Neoadjuvant Nivolumab plus Chemotherapy in Resectable Lung Cancer.

BACKGROUND: Neoadjuvant or adjuvant chemotherapy confers a modest benefit over surgery alone for resectable non-small-cell lung cancer (NSCLC). In early-phase trials, nivolumab-based neoadjuvant regimens have shown promising clinical activity; however, data from phase 3 trials are needed to confirm these findings. METHODS: In this open-label, phase 3 trial, we randomly assigned patients with stage IB to IIIA resectable NSCLC to receive nivolumab plus platinum-based chemotherapy or platinum-based chemotherapy alone, followed by resection. The primary end points were event-free survival and pathological complete response (0% viable tumor in resected lung and lymph nodes), both evaluated by blinded independent review. Overall survival was a key secondary end point. Safety was assessed in all treated patients. RESULTS: The median event-free survival was 31.6 months (95% confidence interval [CI], 30.2 to not reached) with nivolumab plus chemotherapy and 20.8 months (95% CI, 14.0 to 26.7) with chemotherapy alone (hazard ratio for disease progression, disease recurrence, or death, 0.63; 97.38% CI, 0.43 to 0.91; P = 0.005). The percentage of patients with a pathological complete response was 24.0% (95% CI, 18.0 to 31.0) and 2.2% (95% CI, 0.6 to 5.6), respectively (odds ratio, 13.94; 99% CI, 3.49 to 55.75; P<0.001). Results for event-free survival and pathological complete response across most subgroups favored nivolumab plus chemotherapy over chemotherapy alone. At the first prespecified interim analysis, the hazard ratio for death was 0.57 (99.67% CI, 0.30 to 1.07) and did not meet the criterion for significance. Of the patients who underwent randomization, 83.2% of those in the nivolumab-plus-chemotherapy group and 75.4% of those in the chemotherapy-alone group underwent surgery. Grade 3 or 4 treatment-related adverse events occurred in 33.5% of the patients in the nivolumab-plus-chemotherapy group and in 36.9% of those in the chemotherapy-alone group. CONCLUSIONS: In patients with resectable NSCLC, neoadjuvant nivolumab plus chemotherapy resulted in significantly longer event-free survival and a higher percentage of patients with a pathological complete response than chemotherapy alone. The addition of nivolumab to neoadjuvant chemotherapy did not increase the incidence of adverse events or impede the feasibility of surgery. (Funded by Bristol Myers Squibb; CheckMate 816 ClinicalTrials.gov number, NCT02998528.).

Plain language summary of the CheckMate 816 study results: nivolumab plus chemotherapy given before surgery for non-small-cell lung cancer.

WHAT IS THIS SUMMARY ABOUT?: In this article, we summarize results from the ongoing phase 3 CheckMate 816 clinical study that were published in The New England Journal of Medicine in 2022. The goal of CheckMate 816 was to find out if nivolumab, an immunotherapy that activates a person's immune system (the body's natural defense system) to fight cancer, plus chemotherapy works better than chemotherapy alone when given before surgery in people with non-small-cell lung cancer (NSCLC) that can be removed surgically (resectable NSCLC). WHAT HAPPENED IN THE STUDY?: Adults who had not previously taken medications to treat NSCLC and whose cancer could be removed with surgery were included in CheckMate 816. During this study, a computer randomly assigned the treatment each person would receive before surgery for NSCLC. In total, 179 people were randomly assigned to receive nivolumab plus chemotherapy, and 179 people were randomly assigned to receive chemotherapy alone. The researchers assessed whether people who received nivolumab plus chemotherapy lived longer without the cancer geting worse or coming back and whether there were any cancer cells left in the tumor and lymph nodes removed by surgery. The researchers also assessed how adding nivolumab to chemotherapy affected the timing and outcomes of surgery and whether the combination of these drugs was safe. WHAT WERE THE RESULTS?: Researchers found that people who took nivolumab plus chemotherapy lived longer without the cancer getting worse or coming back compared with those who took chemotherapy alone. More people in the nivolumab plus chemotherapy group had no cancer cells left in the tumor and lymph nodes removed by surgery. Most people went on to have surgery in both treatment groups; the people who took nivolumab plus chemotherapy instead of chemotherapy alone had less extensive surgeries and were more likely to have good outcomes after less extensive surgeries. Adding nivolumab to chemotherapy did not lead to an increase in the rate of side effects compared with chemotherapy alone, and side effects were generally mild and manageable. WHAT DO THE RESULTS OF THE STUDY MEAN?: Results from CheckMate 816 support the benefit of using nivolumab plus chemotherapy before surgery for people with resectable NSCLC. Clinical Trial Registration: NCT02998528 (ClinicalTrials.gov).

Homologous Recombination Deficiency: Concepts, Definitions, and Assays.

