Search

Supplemental Methods. Supplemental Table 1: ââ,¬â€¹Genomic Data Characterization by Center. Supplemental Table 2: ââ,¬â€¹Gene Panels Submitted by Each Center. Figure S1: Number of putative germline SNPs per sample, before and after uniform germline filtering. Figure S2ââ,¬â€¹. Distribution of total somatic mutation burden per sample stratified by sequencing panel. Figure S3: ââ,¬â€¹Log-scale comparison of mutation frequencies at hotspot sites between GENIE (data aggregated from all sequencing panels) and cancerhotspots.org (CHS) using a binomial test. Figure S4:ââ,¬â€¹ Comparison of mutation frequencies at hotspot sites in each GENIE sequencing panel with cancerhotspots.org (CHS) using a binomial test.

Table S4

Univariate and multivariate survival analysis.

AACR GENIE Data Guide

S1 shows intratumor mutation heterogeneity between two specimens of one tumor. S2 describes mutational load of the patient cohort. S3 shows survival difference based on histology. S4 shows mutational signature.

Malignant gliomas are a group of intracranial cancers associated with disproportionately high mortality and morbidity. Here, we report ultradeep targeted sequencing of a prospective cohort of 237 tumors from 234 patients consisting of both glioblastoma (GBM) and lower-grade glioma (LGG) using our customized gene panels. We identified 2,485 somatic mutations, including single-nucleotide substitutions and small indels, using a validated in-house protocol. Sixty-one percent of the mutations were contributed by 12 hypermutators. The hypermutators were enriched for recurrent tumors and had comparable outcome, and most were associated with temozolomide exposure. TP53 was the most frequently mutated gene in our cohort, followed by IDH1 and EGFR. We detected at least one EGFR mutation in 23% of LGGs, which was significantly higher than 6% seen in The Cancer Genome Atlas, a pattern that can be partially explained by the different patient composition and sequencing depth. IDH hotspot mutations were found with higher frequencies in LGG (83%) and secondary GBM (77%) than primary GBM (9%). Multivariate analyses controlling for age, histology, and tumor grade confirm the prognostic value of IDH mutation. We predicted 1p/19q status using the panel sequencing data and received only modest performance by benchmarking the prediction to FISH results of 50 tumors. Targeted therapy based on the sequencing data resulted in three responders out of 14 participants. In conclusion, our study suggests ultradeep targeted sequencing can recapitulate previous findings and can be a useful approach in the clinical setting.

Supplemental Tables 1, 2, 3 and Supplemental Figure 1

Purpose:Molecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies.Experimental Design:We analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA).Results:A total of 153/206 (74.3%) were men; median age, 62 years (range, 18–91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1–13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%–55.03%). TP53 was the common altered gene [>120 alterations (non-unique)] followed by EGFR, MET, ARID1A, MYC, NF1, BRAF, and ERBB2 [20–38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in MYC, EGFR, ERBB2, BRAF, CCNE1, MET, PIK3CA, ARID1A, CDK6, and KRAS. In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have ERBB2 alterations, 35.7% (5/14) versus 8.8% HBV-negative (P = 0.04).Conclusions:This study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology.

Purpose:Physicians are expected to assess prognosis both for patient counseling and for determining suitability for clinical trials. Increasingly, cell-free circulating tumor DNA (cfDNA) sequencing is being performed for clinical decision making. We sought to determine whether variant allele frequency (VAF) in cfDNA is associated with prognosis.Experimental Design:We performed a retrospective analysis of 298 patients with metastatic disease who underwent clinical comprehensive cfDNA analysis and assessed association between VAF and overall survival.Results:cfDNA mutations were detected in 240 patients (80.5%). Median overall survival (OS) was 11.5 months. cfDNA mutation detection and number of nonsynonymous mutations (NSM) significantly differed between tumor types, being lowest in appendiceal cancer and highest in colon cancer. Having more than one NSM detected was associated with significantly worse OS (HR = 2.3; P < 0.0001). VAF was classified by quartiles, Q1 lowest, Q4 highest VAF. Higher VAF levels were associated with a significantly worse overall survival (VAF Q3 HR 2.3, P = 0.0069; VAF Q4 HR = 3.8, P < 0.0001) on univariate analysis. On multivariate analysis, VAF Q4, male sex, albumin level 0 and number of prior therapies >4 were independent predictors of worse OS.Conclusions:Higher levels of cfDNA VAF and a higher number of NSMs were associated with worse OS in patients with metastatic disease. Further study is needed to determine optimal VAF thresholds for clinical decision making and the utility of cfDNA VAF as a prognostic marker in different tumor types.

Supplementary Figure 2

Supplemental table 1

Purpose: Physicians are expected to assess prognosis both for patient counseling and for determining suitability for clinical trials. Increasingly, cell-free circulating tumor DNA (cfDNA) sequencing is being performed for clinical decision making. We sought to determine whether variant allele frequency (VAF) in cfDNA is associated with prognosis. Experimental Design: We performed a retrospective analysis of 298 patients with metastatic disease who underwent clinical comprehensive cfDNA analysis and assessed association between VAF and overall survival. Results: cfDNA mutations were detected in 240 patients (80.5%). Median overall survival (OS) was 11.5 months. cfDNA mutation detection and number of nonsynonymous mutations (NSM) significantly differed between tumor types, being lowest in appendiceal cancer and highest in colon cancer. Having more than one NSM detected was associated with significantly worse OS (HR = 2.3; P < 0.0001). VAF was classified by quartiles, Q1 lowest, Q4 highest VAF. Higher VAF levels were associated with a significantly worse overall survival (VAF Q3 HR 2.3, P = 0.0069; VAF Q4 HR = 3.8, P < 0.0001) on univariate analysis. On multivariate analysis, VAF Q4, male sex, albumin level 0 and number of prior therapies >4 were independent predictors of worse OS. Conclusions: Higher levels of cfDNA VAF and a higher number of NSMs were associated with worse OS in patients with metastatic disease. Further study is needed to determine optimal VAF thresholds for clinical decision making and the utility of cfDNA VAF as a prognostic marker in different tumor types.

Acquired resistance to molecular targeted therapy is a significant challenge of the precision medicine era. The ability to understand these mechanisms of resistance may improve patient selection and allow for the development of rationally designed next-line or combination treatment strategies and improved patient outcomes. AKT is a critical effector of the phosphoinositide 3-kinase signaling cascade, one of the most commonly activated pathways in human cancer. Deregulation of signaling pathways, such as RAF/MEK/ERK are previously described mechanisms of resistance to AKT/PI3K inhibitors. Mutations in the mTOR gene, however, are exceedingly rare. We present a case of acquired mTOR resistance, following targeted AKT inhibition, and subsequent response to mTOR1/2 inhibitor in a patient with metastatic endometrial cancer, the first documented response to ATP-competitive mTOR inhibition in this setting. This case supports mTOR mutation as a mechanism of resistance, and underscores the importance of tumor molecular profiling, exemplifying precision medicine in action.

