customizing disease prevention and treatment strategies. Under the precision medicine paradigm, each patient has unique pathologic processes resulting from cellular genomic, epigenomic, proteomic, and metabolomic alterations, which are influenced by pharmacological, environmental, microbial, dietary, and lifestyle factors. Hence, to realize the promise of precision medicine, multi-level research methods that can comprehensively analyze many of these variables are needed. In order to address this gap, the integrative field of molecular pathology and population data science (i.e., molecular pathological epidemiology) has been developed to enable such multi-level analyses, especially in gastrointestinal cancer research. Further integration of pharmacology can improve our understanding of drug effects, and inform decision-making of drug use at both the individual and population levels. Such integrative research demonstrated potential benefits of aspirin in colorectal carcinoma with PIK3CA mutations, providing the basis for new clinical trials. Evidence also suggests that HPGD (15-PDGH) expression levels in normal colon and the germline rs6983267 polymorphism that relates to tumor CTNNB1 (β-catenin)/WNT signaling status may predict the efficacy of aspirin for cancer chemoprevention. As immune checkpoint blockade targeting the CD274 (PD-L1)/PDCD1 (PD-1) pathway for microsatellite instability-high (or mismatch repair-deficient) metastatic gastrointestinal or other tumors has become standard of care, potential modifying effects of dietary, lifestyle, microbial, and environmental factors on immunotherapy need to be studied to further optimize treatment strategies. With its broad applicability, our integrative approach can provide insights into the interactive role of medications, exposures, and molecular pathology, and guide the development of precision medicine.

Evidence suggests that nonsteroidal anti-inflammatory drug aspirin (acetylsalicylic acid) may improve patient survival in PIK3CA-mutant colorectal carcinoma, but not in PIK3CA-wild-type carcinoma. However, whether aspirin directly influences the viability of PIK3CA-mutant colon cancer cells is poorly understood. We conducted in vitro experiments to test our hypothesis that the anti-proliferative activity of aspirin might be stronger for PIK3CA-mutant colon cancer cells than for PIK3CA-wild-type colon cancer cells. We measured the anti-proliferative effect of aspirin at physiologic concentrations in seven PIK3CA-mutant and six PIK3CA-wild-type human colon cancer cell lines. After exposure to aspirin, the apoptotic index and cell cycle phase of colon cancer cells were assessed. In addition, the effect of aspirin was examined in parental SW48 cells and SW48 cell clones with individual knock-in PIK3CA mutations of either c.3140A>G (p.H1047R) or c.1633G>A (p.E545K). Aspirin induced greater dose-dependent loss of cell viability in PIK3CA-mutant cells than in PIK3CA-wild-type cells after treatment for 48 and 72 hours. Aspirin treatment also led to higher proportions of apoptotic cells and G0/G1 phase arrest in PIK3CA-mutant cells than in PIK3CA-wild-type cells. Aspirin treatment of isogenic SW48 cells carrying a PIK3CA mutation, either c.3140A>G (p.H1047R) or c.1633G>A (p. E545K), resulted in a more significant loss of cell viability compared to wild-type controls. Our findings indicate that aspirin causes cell cycle arrest, induces apoptosis, and leads to loss of cell viability more profoundly in PIK3CA-mutated colon cancer cells than in PIK3CA-wild-type colon cancer cells. These findings support the use of aspirin to treat patients with PIK3CA-mutant colon cancer.

Objectives: Evidence suggests a possible role of Fusobacterium nucleatum in colorectal carcinogenesis, especially in right-sided proximal colorectum. Considering a change in bowel contents and microbiome from proximal to distal colorectal segments, we hypothesized that the proportion of colorectal carcinoma enriched with F. nucleatum might gradually increase along the bowel subsites from rectum to cecum. Methods: A retrospective, cross-sectional analysis was conducted on 1,102 colon and rectal carcinomas in molecular pathological epidemiology databases of the Nurses’ Health Study and the Health Professionals Follow-up Study. We measured the amount of F. nucleatum DNA in colorectal tumor tissue using a quantitative PCR assay and equally dichotomized F. nucleatum-positive cases (high vs. low). We used multivariable logistic regression analysis to examine the relationship of a bowel subsite variable (rectum, rectosigmoid junction, sigmoid colon, descending colon, splenic flexure, transverse colon, hepatic flexure, ascending colon, and cecum) with the amount of F. nucleatum. Results: The proportion of F. nucleatum-high colorectal cancers gradually increased from rectal cancers (2.5% 4/157) to cecal cancers (11% 19/178), with a statistically significant linear trend along all subsites (P<0.0001) and little evidence of non-linearity. The proportion of F. nucleatum-low cancers was higher in rectal, ascending colon, and cecal cancers than in cancers of middle segments. Conclusions: The proportion of F. nucleatum-high colorectal cancers gradually increases from rectum to cecum. Our data support the colorectal continuum model that reflects pathogenic influences of the gut microbiota on neoplastic and immune cells and challenges the prevailing two-colon (proximal vs. distal) dichotomy paradigm.

Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography–mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism.

Synthetic lethality, an interaction whereby the co-occurrence of two genetic events leads to cell death but one event alone does not, can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets since many cancers exhibit an impaired DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors in homologous recombination-deficient cancers highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair (dMMR). Here we analyzed data from large-scale CRISPR/Cas9 knockout and RNA interference (RNAi) silencing screens and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in microsatellite stable (MSS) models. WRN depletion induced double-strand DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity, but not the exonuclease activity of WRN. These findings expose WRN as a synthetic lethal vulnerability and promising drug target for MSI cancers.