BACKGROUND: Homologous recombination deficiency (HRD) is a phenotype that is characterized by the inability of a cell to effectively repair DNA double-strand breaks using the homologous recombination repair (HRR) pathway. Loss-of-function genes involved in this pathway can sensitize tumors to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy, which target the destruction of cancer cells by working in concert with HRD through synthetic lethality. However, to identify patients with these tumors, it is vital to understand how to best measure homologous repair (HR) status and to characterize the level of alignment in these measurements across different diagnostic platforms. A key current challenge is that there is no standardized method to define, measure, and report HR status using diagnostics in the clinical setting. METHODS: Friends of Cancer Research convened a consortium of project partners from key healthcare sectors to address concerns about the lack of consistency in the way HRD is defined and methods for measuring HR status. RESULTS: This publication provides findings from the group's discussions that identified opportunities to align the definition of HRD and the parameters that contribute to the determination of HR status. The consortium proposed recommendations and best practices to benefit the broader cancer community. CONCLUSION: Overall, this publication provides additional perspectives for scientist, physician, laboratory, and patient communities to contextualize the definition of HRD and various platforms that are used to measure HRD in tumors.

The genomic landscape of response to EGFR blockade in colorectal cancer.

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.

Clinical study of genomic drivers in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer with complex genomes and dense fibrotic stroma. This study was designed to identify clinically relevant somatic aberrations in pancreatic cancer genomes of patients with primary and metastatic disease enrolled and treated in two clinical trials. METHODS: Tumour nuclei were flow sorted prior to whole genome copy number variant (CNV) analysis. Targeted or whole exome sequencing was performed on most samples. We profiled biopsies from 68 patients enrolled in two Stand Up to Cancer (SU2C)-sponsored clinical trials. These included 38 resected chemoradiation naïve tumours (SU2C 20206-003) and metastases from 30 patients who progressed on prior therapies (SU2C 20206-001). Patient outcomes including progression-free survival (PFS) and overall survival (OS) were observed. RESULTS: We defined: (a) CDKN2A homozygous deletions that included the adjacent MTAP gene, only its' 3' region, or excluded MTAP; (b) SMAD4 homozygous deletions that included ME2; (c) a pancreas-specific MYC super-enhancer region; (d) DNA repair-deficient genomes; and (e) copy number aberrations present in PDA patients with long-term (⩾ 40 months) and short-term (⩽ 12 months) survival after surgical resection. CONCLUSIONS: We provide a clinically relevant framework for genomic drivers of PDA and for advancing novel treatments.

Advancing Evidence Generation for Circulating Tumor DNA: Lessons Learned from A Multi-Assay Study of Baseline Circulating Tumor DNA Levels across Cancer Types and Stages.

Circulating tumor DNA (ctDNA) holds promise as a biomarker for predicting clinical responses to therapy in solid tumors, and multiple ctDNA assays are in development. However, the heterogeneity in ctDNA levels prior to treatment (baseline) across different cancer types and stages and across ctDNA assays has not been widely studied. Friends of Cancer Research formed a collaboration across multiple commercial ctDNA assay developers to assess baseline ctDNA levels across five cancer types in early- and late-stage disease. This retrospective study included eight commercial ctDNA assay developers providing summary-level de-identified data for patients with non-small cell lung cancer (NSCLC), bladder, breast, prostate, and head and neck squamous cell carcinoma following a common analysis protocol. Baseline ctDNA levels across late-stage cancer types were similarly detected, highlighting the potential use of ctDNA as a biomarker in these cancer types. Variability was observed in ctDNA levels across assays in early-stage NSCLC, indicative of the contribution of assay analytical performance and methodology on variability. We identified key data elements, including assay characteristics and clinicopathological metadata, that need to be standardized for future meta-analyses across multiple assays. This work facilitates evidence generation opportunities to support the use of ctDNA as a biomarker for clinical response.

Liquid biopsy approaches to capture tumor evolution and clinical outcomes during cancer immunotherapy.

Circulating cell-free tumor DNA (ctDNA) can serve as a real-time biomarker of tumor burden and provide unique insights into the evolving molecular landscape of cancers under the selective pressure of immunotherapy. Tracking the landscape of genomic alterations detected in ctDNA may reveal the clonal architecture of the metastatic cascade and thus improve our understanding of the molecular wiring of therapeutic responses. While liquid biopsies may provide a rapid and accurate evaluation of tumor burden dynamics during immunotherapy, the complexity of antitumor immune responses is not fully captured through single-feature ctDNA analyses. This underscores a need for integrative studies modeling the tumor and the immune compartment to understand the kinetics of tumor clearance in association with the quality of antitumor immune responses. Clinical applications of ctDNA testing in patients treated with immune checkpoint inhibitors have shown both predictive and prognostic value through the detection of genomic biomarkers, such as tumor mutational burden and microsatellite instability, as well as allowing for real-time monitoring of circulating tumor burden and the assessment of early on-therapy responses. These efforts highlight the emerging role of liquid biopsies in selecting patients for cancer immunotherapy, monitoring therapeutic efficacy, determining the optimal duration of treatment and ultimately guiding treatment selection and sequencing. The clinical translation of liquid biopsies is propelled by the increasing number of ctDNA-directed interventional clinical trials in the immuno-oncology space, signifying a critical step towards implementation of liquid biopsies in precision immuno-oncology.