Patients with advanced solid malignancies recurrent or resistant to standard therapy have limited treatment options. The role of molecular biomarkers for predicting immune checkpoint blockade (ICB) efficacy are not well characterized in these patients. Tumor mutational profiles of 490 patients with a variety of advanced solid tumors enrolled in a prospective protocol were analyzed to identify prognostic and predictive biomarkers. ICB therapy was defined as treatment with any CTLA-4, PD-1, and/or PD-L1 monoclonal antibody. ICB treatment was associated with significantly improved overall survival compared to non-ICB therapy. Multivariate regression analysis including the two variables of tumor mutation burden (TMB) and ICB, and their interaction term, showed favorable survival associated with ICB, unfavorable survival associated with TMB without ICB treatment, and improved outcome with increasing TMB in ICB treated patients. Tumor TP53 mutation was associated with worse survival, but these patients still benefitted from ICB. A more comprehensive multivariate analysis including cancer type, specific gene mutations, and TMB revealed that ICB treatment was an independent predictor of improved overall survival. Therefore, ICB-based therapeutic trials are beneficial in patients with advanced solid malignancies, but the most benefit may be restricted to patients with the right combination of TMB and specific tumor histology and genotype.

PURPOSE Many targeted therapies are currently available only via clinical trials. Therefore, routine precision oncology using biomarker-based assignment to drug depends on matching patients to clinical trials. A comprehensive and up-to-date trial database is necessary for optimal patient-trial matching. METHODS We describe processes for establishing and maintaining a clinical trial database, focusing on genomically informed trials. Furthermore, we present OCTANE (Oncology Clinical Trial Annotation Engine), an informatics framework supporting these processes in a scalable fashion. To illustrate how the framework can be applied at an institution, we describe how we implemented an instance of OCTANE at a large cancer center. OCTANE consists of three modules. The data aggregation module automates retrieval, aggregation, and update of trial information. The annotation module establishes the database schema, implements data integration necessary for automation, and provides an annotation interface. The update module monitors trial change logs, identifies critical change events, and alerts the annotators when manual intervention may be needed. RESULTS Using OCTANE, we annotated 5,439 oncology clinical trials (4,438 genomically informed trials) that collectively were associated with 1,453 drugs, 779 genes, and 252 cancer types. To date, we have used the database to screen 4,220 patients for trial eligibility. We compared the update module with expert review, and the module achieved 98.5% accuracy, 0% false-negative rate, and 2.3% false-positive rate. CONCLUSION OCTANE is a general informatics framework that can be helpful for establishing and maintaining a comprehensive database necessary for automating patient-trial matching, which facilitates the successful delivery of personalized cancer care on a routine basis. Several OCTANE components are publically available and may be useful to other precision oncology programs.

PURPOSE Smaller hotspot-based next-generation sequencing (NGS) panels have emerged to support standard of care therapy for patients with cancer. When standard treatments fail, it is unknown whether additional testing using an expanded panel of genes provides any benefit. The purpose of this study was to determine if larger sequencing panels that capture additional actionable genes, coupled with decision support, translates into treatment with matched therapy after frontline therapy has failed. PATIENTS AND METHODS A prospective protocol accrued 521 patients with a wide variety of refractory cancers. NGS testing using a 46- or 50-gene hotspot assay, then a 409-gene whole-exome assay, was sequentially performed in a Clinical Laboratory Improvement Amendments–certified clinical laboratory. A decision-support team annotated somatic alterations in clinically actionable genes for function and facilitated therapeutic matching. Survival and the impact of matched therapy use were determined by Kaplan-Meier estimate, log-rank test, and Cox proportional hazards regression. RESULTS The larger NGS panel identified at least one alteration in an actionable gene not previously identified in the smaller sequencing panel in 214 (41%) of 521 of enrolled patients. After the application of decision support, 41% of the alterations in actionable genes were considered to affect the function of the gene and were deemed actionable. Forty patients (40 of 214 [19%]) were subsequently treated with matched therapy. Treatment with matched therapy was associated with significantly improved overall survival compared with treatment with nonmatched therapy ( P = .017). CONCLUSION Combining decision support with larger NGS panels that incorporate genes beyond those recommended in current treatment guidelines helped to identify patients who were eligible for matched therapy while improving overall treatment selection and survival. This survival benefit was restricted to a small subset of patients.

Since the discovery that DNA alterations initiate tumorigenesis, scientists and clinicians have been exploring ways to counter these changes with targeted therapeutics. The sequencing of tumor DNA was initially limited to highly actionable hot spots—areas of the genome that are frequently altered and have an approved matched therapy in a specific tumor type. Large-scale genome sequencing programs quickly developed technological improvements that enabled the deployment of whole-exome and whole-genome sequencing technologies at scale for pristine sample materials in research environments. However, the turning point for precision medicine in oncology was the innovations in clinical laboratories that improved turnaround time, depth of coverage, and the ability to reliably sequence archived, clinically available samples. Today, tumor genome sequencing no longer suffers from significant technical or financial hurdles, and the next opportunity for improvement lies in the optimal utilization of the technologies and data for many different tumor types.

Malignant gliomas are a group of intracranial cancers associated with disproportionately high mortality and morbidity. Here, we report ultradeep targeted sequencing of a prospective cohort of 237 tumors from 234 patients consisting of both glioblastoma (GBM) and lower-grade glioma (LGG) using our customized gene panels. We identified 2,485 somatic mutations, including single-nucleotide substitutions and small indels, using a validated in-house protocol. Sixty-one percent of the mutations were contributed by 12 hypermutators. The hypermutators were enriched for recurrent tumors and had comparable outcome, and most were associated with temozolomide exposure. TP53 was the most frequently mutated gene in our cohort, followed by IDH1 and EGFR. We detected at least one EGFR mutation in 23% of LGGs, which was significantly higher than 6% seen in The Cancer Genome Atlas, a pattern that can be partially explained by the different patient composition and sequencing depth. IDH hotspot mutations were found with higher frequencies in LGG (83%) and secondary GBM (77%) than primary GBM (9%). Multivariate analyses controlling for age, histology, and tumor grade confirm the prognostic value of IDH mutation. We predicted 1p/19q status using the panel sequencing data and received only modest performance by benchmarking the prediction to FISH results of 50 tumors. Targeted therapy based on the sequencing data resulted in three responders out of 14 participants. In conclusion, our study suggests ultradeep targeted sequencing can recapitulate previous findings and can be a useful approach in the clinical setting.

