Implementation of a prostate cancer-specific targeted sequencing panel for credentialing of patient-derived cell lines and genomic characterization of patient samples.

BACKGROUND: Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer-associated genes as an efficient approach to characterizing patient samples (compared to whole-exome and whole-genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost-effective method for genomic assessment of patient-derived cell lines to evaluate fidelity to initial patient tumor samples. METHODS: We developed a prostate cancer-specific targeted next-generation sequencing (NGS) panel to detect alterations in 62 prostate cancer-associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient-derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors. RESULTS: The prostate cancer-specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell-free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient-derived cell lines; however, no prostate cancer-specific mutations were identified in the tumor-derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells. CONCLUSIONS: We evaluated a prostate cancer-specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh-frozen tumors, cell-free DNA, CTCs, and cell lines. Targeted sequencing of patient-derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer-specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.

Genomic analyses of PMBL reveal new drivers and mechanisms of sensitivity to PD-1 blockade.

Primary mediastinal large B-cell lymphomas (PMBLs) are aggressive tumors that typically present as large mediastinal masses in young women. PMBLs share clinical, transcriptional, and molecular features with classical Hodgkin lymphoma (cHL), including constitutive activation of nuclear factor κB (NF-κB), JAK/STAT signaling, and programmed cell death protein 1 (PD-1)-mediated immune evasion. The demonstrated efficacy of PD-1 blockade in relapsed/refractory PMBLs led to recent approval by the US Food and Drug Administration and underscored the importance of characterizing targetable genetic vulnerabilities in this disease. Here, we report a comprehensive analysis of recurrent genetic alterations -somatic mutations, somatic copy number alterations, and structural variants-in a cohort of 37 newly diagnosed PMBLs. We identified a median of 9 genetic drivers per PMBL, including known and newly identified components of the JAK/STAT and NF-κB signaling pathways and frequent B2M alterations that limit major histocompatibility complex class I expression, as in cHL. PMBL also exhibited frequent, newly identified driver mutations in ZNF217 and an additional epigenetic modifier, EZH2. The majority of these alterations were clonal, which supports their role as early drivers. In PMBL, we identified several previously uncharacterized molecular features that may increase sensitivity to PD-1 blockade, including high tumor mutational burden, microsatellite instability, and an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutational signature. The shared genetic features between PMBL and cHL provide a framework for analyzing the mechanism of action of PD-1 blockade in these related lymphoid malignancies.

Duodenal-type and nodal follicular lymphomas differ by their immune microenvironment rather than their mutation profiles.

Duodenal-type follicular lymphoma (DTFL) is a rare and highly indolent follicular lymphoma (FL) variant. It is morphologically and immunophenotypically indistinguishable from typical FL, characterized by restricted involvement of intestinal mucosa, and lacks extraintestinal manifestations. The molecular determinants of this distinct clinical behavior are largely unknown. Thirty-eight diagnostic biopsies from patients with DTFL were evaluated. The 10-year overall survival rate was 100% in clinically evaluable patients (n = 19). We compared the targeted mutation profile of DTFL (n = 31), limited-stage typical FL (LSTFL; n = 17), and advanced-stage typical FL (ASTFL; n = 241). The mutation frequencies of recurrently mutated genes, including CREBBP, TNFRSF14/HVEM, and EZH2 were not significantly different. However, KMT2D was less commonly mutated in DTFL (52%) and LSTFL (24%) as compared with ASTFL (79%). In ASTFL, 41% of KMT2D-mutated cases harbored multiple mutations in KMT2D, as compared with only 12% in LSTFL (P = .019) and 0% in DTFL (P < .0001). Whole exome and targeted sequencing of DTFL revealed high mutation frequencies of EEF1A1 (35%) and HVCN1 (22%). We compared the immune microenvironment gene expression signatures of DTFL (n = 8) and LSTFL (n = 7). DTFL clearly separated from LSTFL by unsupervised, hierarchical clustering of 147 chemokines and cytokines and was enriched for a chronic inflammation signature. In conclusion, the mutational landscape of DTFL is highly related to typical FL. The lower frequency of multiple mutations in KMT2D in DTFL and LSTFL indicates an increasing selection pressure for complete KMT2D loss in ASTFL pathogenesis. The highly dissimilar immune microenvironment of DTFL suggests a central role in the biology of this disease.