RNA Sequencing Identifies Novel NRG1 Fusions in Solid Tumors that Lack Co-Occurring Oncogenic Drivers.

NRG1 gene fusions are rare, therapeutically relevant, oncogenic drivers that occur across solid tumor types. To understand the landscape of NRG1 gene fusions, 4397 solid tumor formalin-fixed, paraffin-embedded samples consecutively tested by comprehensive genomic and immune profiling during standard care were analyzed. Nineteen NRG1 fusions were found in 17 unique patients, across multiple tumor types, including non-small-cell lung (n = 7), breast (n = 2), colorectal (n = 3), esophageal (n = 2), ovarian (n = 1), pancreatic (n = 1), and unknown primary (n = 1) carcinomas, with a cumulative incidence of 0.38%. Fusions were identified with breakpoints across four NRG1 introns spanning 1.4 megabases, with a mixture of known (n = 8) and previously unreported (n = 11) fusion partners. Co-occurring driver alterations in tumors with NRG1 fusions were uncommon, except colorectal carcinoma, where concurrent alterations in APC, BRAF, and ERBB2 were present in a subset of cases. The overall lack of co-occurring drivers highlights the importance of identifying NRG1 gene fusions, as these patients are unlikely to harbor other targetable alterations. In addition, RNA sequencing is important to identify NRG1 gene fusions given the variety of fusion partners and large genomic areas where breakpoints can occur.

Validation of the Labcorp Plasma Focus Test to Facilitate Precision Oncology Through Cell-Free DNA Genomic Profiling of Solid Tumors.

Genomic profiling is critical for precision oncology to guide treatment decisions. Liquid biopsy testing is a complementary approach to tissue testing, particularly when tissue is not readily available. The Labcorp Plasma Focus test is a circulating cell-free DNA genomic profiling test that identifies actionable variants in solid cancers, including non-small-cell lung, colorectal, melanoma, breast, esophageal, gastroesophageal junction, and gastric cancers. This study highlights the analytical validation of the test, including accuracy compared with orthogonal methods, as well as sensitivity, specificity, precision, reproducibility, and repeatability. Concordance with orthogonal methods showed percent positive agreement of 98.7%, 89.3%, and 96.2% for single nucleotide variants (SNVs), insertion/deletions (indels), and copy number amplifications (CNAs), respectively, and 100.0% for translocations and microsatellite instability (MSI). Analytical sensitivity revealed a median limit of detection of 0.7% and 0.6% for SNVs and indels, 1.4-fold for CNAs, 0.5% variant allele frequency for translocations, and 0.6% for MSI. Specificity was >99% for SNVs/indels and 100% for CNAs, translocations, and MSI. Average positive agreement from precision, reproducibility, and repeatability experiments was 97.5% and 88.9% for SNVs/indels and CNAs, and 100% for translocations and MSI. Taken together, these data show that the Labcorp Plasma Focus test is a highly accurate, sensitive, and specific approach for cell-free DNA genomic profiling to supplement tissue testing and inform treatment decisions.

ctDNA response after pembrolizumab in non-small cell lung cancer: phase 2 adaptive trial results.

Circulating tumor DNA (ctDNA) has shown promise in capturing primary resistance to immunotherapy. BR.36 is a multi-center, randomized, ctDNA-directed, phase 2 trial of molecular response-adaptive immuno-chemotherapy for patients with lung cancer. In the first of two independent stages, 50 patients with advanced non-small cell lung cancer received pembrolizumab as standard of care. The primary objectives of stage 1 were to ascertain ctDNA response and determine optimal timing and concordance with radiologic Response Evaluation Criteria in Solid Tumors (RECIST) response. Secondary endpoints included the evaluation of time to ctDNA response and correlation with progression-free and overall survival. Maximal mutant allele fraction clearance at the third cycle of pembrolizumab signified molecular response (mR). The trial met its primary endpoint, with a sensitivity of ctDNA response for RECIST response of 82% (90% confidence interval (CI): 52-97%) and a specificity of 75% (90% CI: 56.5-88.5%). Median time to ctDNA response was 2.1 months (90% CI: 1.5-2.6), and patients with mR attained longer progression-free survival (5.03 months versus 2.6 months) and overall survival (not reached versus 7.23 months). These findings are incorporated into the ctDNA-driven interventional molecular response-adaptive second stage of the BR.36 trial in which patients at risk of progression are randomized to treatment intensification or continuation of therapy. ClinicalTrials.gov ID: NCT04093167 .