PURPOSE In pancreatic cancer (PC), the RAF family alterations define a rare subset of patients that may predict response to inhibition of the BRAF/MEK/ERK signaling pathway. A comprehensive understanding of the molecular and clinical characteristics of RAF-mutated PC may support future development of RAF-directed strategies. METHODS Clinical outcomes were assessed across a multi-institutional case series of 81 patients with RAF family-mutated PC. Mutational subgroups were defined on the basis of RAF alteration hotspots and therapeutic implications. RESULTS The frequency of RAF alterations in PC was 2.2% (84 of 3,781) within a prevalence cohort derived from large molecular databases where BRAF V600E (Exon 15), BRAF ΔNVTAP (Exon 11), and SND1-BRAF fusions were the most common variants. In our retrospective case series, we identified 17 of 81 (21.0%) molecular profiles with a BRAF V600/Exon 15 mutation without any confounding drivers, 25 of 81 (30.9%) with BRAF or RAF1 fusions, and 18 of 81 (22.2%) with Exon 11 mutations. The remaining 21 of 81 (25.9%) profiles had atypical RAF variants and/or multiple oncogenic drivers. Clinical benefit from BRAF/MEK/ERK inhibitors was observed in 3 of 3 subjects within the V600 subgroup (two partial responses), 4 of 6 with fusions (two partial responses), 2 of 6 with Exon 11 mutations (one partial response), and 0 of 3 with confounding drivers. Outcomes analyses also suggested a trend favoring fluorouracil-based regimens over gemcitabine/nab-paclitaxel within the fusion subgroup (P = .027). CONCLUSION Prospective evaluation of RAF-directed therapies is warranted in RAF-mutated PC; however, differential responses to targeted agents or standard regimens for each mutational subgroup should be a consideration when designing clinical trials., In KRAS wild-type PDAC, certain RAF alterations predict benefit from map-kinase targeted therapy

Purpose: Ocular adnexal (OA) sebaceous carcinoma is an aggressive malignancy of the eyelid and ocular adnexa that frequently recurs and metastasizes, and effective therapies beyond surgical excision are lacking. There remains a critical need to define the molecular-genetic drivers of the disease to understand carcinomagenesis and progression and to devise novel treatment strategies. Experimental Design: We present next-generation sequencing of a targeted panel of cancer-associated genes in 42 and whole transcriptome RNA sequencing from eight OA sebaceous carcinomas from 29 patients. Results: We delineate two potentially distinct molecular-genetic subtypes of OA sebaceous carcinoma. The first is defined by somatic mutations impacting TP53 and/or RB1 [20/29 (70%) patients, including 10 patients whose primary tumors contained coexisting TP53 and RB1 mutations] with frequent concomitant mutations affecting NOTCH genes. These tumors arise in older patients and show frequent local recurrence. The second subtype [9/29 (31%) patients] lacks mutations affecting TP53, RB1, or NOTCH family members, but in 44% (4/9) of these tumors, RNA sequencing and in situ hybridization studies confirm transcriptionally active high-risk human papillomavirus. These tumors arise in younger patients and have not shown local recurrence. Conclusions: Together, our findings establish a potential molecular-genetic framework by which to understand the development and progression of OA sebaceous carcinoma and provide key molecular-genetic insights to direct the design of novel therapeutic interventions.

The American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE) is an international data-sharing consortium focused on enabling advances in precision oncology through the gathering and sharing of tumor genetic sequencing data linked with clinical data. The project’s history, operational structure, lessons learned, and institutional perspectives on participation in the data-sharing consortium are reviewed. Individuals involved with the inception and execution of AACR Project GENIE from each member institution described their experiences and lessons learned. The consortium was conceived in January 2014 and publicly released its first data set in January 2017, which consisted of 18,804 samples from 18,324 patients contributed by the eight founding institutions. Commitment and contributions from many individuals at AACR and the member institutions were crucial to the consortium’s success. These individuals filled leadership, project management, informatics, data curation, contracts, ethics, and security roles. Many lessons were learned during the first 3 years of the consortium, including on how to gather, harmonize, and share data; how to make decisions and foster collaboration; and how to set the stage for continued participation and expansion of the consortium. We hope that the lessons shared here will assist new GENIE members as well as others who embark on the journey of forming a genomic data–sharing consortium.

Background Rapid advances in science challenge the timely adoption of evidence-based care in community settings. To bridge the gap between what is possible and what is practiced, we researched approaches to developing an artificial intelligence (AI) application that can provide real-time patient-specific decision support. Materials and methods The Oncology Expert Advisor (OEA) was designed to simulate peer-to-peer consultation with three core functions: patient history summarization, treatment options recommendation, and management advisory. Machine-learning algorithms were trained to construct a dynamic summary of patients cancer history and to suggest approved therapy or investigative trial options. All patient data used were retrospectively accrued. Ground truth was established for approximately 1,000 unique patients. The full Medline database of more than 23 million published abstracts was used as the literature corpus. Results OEA's accuracies of searching disparate sources within electronic medical records to extract complex clinical concepts from unstructured text documents varied, with F1 scores of 90%-96% for non-time-dependent concepts (e.g., diagnosis) and F1 scores of 63%-65% for time-dependent concepts (e.g., therapy history timeline). Based on constructed patient profiles, OEA suggests approved therapy options linked to supporting evidence (99.9% recall; 88% precision), and screens for eligible clinical trials on ClinicalTrials.gov (97.9% recall; 96.9% precision). Conclusion Our results demonstrated technical feasibility of an AI-powered application to construct longitudinal patient profiles in context and to suggest evidence-based treatment and trial options. Our experience highlighted the necessity of collaboration across clinical and AI domains, and the requirement of clinical expertise throughout the process, from design to training to testing. Implications for practice Artificial intelligence (AI)-powered digital advisors such as the Oncology Expert Advisor have the potential to augment the capacity and update the knowledge base of practicing oncologists. By constructing dynamic patient profiles from disparate data sources and organizing and vetting vast literature for relevance to a specific patient, such AI applications could empower oncologists to consider all therapy options based on the latest scientific evidence for their patients, and help them spend less time on information "hunting and gathering" and more time with the patients. However, realization of this will require not only AI technology maturation but also active participation and leadership by clincial experts.

Purpose To determine the significant genomic alterations in patients with metastatic breast cancer (MBC) and survival outcomes in common genotypes. Patients and Methods High-depth next-generation sequencing was performed for 202 genes in tumor and normal DNA from 257 patients with MBC, including 165 with estrogen receptor/progesterone receptor–positive and human epidermal growth factor receptor 2 (HER2 [hormone receptor positive (HR+)])–positive, 32 with HER2-positive, and 60 with triple-negative (estrogen receptor/progesterone receptor–negative and HER2-negative) disease. Kaplan-Meier survival analysis was performed in the discovery set, in patients with breast cancer analyzed in The Cancer Genome Atlas, and in a separate cohort of 98 patients with MBC who underwent clinical genomic testing. Results Significantly mutated genes (SMGs) varied by histology and tumor subtype, but TP53 was an SMG in all three subtypes. The most SMGs in patients with HR+ cancer were PIK3CA (32%), TP53 (29%), GATA3 (15%), CDH1 (8%), MAP3K1 (8%), PTEN (5%), TGFBR2 (4%), AKT1 (4%), and MAP2K4 (4%). TP53 mutations were associated with shorter recurrence-free survival ( P = .004), progression-free survival ( P < .001), and overall survival ( P = .003). Furthermore, TP53 status was prognostic among patients with HR+ cancer with PIK3CA mutations. TP53 mutations were associated with poorer overall survival in the 442 patients with HR+ breast cancer analyzed in The Cancer Genome Atlas ( P = .042) and in an independent set of 96 patients with HR+ MBC who underwent clinical sequencing ( P < .001). Conclusion SMGs differ by tumor subtype, but TP53 is significantly mutated in all three breast cancer subtypes. TP53 mutations are associated with poor prognosis in HR+ breast cancer and should be considered in the design and interpretation of precision oncology trials.

