T cell lymphoma and secondary primary malignancy risk after commercial CAR T cell therapy.

We report a T cell lymphoma (TCL) occurring 3 months after anti-CD19 chimeric antigen receptor (CAR) T cell immunotherapy for non-Hodgkin B cell lymphoma. The TCL was diagnosed from a thoracic lymph node upon surgery for lung cancer. The TCL exhibited CD8+ cytotoxic phenotype and a JAK3 variant, while the CAR transgene was very low. The T cell clone was identified at low levels in the blood before CAR T infusion and in lung cancer. To assess the overall risk of secondary primary malignancy after commercial CAR T (CD19, BCMA), we analyzed 449 patients treated at the University of Pennsylvania. At a median follow-up of 10.3 months, 16 patients (3.6%) had a secondary primary malignancy. The median onset time was 26.4 and 9.7 months for solid and hematological malignancies, respectively. The projected 5-year cumulative incidence is 15.2% for solid and 2.3% for hematological malignancies. Overall, one case of TCL was observed, suggesting a low risk of TCL after CAR T.

Molecular Neuropathology in Practice: Clinical Profiling and Integrative Analysis of Molecular Alterations in Glioblastoma.

Molecular profiling of glioblastoma has revealed complex cytogenetic, epigenetic, and molecular abnormalities that are necessary for diagnosis, prognosis, and treatment. Our neuro-oncology group has developed a data-driven, institutional consensus guideline for efficient and optimal workup of glioblastomas based on our routine performance of molecular testing. We describe our institution's testing algorithm, assay development, and genetic findings in glioblastoma, to illustrate current practices and challenges in neuropathology related to molecular and genetic testing. We have found that coordination of test requisition, tissue handling, and incorporation of results into the final pathologic diagnosis by the neuropathologist improve patient care. Here, we present analysis of O6-methylguanine-DNA-methyltransferase promoter methylation and next-generation sequencing results of 189 patients, obtained utilizing our internal processes led by the neuropathology team. Our institutional pathway for neuropathologist-driven molecular testing has streamlined the management of glioblastoma samples for efficient return of results for incorporation of genomic data into the pathological diagnosis and optimal patient care.

Identification and Confirmation of Potentially Actionable Germline Mutations in Tumor-Only Genomic Sequencing.

PURPOSE: Tumor-only genomic profiling (TGP) is increasingly advocated for all patients with cancer given the possible therapeutic implications. It is critical to develop clinical algorithms to identify and address potentially actionable germline findings identified by TGP. METHODS: A multidisciplinary team analyzed publicly available data for genes in which mutations are implicated in germline cancer susceptibility and established a pipeline to automate clinical referral for evaluation of TGP findings. RESULTS: A total of 2,308 patients underwent TGP, with 81 patients (3.5%) identified by the automatic referral pipeline; 37 patients (1.6%) were referred outside the pipeline based on concerns by the molecular geneticist, pathologist, or oncologist regarding genotype-phenotype correlation. Thirty-one patients (38%) and 17 patients (46%) underwent germline testing from the automatic pipeline and other referrals, respectively, and of these patients, 23 (72%) and four (24%) had confirmed germline pathogenic variants (GPVs), respectively. The majority of confirmed GPVs were in automatic referral genes, with BRCA2 being most common (confirmed GPVs in 11 [85%] of 13 patients tested), followed by PALB2 (five [67%] of six patients), BRCA1 (two [40%] of five patients), MSH6 (two of three patients), and MLH1 (two of two patients). Forty-eight percent of confirmed GPVs were found in tumors known to be associated with germline mutations in the gene. Germline testing was not performed in 50 (62%) of 81 patients identified by automatic referral as a result of poor patient health or death (30%), lack of follow-up (30%), and patient refusal (30%). CONCLUSION: Of patients undergoing TGP, 5% had somatic findings triggering referral, and implementation of an automatic referral pipeline based solely on gene versus other clinical or molecular features resulted in a 74% germline confirmation. However, only 41% of referred patients underwent germline testing. Systems-based approaches are needed to identify carriers of actionable germline cancer susceptibility mutations identified by TGP.