Detection of Mutations in Barrett's Esophagus Before Progression to High-Grade Dysplasia or Adenocarcinoma.

BACKGROUND & AIMS: Barrett's esophagus (BE) is the greatest risk factor for esophageal adenocarcinoma (EAC), but only a small proportion of patients with BE develop cancer. Biomarkers might be able to identify patients at highest risk of progression. We investigated genomic differences in surveillance biopsies collected from patients whose BE subsequently progressed compared to patients whose disease did not progress. METHODS: We performed a retrospective case-control study of 24 patients with BE that progressed to high-grade dysplasia (HGD, n = 14) or EAC (n = 10). The control group (n = 73, called non-progressors) comprised patients with BE and at least 5 years of total endoscopic biopsy surveillance without progression to HGD or EAC. From each patient, we selected a single tissue sample obtained more than 1 year before progression (cases) or more than 2 years before the end of follow-up (controls). Pathogenic mutations, gene copy numbers, and ploidy were compared between samples from progressors and non-progressors. RESULTS: TP53 mutations were detected in 46% of samples from progressors and 5% of non-progressors. In this case-control sample set, TP53 mutations in BE tissues increased the adjusted risk of progression 13.8-fold (95% confidence interval, 3.2-61.0) (P < .001). We did not observe significant differences in ploidy or copy-number profile between groups. We identified 147 pathogenic mutations in 57 distinct genes-the average number of pathogenic mutations was higher in samples from progressors (n = 2.5) than non-progressors (n = 1.2) (P < .001). TP53 and other somatic mutations were recurrently detected in samples with limited copy-number changes (aneuploidy). CONCLUSIONS: In genomic analyses of BE tissues from patients with or without later progression to HGD or EAC, we found significantly higher numbers of TP53 mutations in BE from patients with subsequent progression. These mutations were frequently detected before the onset of dysplasia or substantial changes in copy number.

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

PURPOSE: Germline 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. METHODS: Using 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. RESULTS: We 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). CONCLUSION: Nearly 10% of PDAC patients harbor germline variants, although the majority lack somatic second hits, the therapeutic significance of which warrants further study.

Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing.

BACKGROUND: Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. RESULTS: Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. CONCLUSIONS: For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications.

Clinical and mutational spectrum of highly differentiated, paired box 3:forkhead box protein o1 fusion-negative rhabdomyosarcoma: A report from the Children's Oncology Group.

BACKGROUND: Pediatric paired box 3:forkhead box protein O1 fusion-negative (PF-) rhabdomyosarcoma (RMS) represents a diverse spectrum of tumors with marked differences in histology, myogenic differentiation, and clinical behavior. METHODS: This study sought to evaluate the clinical and mutational spectrum of 24 pediatric PF- human RMS tumors with high levels of myogenic differentiation. Tumors were sequenced with OncoPanel v.2, a panel consisting of the coding regions of 504 genes previously linked to human cancer. RESULTS: Most of the tumors (19 of 24) arose at head/neck or genitourinary sites, and the overall survival rate was 100% with a median follow-up time of 4.6 years (range, 1.4-8.6 years). RAS pathway gene mutations were the most common mutations in PF-, highly differentiated RMS tumors. In addition, Hedgehog (Hh) and mechanistic target of rapamycin (mTOR) gene mutations with evidence for functional relevance (high-impact) were identified in subsets of tumors. The presence of Hh and mTOR pathway gene mutations was mutually exclusive and was associated with high-impact RAS pathway gene mutations in 3 of 4 Hh-mutated tumors and in 1 of 6 mTOR-mutated tumors. CONCLUSIONS: Interestingly, Hh and mTOR gene mutations were previously associated with rhabdomyomas, which are also known to preferentially arise at head/neck and genitourinary sites. Findings from this study further support the idea that PF-, highly differentiated RMS tumors and rhabdomyomas may represent a continuous spectrum of tumors. Cancer 2018;124:1973-81. © 2018 American Cancer Society.

Diffuse large B-cell lymphoma patient-derived xenograft models capture the molecular and biological heterogeneity of the disease.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition.

Targetable genetic features of primary testicular and primary central nervous system lymphomas.

Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) are extranodal large B-cell lymphomas (LBCLs) with inferior responses to current empiric treatment regimens. To identify targetable genetic features of PCNSL and PTL, we characterized their recurrent somatic mutations, chromosomal rearrangements, copy number alterations (CNAs), and associated driver genes, and compared these comprehensive genetic signatures to those of diffuse LBCL and primary mediastinal large B-cell lymphoma (PMBL). These studies identify unique combinations of genetic alterations in discrete LBCL subtypes and subtype-selective bases for targeted therapy. PCNSLs and PTLs frequently exhibit genomic instability, and near-uniform, often biallelic, CDKN2A loss with rare TP53 mutations. PCNSLs and PTLs also use multiple genetic mechanisms to target key genes and pathways and exhibit near-uniform oncogenic Toll-like receptor signaling as a result of MYD88 mutation and/or NFKBIZ amplification, frequent concurrent B-cell receptor pathway activation, and deregulation of BCL6. Of great interest, PCNSLs and PTLs also have frequent 9p24.1/PD-L1/PD-L2 CNAs and additional translocations of these loci, structural bases of immune evasion that are shared with PMBL.

BreaKmer: detection of structural variation in targeted massively parallel sequencing data using kmers.

Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for 'targeted' resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a 'kmer' strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings.

Somatic activating ARAF mutations in Langerhans cell histiocytosis.

The extracellular signal-regulated kinase (ERK) signaling pathway is activated in Langerhans cell histiocytosis (LCH) histiocytes, but only 60% of cases carry somatic activating mutations of BRAF. To identify other genetic causes of ERK pathway activation, we performed whole exome sequencing on purified LCH cells in 3 cases. One patient with wild-type BRAF alleles in his histiocytes had compound mutations in the kinase domain of ARAF. Unlike wild-type ARAF, this mutant was a highly active mitogen-activated protein kinase kinase in vitro and was capable of transforming mouse embryo fibroblasts. Mutant ARAF activity was inhibited by vemurafenib, a BRAF inhibitor, indicating the importance of fully evaluating ERK pathway abnormalities in selecting LCH patients for targeted inhibitor therapy.

Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factor MYBL1.

Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

MAP2K1 and MAP3K1 mutations in Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic activating mutations of BRAF. However, the extracellular signal-related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a targeted massively parallel sequencing panel to 30 LCH samples to test for the presence of additional genetic alterations that might cause ERK pathway activation. In 20 BRAF wild type samples, we found 3 somatic mutations in MAP2K1 (MEK1) including C121S and C121S/G128D in the kinase domain, and 56_61QKQKVG>R, an in-frame deletion in the N-terminal regulatory domain. All three variant proteins constitutively phosphorylated ERK in in vitro kinase assays. The C121S/G128D and 56_61QKQKVG>R variants were resistant to the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib in vitro. Within the entire sample set, we found 3 specimens with mutations in MAP3K1 (MEKK1), including two truncation mutants, T779fs and T1481fs; T1481fs encoded an unstable and nonfunctional protein when expressed in vitro. T779fs was present in a specimen carrying BRAF V600E. The third variant was a single nucleotide substitution, E1286V, which was fully functional and is likely a germline polymorphism. These results indicate that LCH cells can harbor additional genetic alterations in the RAS-RAF-MEK pathway which, in the case of MAP2K1, may be responsible for ERK activation in a wild type BRAF setting. The resistance of some of these variants to trametinib may also have clinical implications for the combined use of RAF and MEK inhibitors in LCH.

Integration of gene mutations in risk prognostication for patients receiving first-line immunochemotherapy for follicular lymphoma: a retrospective analysis of a prospective clinical trial and validation in a population-based registry.