// Vikas Dembla 1 , Neeta Somaiah 2 , Pedro Barata 3 , Kenneth Hess 4 , Siqing Fu 1 , Filip Janku 1 , Daniel D. Karp 1 , Aung Naing 1 , Sarina Anne Piha-Paul 1 , Vivek Subbiah 1 , Apostolia M. Tsimberidou 1 , Kenna Shaw 5 , Funda Meric-Bernstam 1 and David S. Hong 1 1 Department of Investigational Cancer Therapeutics (Phase 1 Program), The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 Department of Sarcoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Department of Solid Tumors, Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio, USA 4 Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 5 Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA Correspondence to: David S. Hong, email: dshong@mdanderson.org Keywords: MDM2 amplification; phase I trials; solid tumors; TP53 mutation; CDK4 amplification Received: January 04, 2018 Accepted: August 20, 2018 Published: September 04, 2018 ABSTRACT Background: TP53 is the most commonly mutated gene in cancer and codes for the best studied tumor suppressor, p53. MDM2 is involved in the negative regulation of p53 and itself serves as an oncogene, reported to be overexpressed in several cancer tumor types. In this retrospective study, we assessed the occurrence of MDM2 amplification among patients with various types of cancers and its association with clinical factors, other genetic aberrations, and response to targeted therapy in a phase I clinical trial setting. Methods: Samples from patients with advanced solid tumors who had been referred to the MD Anderson phase I clinical trials program between January 2011 and January 2016 were collected and analyzed for MDM2 amplification using FoundationOne’s genomic profiling assay. Patients whose tumors expressed MDM2 amplification were compared to those with tumors of the same histologic types without MDM2 amplification. Results: We tested tumors from 523 patients, of which 23 (4.4%) had MDM2 amplification. The highest prevalence of MDM2 amplification was in sarcoma (57%), breast cancer (13%) and bladder cancer (9%). Six patients with liposarcoma were treated on phase I protocol with an MDM2 inhibitor. The most common molecular aberrations co-occurring with MDM2 amplification was CDK4 amplification (70%). TP53 mutation was also detected in 7 patients (30%). Conclusion: MDM2 amplification was most commonly associated with liposarcoma. Concomitant alterations in additional genes such as CDK4 amplification and TP53 mutations, along with variable responses to targeted therapies including MDM2 inhibitors, suggest that further combinational studies are needed to target this population.

Well-differentiated/dedifferentiated liposarcoma is a common soft tissue sarcoma with approximately 1500 new cases per year. Surgery is the mainstay of treatment but recurrences are frequent and systemic options are limited. ‘Tumor genotyping’ is becoming more common in clinical practice as it offers the hope of personalized targeted therapy. We wanted to evaluate the results and the clinical utility of available next-generation sequencing panels in WD/DD liposarcoma. Patients who had their tumor sequenced by either FoundationOne (n = 13) or the institutional T200/T200.1 panels (n = 7) were included in this study. Significant copy number alterations were identified, but mutations were infrequent. Out of the 27 mutations detected in 7 samples, 8 (CTNNB1, MECOM, ZNF536, EGFR, EML4, CSMD3, PBRM1, PPP1R3A) were identified as deleterious (on Condel, PolyPhen and SIFT) and a truncating mutation was found in NF2. Of these, EGFR and NF2 are potential driver mutations and have not been reported previously in liposarcoma. MDM2 and CDK4 amplification was universally present in all the tested samples and multiple other recurrent genes with high amplification or high deletion were detected. Many of these targets are potentially actionable. Eight patients went on to receive an MDM2 inhibitor with a median time to progression of 23 months (95% CI: 10-83 months).

Purpose: Colorectal cancers are classified as right/left-sided based on whether they occur before/after the splenic flexure, with established differences in molecular subtypes and outcomes. However, it is unclear if this division is optimal and whether precise tumor location provides further information. Experimental Design: In 1,876 patients with colorectal cancer, we compared mutation prevalence and overall survival (OS) according to side and location. Consensus molecular subtype (CMS) was compared in a separate cohort of 608 patients. Results: Mutation prevalence differed by side and location for TP53, KRAS, BRAFV600, PIK3CA, SMAD4, CTNNB1, GNAS, and PTEN. Within left- and right-sided tumors, there remained substantial variations in mutation rates. For example, within right-sided tumors, RAS mutations decreased from 70% for cecal, to 43% for hepatic flexure location (P = 0.0001), while BRAFV600 mutations increased from 10% to 22% between the same locations (P < 0.0001). Within left-sided tumors, the sigmoid and rectal region had more TP53 mutations (P = 0.027), less PIK3CA (P = 0.0009), BRAF (P = 0.0033), or CTNNB1 mutations (P < 0.0001), and less MSI (P < 0.0001) than other left-sided locations. Despite this, a left/right division preceding the transverse colon maximized prognostic differences by side and transverse colon tumors had K-modes mutation clustering that appeared more left than right sided. CMS profiles showed a decline in CMS1 and CMS3 and rise in CMS2 prevalence moving distally. Conclusions: Current right/left classifications may not fully recapitulate regional variations in tumor biology. Specifically, the sigmoid-rectal region appears unique and the transverse colon is distinct from other right-sided locations. Clin Cancer Res; 24(5); 1062–72. ©2017 AACR. See related commentary by Dienstmann, p. 989

Context.— Optical imaging techniques are currently available for imaging tissues without the need for any type of extensive tissue preparation. There are several applications for their potential use in surgical pathology practice. Objective.— To evaluate the feasibility of using a confocal fluorescence microscopy (CFM) platform for ex vivo examination of tissues obtained from surgical resections of breast, lung, kidney, and liver. Design.— Tissue fragments (0.5–1.0 cm) were immersed in 0.6 mM acridine orange for 6 seconds and imaged using a CFM platform at a 488-nm wavelength. The imaged tissues were subsequently fixed in formalin and processed routinely to generate hematoxylin-eosin–stained tissue sections. Mosaics of the grayscale CFM images were studied at different magnifications for recognition of the tissue and were compared with conventional histopathologic examination of hematoxylin-eosin tissue sections. Results.— We imaged 55 tissue fragments obtained from 16 breast (29%), 18 lung (33%), 14 kidney (25%), and 7 liver (13%) surgical excision specimens. Acridine orange labeled the nuclei, creating the contrast between nucleus and cytoplasm and thereby recapitulating the tissue architecture. We could obtain CFM images of good quality within 5 to 10 minutes that allowed recognition of the cytomorphologic details for categorization of the imaged tissue and were similar to histologic examination of hematoxylin-eosin tissue sections. Conclusions.— The ease and speed of acquisition of CFM images together with the resolution and resemblance of the CFM images to hematoxylin-eosin sections suggest that the CFM platform has excellent potential for use in surgical pathology practice.