Clinical Implications of Plasma-Based Genotyping With the Delivery of Personalized Therapy in Metastatic Non-Small Cell Lung Cancer.

Importance: The clinical implications of adding plasma-based circulating tumor DNA next-generation sequencing (NGS) to tissue NGS for targetable mutation detection in non-small cell lung cancer (NSCLC) have not been formally assessed. Objective: To determine whether plasma NGS testing was associated with improved mutation detection and enhanced delivery of personalized therapy in a real-world clinical setting. Design, Setting, and Participants: This prospective cohort study enrolled 323 patients with metastatic NSCLC who had plasma testing ordered as part of routine clinical management. Plasma NGS was performed using a 73-gene commercial platform. Patients were enrolled at the Hospital of the University of Pennsylvania from April 1, 2016, through January 2, 2018. The database was locked for follow-up and analyses on January 2, 2018, with a median follow-up of 7 months (range, 1-21 months). Main Outcomes and Measures: The number of patients with targetable alterations detected with plasma and tissue NGS; the association between the allele fractions (AFs) of mutations detected in tissue and plasma; and the association of response rate with the plasma AF of the targeted mutations. Results: Among the 323 patients with NSCLC (60.1% female; median age, 65 years [range, 33-93 years]), therapeutically targetable mutations were detected in EGFR, ALK, MET, BRCA1, ROS1, RET, ERBB2, or BRAF for 113 (35.0%) overall. Ninety-four patients (29.1%) had plasma testing only at the discretion of the treating physician or patient preference. Among the 94 patients with plasma testing alone, 31 (33.0%) had a therapeutically targetable mutation detected, thus obviating the need for an invasive biopsy. Among the remaining 229 patients who had concurrent plasma and tissue NGS or were unable to have tissue NGS, a therapeutically targetable mutation was detected in tissue alone for 47 patients (20.5%), whereas the addition of plasma testing increased this number to 82 (35.8%). Thirty-six of 42 patients (85.7%) who received a targeted therapy based on the plasma result achieved a complete or a partial response or stable disease. The plasma-based targeted mutation AF had no correlation with depth of Response Evaluation Criteria in Solid Tumors response (r = -0.121; P = .45). Conclusions and Relevance: Integration of plasma NGS testing into the routine management of stage IV NSCLC demonstrates a marked increase of the detection of therapeutically targetable mutations and improved delivery of molecularly guided therapy.

Validation of a next-generation sequencing oncology panel optimized for low input DNA.

One caveat of next-generation sequencing (NGS)-based clinical oncology testing is the high amount of input DNA required. We sought to develop a focused NGS panel that could capture hotspot regions in relevant genes requiring 0.5-10 ng input DNA. The resulting Penn Precision Panel (PPP) targeted 20 genes containing clinically significant variants relevant to many cancers. One hundred twenty-three samples were analyzed, including 83 solid tumor specimens derived from FFPE. Various input quantities of DNA (0.5-10 ng) were amplified with content-specific PCR primer pools, then sequenced on a MiSeq instrument (Illumina, Inc.) via paired-end, 2 × 186 base pair reads to an average read depth of greater than 6500x. Variants were detected using an in-house analysis pipeline. Clinical sensitivity and specificity were assessed using results from our previously validated solid tumor NGS panel; sensitivity of the PPP is 96.75% (387/400 variants) and specificity is 99.9% (8427/8428 base pairs). Variant allele frequencies (VAFs) are highly concordant across both assays (r = 0.98 p < 0.0001). The PPP is a robust, clinically validated test optimized for low-yield solid tumor specimens, capturing a high percentage of clinically relevant variants found by larger commercially available NGS panels while using only 0.5-10 ng of input DNA.

Feasibility of monitoring advanced melanoma patients using cell-free DNA from plasma.