BACKGROUND: Follicular lymphoma is a clinically and genetically heterogeneous disease, but the prognostic value of somatic mutations has not been systematically assessed. We aimed to improve risk stratification of patients receiving first-line immunochemotherapy by integrating gene mutations into a prognostic model. METHODS: We did DNA deep sequencing to retrospectively analyse the mutation status of 74 genes in 151 follicular lymphoma biopsy specimens that were obtained from patients within 1 year before beginning immunochemotherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These patients were recruited between May 4, 2000, and Oct 20, 2010, as part of a phase 3 trial (GLSG2000). Eligible patients had symptomatic, advanced stage follicular lymphoma and were previously untreated. The primary endpoints were failure-free survival (defined as less than a partial remission at the end of induction, relapse, progression, or death) and overall survival calculated from date of treatment initiation. Median follow-up was 7·7 years (IQR 5·5-9·3). Mutations and clinical factors were incorporated into a risk model for failure-free survival using multivariable L1-penalised Cox regression. We validated the risk model in an independent population-based cohort of 107 patients with symptomatic follicular lymphoma considered ineligible for curative irradiation. Pretreatment biopsies were taken between Feb 24, 2004, and Nov 24, 2009, within 1 year before beginning first-line immunochemotherapy consisting of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP). Median follow-up was 6·7 years (IQR 5·7-7·6). FINDINGS: We established a clinicogenetic risk model (termed m7-FLIPI) that included the mutation status of seven genes (EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), the Follicular Lymphoma International Prognostic Index (FLIPI), and Eastern Cooperative Oncology Group (ECOG) performance status. In the training cohort, m7-FLIPI defined a high-risk group (28%, 43/151) with 5-year failure-free survival of 38·29% (95% CI 25·31-57·95) versus 77·21% (95% CI 69·21-86·14) for the low-risk group (hazard ratio [HR] 4·14, 95% CI 2·47-6·93; p<0·0001; bootstrap-corrected HR 2·02), and outperformed a prognostic model of only gene mutations (HR 3·76, 95% CI 2·10-6·74; p<0·0001; bootstrap-corrected HR 1·57). The positive predictive value and negative predictive value for 5-year failure-free survival were 64% and 78%, respectively, with a C-index of 0·80 (95% CI 0·71-0·89). In the validation cohort, m7-FLIPI again defined a high-risk group (22%, 24/107) with 5-year failure-free survival of 25·00% (95% CI 12·50-49·99) versus 68·24% (58·84-79·15) in the low-risk group (HR 3·58, 95% CI 2·00-6·42; p<0.0001). The positive predictive value for 5-year failure-free survival was 72% and 68% for negative predictive value, with a C-index of 0·79 (95% CI 0·69-0·89). In the validation cohort, risk stratification by m7-FLIPI outperformed FLIPI alone (HR 2·18, 95% CI 1·21-3·92), and FLIPI combined with ECOG performance status (HR 2·03, 95% CI 1·12-3·67). INTERPRETATION: Integration of the mutational status of seven genes with clinical risk factors improves prognostication for patients with follicular lymphoma receiving first-line immunochemotherapy and is a promising approach to identify the subset at highest risk of treatment failure. FUNDING: Deutsche Krebshilfe, Terry Fox Research Institute.

Genomic aberrations in cervical adenocarcinomas in Hong Kong Chinese women.

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole-exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

Molecular Characterization and Therapeutic Targeting of Colorectal Cancers Harboring Receptor Tyrosine Kinase Fusions.

PURPOSE: Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion-associated colorectal cancer. EXPERIMENTAL DESIGN: We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results. RESULTS: We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair-deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E-mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy. CONCLUSIONS: RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target.

Dissection of PIK3CA Aberration for Cervical Adenocarcinoma Outcomes.

Personalized treatment of genetically stratified subgroups has the potential to improve outcomes in many malignant tumors. This study distills clinically meaningful prognostic/predictive genomic marker for cervical adenocarcinoma using signature genomic aberrations and single-point nonsynonymous mutation-specific droplet digital PCR (ddPCR). Mutations in PIK3CA E542K, E545K, or H1047R were detected in 41.7% of tumors. PIK3CA mutation detected in the patient's circulating DNA collected before treatment or during follow-up was significantly associated with decreased progression-free survival or overall survival. PIK3CA mutation in the circulating DNA during follow-up after treatment predicted recurrence with 100% sensitivity and 64.29% specificity. It is the first indication of the predictive power of PIK3CA mutations in cervical adenocarcinoma. The work contributes to the development of liquid biopsies for follow up surveillance and a possibility of tailoring management of this particular women's cancer.

OncoTree: A Cancer Classification System for Precision Oncology.