BACKGROUND Genomic testing is increasingly performed in oncology, but concerns remain regarding the clinician's ability to interpret results. In the current study, the authors sought to determine the agreement between physicians and genomic annotators from the Precision Oncology Decision Support (PODS) team at The University of Texas MD Anderson Cancer Center in Houston regarding actionability and the clinical use of test results. METHODS On a prospective protocol, patients underwent clinical genomic testing for hotspot mutations in 46 or 50 genes. Six months after sequencing, physicians received questionnaires for patients who demonstrated a variant in an actionable gene, investigating their perceptions regarding the actionability of alterations and clinical use of these findings. Genomic annotators independently classified these variants as actionable, potentially actionable, unknown, or not actionable. RESULTS Physicians completed 250 of 288 questionnaires (87% response rate). Physicians considered 168 of 250 patients (67%) as having an actionable alteration; of these, 165 patients (98%) were considered to have an actionable alteration by the PODS team and 3 were of unknown significance. Physicians were aware of genotype-matched therapy available for 119 patients (71%) and 48 of these 119 patients (40%) received matched therapy. Approximately 46% of patients in whom physicians regarded alterations as not actionable (36 of 79 patients) were classified as having an actionable/potentially actionable mutation by the PODS team. However, many of these were only theoretically actionable due to limited trials and/or therapies (eg, KRAS). CONCLUSIONS Physicians are aware of recurrent mutations in actionable genes on “hotspot” panels. As larger genomic panels are used, there may be a growing need for annotation of actionability. Decision support to increase awareness of genomically relevant trials and novel treatment options for recurrent mutations (eg, KRAS) also are needed. Cancer 2017. © 2017 American Cancer Society.

Squamous cell carcinoma of the anal canal (SCCA) is a rare gastrointestinal malignancy with an increasing annual incidence globally. The majority of cases are linked to prior infection with the human papillomavirus (HPV). For patients with metastatic SCCA, no consensus standard treatment exists. Identification of relevant targeted agents as novel therapeutic approaches for metastatic SCCA has been limited by a lack of comprehensive molecular profiling. We performed whole-exome sequencing on tumor–normal pairs from 24 patients with metastatic SCCA. Tumor tissue from 17 additional patients was analyzed using a 263-gene panel as a validation cohort. Gene expression profiling was performed on available frozen tissue to assess for differential expression patterns. Based on these findings, patient-derived xenograft (PDX) models of SCCA were generated to test targeted therapies against PI3K and EGFR. Despite a low mutation burden, mutations in PIK3CA, MLL2, and MLL3 were among the most commonly mutated genes. An association between TP53 mutations and HPV-negative SCCA tumors was observed. Gene expression analysis suggested distinct tumor subpopulations harboring PIK3CA mutations and for which HPV had integrated into the host genome. In vivo studies demonstrated improvement with anti-EGFR treatment. Gene mutation frequencies, tumor mutation burden, and gene expression patterns for metastatic SCCA appear similar to other HPV-associated malignancies. Implications: This first comprehensive genomic characterization for patients with metastatic SCCA provides further rationale for the integration of SCCA into the development of novel targeted therapies across HPV-related cancers. Mol Cancer Res; 15(11); 1542–50. ©2017 AACR.

We present the case of a 53-year-old male with metastatic rectal cancer who was treatment resistant to FOLFOX and FOLFOXIRI. Due to a Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, regorafenib was given in the third line setting. Surprisingly, the patient had a prolonged partial response that lasted 27 months. Mutational status was extensively evaluated to identify potential alterations that might play a role as predictive markers for this unusual event. A poorly characterized but nontransforming mutation in Fms-like tyrosine kinase 4 (FLT4) was present in the tumor. Prior to and at the time of clinical progression, we found amplification of fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR), loss of the FLT4 mutation, and gain of KIT proto-oncogene receptor tyrosine kinase (KIT) G961S suggesting potential roles in acquired resistance.

The tropomyosin receptor kinase (TRK) family includes TRKA, TRKB, and TRKC proteins, which are encoded by NTRK1, NTRK2 and NTRK3 genes, respectively. Binding of neurotrophins to TRK proteins induces receptor dimerization, phosphorylation, and activation of the downstream signaling cascades via PI3K, RAS/MAPK/ERK, and PLC-gamma. TRK pathway aberrations, including gene fusions, protein overexpression, and single nucleotide alterations, have been implicated in the pathogenesis of many cancer types, with NTRK gene fusions being the most well validated oncogenic events to date. Although the NTRK gene fusions are infrequent in most cancer types, certain rare tumor types are predominately driven by these events. Conversely, in more common histologies, such as lung and colorectal cancers, prevalence of the NTRK fusions is well below 5%. Selective inhibition of TRK signaling may therefore be beneficial among patients whose tumors vary in histologies, but share underlying oncogenic NTRK gene alterations. Currently, several TRK-targeting compounds are in clinical development. The ongoing Phase 2 trials with entrectinib and LOXO-101, two of the leading TRK inhibitors, are designed as 'basket trials', inclusive of patients whose tumors harbor NTRK gene fusions, independent of histology. Additional Phase 1 studies of other TRK inhibitors, including MGCD516, PLX7486, DS-6051b, and TSR-011, are underway. Interim data examining NTRK-rearranged tumors treated with entrectinib or LOXO-101 demonstrate encouraging activity, with patients achieving rapid and durable responses. Consequently, both drugs have achieved orphan designation from regulatory agencies, and efforts are underway to further expedite their development.

// Zhijie Wang 1, 6 , Sarina Piha-Paul 1 , Filip Janku 1 , Vivek Subbiah 1 , Naiyi Shi 1 , Jing Gong 1 , Chetna Wathoo 2 , Kenna Shaw 2 , Kenneth Hess 3 , Russell Broaddus 4 , Aung Naing 1 , David Hong 1 , Apostolia M. Tsimberidou 1 , Daniel Karp 1 , James Yao 5 , Funda Meric-Bernstam 1 and Siqing Fu 1 1 Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 Institute of Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 5 Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 6 National Cancer Center/Cancer Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China Correspondence to: Siqing Fu, email: siqingfu@mdanderson.org Keywords: KRAS, TP53, chronic inflammation, phase I trial, gene aberration-related therapy Received: October 15, 2016 Accepted: March 16, 2017 Published: April 05, 2017 ABSTRACT Purpose: Genetic alterations such as activating KRAS and/or inactivating TP53 are thought to be the most common drivers to tumorigenesis. Therefore, we assessed phase I cancer patients with KRAS+ / TP53+ mutations. Results: Approximately 8% of patients referred to phase I clinical trials harbored concurrent KRAS and TP53 mutations. Patients who received a phase I trial therapy (n = 57) had a median OS of 12 months, compared with 4.6 months in those who were not treated (n = 106; p = 0.003). KRAS G13 and TP53 R273 mutations were associated with poor overall survival (OS), while antiangiogenesis and gene aberration-related therapies were associated with prolonged OS. A prognostic model using neutrophilia, thrombocytosis, hypoalbuminemia, body mass index