To determine the feasibility of liquid biopsy for monitoring of patients with advanced melanoma, cell-free DNA was extracted from plasma for 25 Stage III/IV patients, most (84.0%) having received previous therapy. DNA concentrations ranged from 0.6 to 390.0 ng/ml (median = 7.8 ng/ml) and were positively correlated with tumor burden as measured by imaging (Spearman rho = 0.5435, p = .0363). Using ultra-deep sequencing for a 61-gene panel, one or more mutations were detected in 12 of 25 samples (48.0%), and this proportion did not vary significantly for patients on or off therapy at the time of blood draw (52.9% and 37.5% respectively; p = .673). Sixteen mutations were detected in eight different genes, with the most frequent mutations detected in BRAF, NRAS, and KIT. Allele fractions ranged from 1.1% to 63.2% (median = 29.1%). Among patients with tissue next-generation sequencing, nine of 11 plasma mutations were also detected in matched tissue, for a concordance of 81.8%.

Building a Robust Tumor Profiling Program: Synergy between Next-Generation Sequencing and Targeted Single-Gene Testing.

Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (EGFR, KRAS, BRAF, NPM1, FLT3, and JAK2). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two FLT3 internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

A novel approach for next-generation sequencing of circulating tumor cells.

BACKGROUND: Next-generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low-input DNA available from rare CTCs. Moreover, whole genome amplification (WGA) approaches used to generate sufficient input DNA are often incompatible with blood collection tube preservatives used to facilitate clinical sample batching. METHODS: To address this, we have developed a novel approach combining tumor cell isolation from preserved blood with Repli-G WGA and Illumina TruSeq Amplicon Cancer Panel-based NGS. We purified cell pools ranging from 10 to 1000 cells from three different cell lines, and quantitatively demonstrate comparable quality of DNA extracted from preserved versus unpreserved samples. RESULTS: Preservation and WGA were compatible with the generation of high-quality libraries. Known point mutations and gene amplification were detected for libraries that had been prepared from amplified DNA from preserved blood. CONCLUSION: These spiking experiments provide proof of concept of a clinically applicable workflow for real-time monitoring of patient tumor using noninvasive liquid biopsies.

Detection of Therapeutically Targetable Driver and Resistance Mutations in Lung Cancer Patients by Next-Generation Sequencing of Cell-Free Circulating Tumor DNA.

PURPOSE: The expanding number of targeted therapeutics for non-small cell lung cancer (NSCLC) necessitates real-time tumor genotyping, yet tissue biopsies are difficult to perform serially and often yield inadequate DNA for next-generation sequencing (NGS). We evaluated the feasibility of using cell-free circulating tumor DNA (ctDNA) NGS as a complement or alternative to tissue NGS. EXPERIMENTAL DESIGN: A total of 112 plasma samples obtained from a consecutive study of 102 prospectively enrolled patients with advanced NSCLC were subjected to ultra-deep sequencing of up to 70 genes and matched with tissue samples, when possible. RESULTS: We detected 275 alterations in 45 genes, and at least one alteration in the ctDNA for 86 of 102 patients (84%), with EGFR variants being most common. ctDNA NGS detected 50 driver and 12 resistance mutations, and mutations in 22 additional genes for which experimental therapies, including clinical trials, are available. Although ctDNA NGS was completed for 102 consecutive patients, tissue sequencing was only successful for 50 patients (49%). Actionable EGFR mutations were detected in 24 tissue and 19 ctDNA samples, yielding concordance of 79%, with a shorter time interval between tissue and blood collection associated with increased concordance (P = 0.038). ctDNA sequencing identified eight patients harboring a resistance mutation who developed progressive disease while on targeted therapy, and for whom tissue sequencing was not possible. CONCLUSIONS: Therapeutically targetable driver and resistance mutations can be detected by ctDNA NGS, even when tissue is unavailable, thus allowing more accurate diagnosis, improved patient management, and serial sampling to monitor disease progression and clonal evolution. Clin Cancer Res; 22(23); 5772-82. ©2016 AACR.