PURPOSE: Cancer classification is foundational for patient care and oncology research. Systems such as International Classification of Diseases for Oncology (ICD-O), Systematized Nomenclature of Medicine Clinical Terms (SNOMED-CT), and National Cancer Institute Thesaurus (NCIt) provide large sets of cancer classification terminologies but they lack a dynamic modernized cancer classification platform that addresses the fast-evolving needs in clinical reporting of genomic sequencing results and associated oncology research. METHODS: To meet these needs, we have developed OncoTree, an open-source cancer classification system. It is maintained by a cross-institutional committee of oncologists, pathologists, scientists, and engineers, accessible via an open-source Web user interface and an application programming interface. RESULTS: OncoTree currently includes 868 tumor types across 32 organ sites. OncoTree has been adopted as the tumor classification system for American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE), a large genomic and clinical data-sharing consortium, and for clinical molecular testing efforts at Memorial Sloan Kettering Cancer Center and Dana-Farber Cancer Institute. It is also used by precision oncology tools such as OncoKB and cBioPortal for Cancer Genomics. CONCLUSION: OncoTree is a dynamic and flexible community-driven cancer classification platform encompassing rare and common cancers that provides clinically relevant and appropriately granular cancer classification for clinical decision support systems and oncology research.

ViroPanel: Hybrid Capture and Massively Parallel Sequencing for Simultaneous Detection and Profiling of Oncogenic Virus Infection and Tumor Genome.

Precision cancer medicine aims to classify tumors by site, histology, and molecular testing to determine an individualized profile of cancer alterations. Viruses are a major contributor to oncogenesis, causing 12% to 20% of all human cancers. Several viruses are causal of specific types of cancer, promoting dysregulation of signaling pathways and resulting in carcinogenesis. In addition, integration of viral DNA into the host (human) genome is a hallmark of some viral species. Tests for the presence of viral infection used in the clinical setting most often use quantitative PCR or immunohistochemical staining. Both approaches have limitations and need to be interpreted/scored appropriately. In some cases, results are not binary (virus present/absent), and it is unclear what to do with a weakly or partially positive result. In addition, viral testing of cancers is performed separately from tests to detect human genomic alterations and can thus be time-consuming and use limited valuable specimen. We present a hybrid-capture and massively parallel sequencing approach to detect viral infection that is integrated with targeted genomic analysis to provide a more complete tumor profile from a single sample.

Insulin-Like Growth Factor-1 Receptor Expression and Disease Recurrence and Survival in Patients with Resected Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Insulin-like growth factor-1 receptor (IGF1R) signaling is important in pancreatic ductal adenocarcinoma (PDAC) biology, but little is known regarding IGF1R expression and patient characteristics and outcomes. METHODS: In 365 patients with resected PDAC, we evaluated IGF1R protein expression using IHC on whole-slide sections and IGF1R genomic status using next-generation sequencing. Associations of IGF1R expression, measured by H-scores incorporating staining intensity and proportion of positive tumor cells, with disease-free survival (DFS) and overall survival (OS) were evaluated in 317 and 321 patients, respectively, using Cox regression adjusting for known prognostic factors. RESULTS: Higher IGF1R expression in tumor cells was associated with worse DFS comparing highest versus lowest expression tertiles [median DFS, 10.8 vs. 16.1 months; adjusted hazard ratio (HR), 1.73; 95% confidence interval (CI), 1.24-2.44; P trend = 0.002] and worse OS (median OS, 17.4 vs. 25.8 months; HR, 1.39; 95% CI, 1.00-1.92; P trend = 0.046). The association between high IGF1R expression and reduced DFS was identified primarily among patients with a preoperative body mass index ≥25 kg/m2 (HR, 4.27; 95% CI, 2.03-8.96, comparing extreme tertiles; P interaction = 0.032). KRAS-mutant tumors had greater IGF1R expression, and IGF1R expression in tumor epithelium was inversely correlated with that in stromal cells. Mutations in IGF1R were infrequent, and no overt loss-of-function alterations were identified. Higher IGF1R expression was modestly associated with higher gene copy number (Pearson correlation coefficient = 0.26, P < 0.001). CONCLUSIONS: Higher IGF1R protein expression was associated with worse patient outcomes in resected PDAC. IMPACT: IGF1R expression in PDAC represents a potential biomarker to guide patient selection for more aggressive, multidrug regimens in the adjuvant setting.