This diagnostic study investigates real-time acquisition of fluorescence confocal microscopy of interventional radiology–guided specimens in the radiology suite and compares accuracy of diagnoses with hematoxylin-eosin (H&E)–stained sections among patients undergoing core-needle biopsy., Key Points Question Can fluorescence confocal microscopy be used for immediate evaluation of interventional radiology–guided core-needle biopsy in real time at the bedside in the radiology suite? Findings In this diagnostic study of 105 patients undergoing core-needle biopsy, fluorescence confocal microscopy images were acquired in a mean of 7 minutes, were similar to hematoxylin-eosin–stained tissue sections, and were of acceptable quality in 101 (96.2%). The accuracy of fluorescence confocal microscopy diagnosis compared with the criterion standard of hematoxylin-eosin–stained tissue sections was 95.2% for pathologist 1, 85.7% for pathologist 2, and 96.2% for consensus between them. Meaning These findings suggest that fluorescence confocal microscopy can be used for real-time bedside evaluation of interventional radiology–guided core-needle biopsy with high accuracy in a range of medical settings for procuring high-quality specimens in 1 hospital visit., Importance Strategies to procure high-quality core-needle biopsy (CNB) specimens are critical for making basic tissue diagnoses and for ancillary testing. Objectives To investigate acquisition of fluorescence confocal microscopy (FCM) images of interventional radiology (IR)–guided CNB in real time in the radiology suite and to compare the accuracy of FCM diagnoses with those of hematoxylin-eosin (H&E)–stained CNB sections. Design, Setting, and Participants In this diagnostic study, FCM imaging of IR-guided CNBs was performed in the radiology suite at a major cancer center for patients with an imaging abnormality from August 1, 2016, to April 30, 2019. The time taken to acquire FCM images and the quality of FCM images based on percentage of interpretable tissue with optimal resolution was recorded. The FCM images were read by 2 pathologists and categorized as nondiagnostic, benign/atypical, or suspicious/malignant; these diagnoses were compared with those made using H&E-stained tissue sections. Cases with discrepant diagnosis were reassessed by the pathologists together for a consensus diagnosis. Data were analyzed from June 3 to July 19, 2019. Interventions Each IR-guided CNB was stained with 0.6mM acridine orange, subjected to FCM imaging, and then processed to generate H&E-stained sections. Main Outcomes and Measures Mean time taken for acquisition of FCM images, quality of FCM images based on interpretable percentage of the image, and accuracy of diagnostic categorization based on FCM images compared with H&E-stained sections. Results A total of 105 patients (57 male [54.3%]; mean [SD] age, 63 [13] years) underwent IR-guided CNBs in a mean (SD) of 7 (2) minutes each. The FCM images showed at least 20% of the tissue with optimal quality in 101 CNB specimens (96.2%). The FCM images were accurately interpreted by the 2 pathologists in 100 of 105 cases (95.2%) (2 false-positive and 3 false-negative) and 90 of 105 cases (85.7%) (6 false-positive and 9 false-negative). A reassessment of 14 discordant diagnoses resulted in consensus diagnoses that were accurate in 101 of 105 cases (96.2%) (1 false-positive and 3 false-negative). Conclusions and Relevance The ease of acquisition of FCM images of acceptable quality and the high accuracy of the diagnoses suggest that FCM may be useful for rapid evaluation of IR-guided CNBs. This approach warrants further investigation.

Purpose: Molecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies. Experimental Design: We analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA). Results: A total of 153/206 (74.3%) were men; median age, 62 years (range, 18–91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1–13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%–55.03%). TP53 was the common altered gene [>120 alterations (non-unique)] followed by EGFR, MET, ARID1A, MYC, NF1, BRAF, and ERBB2 [20–38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in MYC, EGFR, ERBB2, BRAF, CCNE1, MET, PIK3CA, ARID1A, CDK6, and KRAS. In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have ERBB2 alterations, 35.7% (5/14) versus 8.8% HBV-negative (P = 0.04). Conclusions: This study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology.

PURPOSE Cell-free DNA (cfDNA) next-generation sequencing is a noninvasive approach for genomic testing. We report the frequency of identifying alterations and their clinical actionability in patients with advanced/metastatic cancer. PATIENTS AND METHODS Prospectively consented patients had cfDNA testing performed. Alterations were assessed for therapeutic implications. RESULTS We enrolled 575 patients with 37 tumor types. Of these patients, 438 (76.2%) had at least one alteration detected, and 205 (35.7%) had one or more alterations of high potential for clinical action. In diseases with 10 or more patients enrolled, 50% or more had at least one alteration deemed of high potential for clinical action. Trials were identified in 80% of patients (286 of 357) with any alteration and in 92% of patients (188 of 205) with one or more alterations of high potential for clinical action of whom 57.6% (118 of 205) had 6 or more months of follow-up available. Of these patients, 10% (12 of 118) had received genomically matched therapy through enrollment in clinical trials (n = 8), off-label drug use (n = 3), or standard of care (n = 1). Although 88.6% of all patients had a performance status of 0 or 1 upon enrollment, the primary reason for not acting on alterations was poor performance status at next treatment change (28.1%; 27 of 96). CONCLUSION cfDNA testing represents a readily accessible method for genomic testing and allows for detection of genomic alterations in most patients with advanced disease. Utility may be higher in patients interested in investigational therapeutics with adequate performance status. Additional study is needed to determine whether utility is enhanced by testing earlier in the treatment course.

Purpose: Biliary tract cancers (BTCs), including intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma (ECC), and gallbladder cancer (GBC), are rare malignancies which frequently present at advanced stage and have a poor survival. Incorporation of next-generation sequencing into clinical care has provided an avenue for the development of targeted therapies in BTCs which are rich in actionable mutations. However, clinically relevant models for BTC are limited, representing a major challenge in the field. We sought to develop a catalog of BTC patient-derived xenograft (PDX) models and representing diverse molecular profiles to create a resource for the modeling of precision oncology. Methods: Tumor tissue from surgical specimens or image-guided biopsies was collected from consenting patients at MD Anderson Cancer Center with histologically-confirmed BTCs for PDX development. Tumor fragments were implanted in the flank of NSG mice and passaged into nude mice. Whole-exome sequencing (WES) was performed on tumors from early-passage (P1-P2) PDXs and matching patient blood to determine somatic mutations/indels and copy number variations. Functional alterations in cancer-related genes that are targetable directly or indirectly with approved or investigational agents were considered “actionable”. Results: Samples were obtained from 97 patients undergoing image-guided biopsy (86/97, 89%) or surgical resection (11/97, 11%). Out of 97 tumors implanted, 33 (34%) PDXs from 30 patients were successfully established: 74% (24/33) ICC, 15% (5/33) ECC, and 12% (4/33) GBC. Relative take rates for PDX development were 34% (29/86) for those developed from biopsy samples and 36% (4/11) for those developed from surgical samples. In two patients, multiple PDXs were developed from biopsies at different time points during their care. WES demonstrated that the PDXs captured several key actionable alterations including alterations in ERRB2, FGFR2, IDH1, ATM, PIK3CA, PTEN and KRAS. Conclusion: Here, we describe a unique collection of clinically and molecularly annotated BTC PDX models which reflect the genomic heterogeneity present in ICC, ECC, and GBC. WES of early-passage PDXs provides evidence that these models capture the variety of actionable alterations harbored in the parental tumor and may enable the development of novel, biomarker-driven treatment strategies and rational combination therapies. To date, this is one of the largest collections of BTC PDX models available and will serve as an invaluable resource to guide the development of personalized treatments for patients with these aggressive malignancies. Citation Format: Timothy Philip DiPeri, Kurt W. Evans, Ching-Wei D. Tzeng, Lawrence Kwong, Michael P. Kahle, Xiaofeng Zheng, Dali Li, Hop S. Tran Cao, Thuy Vu, Sunhee Kim, Fei Su, Bryce Kirby, Chetna Wathoo, Gabriela Raso, Yasmin Rizvi, Huamin Wang, Filip Janku, Kenna Shaw, Timothy Yap, Milind Javle, Jordi Rodon, Funda Meric-Bernstam. The MD Anderson patient-derived xenograft series for modeling precision oncology in biliary tract cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2710.

// Natalia Paez Arango 1 , Lauren Brusco 2 , Kenna R. Mills Shaw 2 , Ken Chen 3 , Agda Karina Eterovic 4 , Vijaykumar Holla 2 , Amber Johnson 2 , Beate Litzenburger 2 , Yekaterina B. Khotskaya 2 , Nora Sanchez 2 , Ann Bailey 2 , Xiaofeng Zheng 3 , Chacha Horombe 2 , Scott Kopetz 5 , Carol J. Farhangfar 6 , Mark Routbort 7 , Russell Broaddus 8 , Elmer V. Bernstam 9,10 , John Mendelsohn 2,11 , Gordon B. Mills 2,4 and Funda Meric-Bernstam 1,2,12 1 Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5 Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 6 Levine Cancer Institute, Carolinas Healthcare System, Charlotte, NC, USA 7 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 8 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 9 School of Biomedical Informatics, The University of Texas Health Science Center at Houston, TX, USA 10 Division of General Internal Medicine, Medical School, The University of Texas Health Science Center at Houston, TX, USA 11 Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 12 Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Funda Meric-Bernstam, email: // Keywords : precision medicine, genomics, somatic mutation, CLIA, clinical trial Received : November 09, 2016 Accepted : February 27, 2017 Published : March 08, 2017 Abstract Purpose: Molecular profiling performed in the research setting usually does not benefit the patients that donate their tissues. Through a prospective protocol, we sought to determine the feasibility and utility of performing broad genomic testing in the research laboratory for discovery, and the utility of giving treating physicians access to research data, with the option of validating actionable alterations in the CLIA environment. Experimental design: 1200 patients with advanced cancer underwent characterization of their tumors with high depth hybrid capture sequencing of 201 genes in the research setting. Tumors were also tested in the CLIA laboratory, with a standardized hotspot mutation analysis on an 11, 46 or 50 gene platform. Results: 527 patients (44%) had at least one likely somatic mutation detected in an actionable gene using hotspot testing. With the 201 gene panel, 945 patients (79%) had at least one alteration in a potentially actionable gene that was undetected with the more limited CLIA panel testing. Sixty-four genomic alterations identified on the research panel were subsequently tested using an orthogonal CLIA assay. Of 16 mutations tested in the CLIA environment, 12 (75%) were confirmed. Twenty-five (52%) of 48 copy number alterations were confirmed. Nine (26.5%) of 34 patients with confirmed results received genotype-matched therapy. Seven of these patients were enrolled onto genotype-matched targeted therapy trials. Conclusion: Expanded cancer gene sequencing identifies more actionable genomic alterations. The option of CLIA validating research results can provide alternative targets for personalized cancer therapy.

The tools of next-generation sequencing (NGS) technology, such as targeted sequencing of candidate cancer genes and whole-exome and -genome sequencing, coupled with encouraging clinical results based on the use of targeted therapeutics and biomarker-guided clinical trials, are fueling further technological advancements of NGS technology. However, NGS data interpretation is associated with challenges that must be overcome to promote the techniques' effective integration into clinical oncology practice. Specifically, sequencing of a patient's tumor often yields 30–65 somatic variants, but most of these variants are “passenger” mutations that are phenotypically neutral and thus not targetable. Therefore, NGS data must be interpreted by multidisciplinary decision-support teams to determine mutation actionability and identify potential “drivers,” so that the treating physician can prioritize what clinical decisions can be pursued in order to provide cancer therapy that is personalized to the patient and his or her unique genome.

// Kanwal Raghav 1 , Van Morris 1 , Chad Tang 2 , Pia Morelli 1 , Hesham M. Amin 3 , Ken Chen 4 , Ganiraju C. Manyam 4 , Bradley Broom 4 , Michael J. Overman 1 , Kenna Shaw 5 , Funda Meric-Bernstam 6 , Dipen Maru 3 , David Menter 1 , Lee M. Ellis 7 , Cathy Eng 1 , David Hong 6 , Scott Kopetz 1 1 Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5 Sheikh Khalifa Bin Zayed Al Nahyan Institute of Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 6 Department of Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 7 Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Scott Kopetz, email: skopetz@mdanderson.org Keywords: amplification, MET, circulating-free DNA, fluorescence in situ hybridization, colorectal cancer Received: March 14, 2016 Accepted: May 05, 2016 Published: July 13, 2016 ABSTRACT Background: MET amplification appears to be a predictive biomarker for MET inhibition. Prior studies reported a MET amplification rate of 9–18% in metastatic colorectal cancer (mCRC) but do not differentiate increased gene copy numbers due to chromosomal level aberrations from focal gene amplifications. Validation of MET amplification rate in mCRC is critical to this field. Results: In tumor tissue-based analyses, overall MET amplification rate was 1.7% (10/590). MET amplification was seen in 0/103 (0%), 4/208 (1.9%) and 6/279 (2.2%) cases, in cohorts 1, 2 and 3, respectively. Rate of MET amplification in cfDNA of cohort 4 patients refractory to anti-EGFR therapy ( n = 53) was 22.6% (12/53) and was significantly higher compared to patients not exposed to anti-EGFR therapy ( p < 0.001). Materials and Methods: We analyzed MET amplification in mCRC ( n = 795) using different methods across multiple cohorts. Cohort 1 ( n = 103) and 2 ( n = 208) included resected liver metastases and tumor biopsies, respectively, tested for MET amplification using fluorescence in-situ hybridization [amplification: MET/CEP7 ratio ≥ 2.0]. Using another tissue-based approach, cohort 3 ( n = 279) included tumor biopsies sequenced with HiSeq (Illumina) with full exome coverage for MET [amplification: ≥ 4 copies identified by an in-house algorithm]. Using a blood-based approach by contrast, cohort 4 ( n = 205) included patients in whom the full exome of MET in circulating-free DNA (cfDNA) was sequenced with HiSeq. Conclusions: Contrary to prior reports, in this large cohort, MET amplification was a rare event in mCRC tissues. In plasma by stark contrast, MET amplification identified by cfDNA occurred in a sizable subset of patients that are refractory to anti-EGFR therapy.

We describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers.

Context.— Rapid advances in the fields of biophotonics, computer science, and instrumentation have allowed for high-resolution imaging of biologic tissues. Objective.— To evaluate the quality of images from an optimized confocal fluorescence microscopy (CFM) platform for rapid evaluation of small fragments of tissue, compared with hematoxylin-eosin staining. Design.— Tissue fragments (up to 1.0 × 0.3 cm) were stained with 0.6 mM acridine orange for 60 seconds and imaged using a CFM platform at 488-nm and 785-nm wavelength. The imaged tissues were then fixed in formalin and processed to generate hematoxylin-eosin–stained tissue sections. The quality of CFM images was scored on a scale of 0 to 3 on the basis of the percentage of the CFM images with recognizable tissue architecture (0, 0%; 1, 50%). The diagnoses made using CFM images were compared with those made using histopathologic analysis of the hematoxylin-eosin–stained tissue sections. Results.— We imaged 118 tissue fragments obtained from 40 breast, 23 lung, 39 kidney, and 16 liver surgical excision specimens. We acquired CFM images in 2 to 3 minutes; 95.8% (113 of 118) of images showed a quality score of 3, and 4.2% (5 of 118) had a score of 2. We achieved a sensitivity of 95.5%, specificity of 97.3%, positive predictive value of 95.5%, and negative predictive value of 97.3%. Conclusions.— Our results demonstrate the suitability of the CFM platform for rapid and accurate evaluation of small tissue fragments in surgical pathology practice.

SummaryInvasive lobular carcinoma (ILC) is the second most prevalent histologic subtype of invasive breast cancer. Here, we comprehensively profiled 817 breast tumors, including 127 ILC, 490 ductal (IDC), and 88 mixed IDC/ILC. Besides E-cadherin loss, the best known ILC genetic hallmark, we identified mutations targeting PTEN, TBX3, and FOXA1 as ILC enriched features. PTEN loss associated with increased AKT phosphorylation, which was highest in ILC among all breast cancer subtypes. Spatially clustered FOXA1 mutations correlated with increased FOXA1 expression and activity. Conversely, GATA3 mutations and high expression characterized luminal A IDC, suggesting differential modulation of ER activity in ILC and IDC. Proliferation and immune-related signatures determined three ILC transcriptional subtypes associated with survival differences. Mixed IDC/ILC cases were molecularly classified as ILC-like and IDC-like revealing no true hybrid features. This multidimensional molecular atlas sheds new light on the genetic bases of ILC and provides potential clinical options.

BACKGROUND: Diffuse low-grade and intermediate-grade gliomas (which together make up the lower-grade gliomas, World Health Organization grades II and III) have highly variable clinical behavior that is not adequately predicted on the basis of histologic class. Some are indolent; others quickly progress to glioblastoma. The uncertainty is compounded by interobserver variability in histologic diagnosis. Mutations in IDH, TP53, and ATRX and codeletion of chromosome arms 1p and 19q (1p/19q codeletion) have been implicated as clinically relevant markers of lower-grade gliomas. METHODS: We performed genomewide analyses of 293 lower-grade gliomas from adults, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA expression, microRNA expression, and targeted protein expression. These data were integrated and tested for correlation with clinical outcomes. RESULTS: Unsupervised clustering of mutations and data from RNA, DNA-copy-number, and DNA-methylation platforms uncovered concordant classification of three robust, nonoverlapping, prognostically significant subtypes of lower-grade glioma that were captured more accurately by IDH, 1p/19q, and TP53 status than by histologic class. Patients who had lower-grade gliomas with an IDH mutation and 1p/19q codeletion had the most favorable clinical outcomes. Their gliomas harbored mutations in CIC, FUBP1, NOTCH1, and the TERT promoter. Nearly all lower-grade gliomas with IDH mutations and no 1p/19q codeletion had mutations in TP53 (94%) and ATRX inactivation (86%). The large majority of lower-grade gliomas without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary glioblastoma. CONCLUSIONS: The integration of genomewide data from multiple platforms delineated three molecular classes of lower-grade gliomas that were more concordant with IDH, 1p/19q, and TP53 status than with histologic class. Lower-grade gliomas with an IDH mutation either had 1p/19q codeletion or carried a TP53 mutation. Most lower-grade gliomas without an IDH mutation were molecularly and clinically similar to glioblastoma. (Funded by the National Institutes of Health.)

The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.

Purpose Determine the characteristics of percutaneous core biopsies that are adequate for a next generation sequencing (NGS) genomic panel. Materials and methods All patients undergoing percutaneous core biopsies in interventional radiology (IR) with samples evaluated for a 46-gene NGS panel during 1-year were included in this retrospective study. Patient and procedure variables were collected. An imaging-based likelihood of adequacy score incorporating targeting and sampling factors was assigned to each biopsied lesion. Univariate and multivariate logistic regression was performed. Results 153 patients were included (58.2% female, average age 59.5 years). The most common malignancy was lung cancer (40.5%), most common biopsied site was lung (36%), and average size of biopsied lesions was 3.8 cm (+/- 2.7). Adequacy for NGS was 69.9%. Univariate analysis showed higher likelihood of adequacy score (p = 0.004), primary malignancy type (p = 0.03), and absence of prior systemic therapy (p = 0.018) were associated with adequacy for NGS. Multivariate analysis showed higher adequacy for lesions with likelihood of adequacy scored 3 (high) versus lesions scored 1 (low) (OR, 7.82; p = 0.002). Melanoma lesions had higher adequacy for NGS versus breast cancer lesions (OR 9.5; p = 0.01). Absence of prior systemic therapy (OR, 6.1; p = 0.02) and systemic therapy 3 months before biopsy yielded greater adequacy for NGS. Lesions

Precision oncology is not an illusion, nor is it the magic bullet that will eradicate all cancers. Precision oncology is simply another weapon in our growing armament against cancer. Rather than honing in on the failures of a relatively young field, one should advocate for integrating its successes into widespread clinical practice, especially for indications, such as: ABL, ALK, BRAF, BRCA1, BRCA2, EGFR, KIT, KRAS, PDGFRA, PDGFRB, ROS1, BCR-ABL, FLT3 and ROS1, where aberrations have been shown to alter responses to US FDA approved drugs – that is, level 1 data. Moreover, to truly assess the promise of precision oncology, we must first begin by defining our expectations for this field. Importantly, we must recognize that the conception of precision oncology arose as an antithesis of the ‘one-size fits all’ cancer therapeutics approach. Consequently, tools used for evaluating these conventional, large-scale trials, are not directly transferable for assessing nonconventional, smaller-scale trials needed for evaluating precision oncology. Hence, a thorough vetting of precision oncology as another tool of the trade, must first begin by reassessing our expectations for this field, as well as current clinical trial designs and end point measurements. Importantly, we must recognize that most targeted therapy approaches are in their infancy, with only monotherapy approaches being assessed and combination therapies likely being necessary to fulfill the promise of precision oncology.