Metastatic Colorectal Cancer Treatment Response Evaluation by Ultra-Deep Sequencing of Cell-Free DNA and Matched White Blood Cells.

PURPOSE: Circulating tumor DNA (ctDNA) has the potential to guide therapy selection and monitor treatment response in patients with metastatic cancer. However, germline and clonal hematopoiesis-associated alterations can confound identification of tumor-specific mutations in cell-free DNA (cfDNA), often requiring additional sequencing of tumor tissue. The current study assessed whether ctDNA-based treatment response monitoring could be performed in a tumor tissue-independent manner by combining ultra-deep targeted sequencing analyses of cfDNA with patient-matched white blood cell (WBC)-derived DNA. EXPERIMENTAL DESIGN: In total, 183 cfDNA and 49 WBC samples, along with 28 tissue samples, from 52 patients with metastatic colorectal cancer participating in the prospective phase III CAIRO5 clinical trial were analyzed using an ultra-deep targeted sequencing liquid biopsy assay. RESULTS: The combined cfDNA and WBC analysis prevented false-positives due to germline or hematopoietic variants in 40% of patients. Patient-matched tumor tissue sequencing did not provide additional information. Longitudinal analyses of ctDNA were more predictive of overall survival than standard-of-care radiological response evaluation. ctDNA mutations related to primary or acquired resistance to panitumumab were identified in 42% of patients. CONCLUSIONS: Accurate calling of ctDNA mutations for treatment response monitoring is feasible in a tumor tissue-independent manner by combined cfDNA and patient-matched WBC genomic DNA analysis. This tissue biopsy-independent approach simplifies sample logistics and facilitates the application of liquid biopsy ctDNA testing for evaluation of emerging therapy resistance, opening new avenues for early adaptation of treatment regimens.

Automated next-generation profiling of genomic alterations in human cancers.

The lack of validated, distributed comprehensive genomic profiling assays for patients with cancer inhibits access to precision oncology treatment. To address this, we describe elio tissue complete, which has been FDA-cleared for examination of 505 cancer-related genes. Independent analyses of clinically and biologically relevant sequence changes across 170 clinical tumor samples using MSK-IMPACT, FoundationOne, and PCR-based methods reveals a positive percent agreement of >97%. We observe high concordance with whole-exome sequencing for evaluation of tumor mutational burden for 307 solid tumors (Pearson r = 0.95) and comparison of the elio tissue complete microsatellite instability detection approach with an independent PCR assay for 223 samples displays a positive percent agreement of 99%. Finally, evaluation of amplifications and translocations against DNA- and RNA-based approaches exhibits >98% negative percent agreement and positive percent agreement of 86% and 82%, respectively. These methods provide an approach for pan-solid tumor comprehensive genomic profiling with high analytical performance.

Changes in Circulating Tumor DNA Reflect Clinical Benefit Across Multiple Studies of Patients With Non-Small-Cell Lung Cancer Treated With Immune Checkpoint Inhibitors.

PURPOSE: As immune checkpoint inhibitors (ICI) become increasingly used in frontline settings, identifying early indicators of response is needed. Recent studies suggest a role for circulating tumor DNA (ctDNA) in monitoring response to ICI, but uncertainty exists in the generalizability of these studies. Here, the role of ctDNA for monitoring response to ICI is assessed through a standardized approach by assessing clinical trial data from five independent studies. PATIENTS AND METHODS: Patient-level clinical and ctDNA data were pooled and harmonized from 200 patients across five independent clinical trials investigating the treatment of patients with non-small-cell lung cancer with programmed cell death-1 (PD-1)/programmed death ligand-1 (PD-L1)-directed monotherapy or in combination with chemotherapy. CtDNA levels were measured using different ctDNA assays across the studies. Maximum variant allele frequencies were calculated using all somatic tumor-derived variants in each unique patient sample to correlate ctDNA changes with overall survival (OS) and progression-free survival (PFS). RESULTS: We observed strong associations between reductions in ctDNA levels from on-treatment liquid biopsies with improved OS (OS; hazard ratio, 2.28; 95% CI, 1.62 to 3.20; P < .001) and PFS (PFS; hazard ratio 1.76; 95% CI, 1.31 to 2.36; P < .001). Changes in the maximum variant allele frequencies ctDNA values showed strong association across different outcomes. CONCLUSION: In this pooled analysis of five independent clinical trials, consistent and robust associations between reductions in ctDNA and outcomes were found across multiple end points assessed in patients with non-small-cell lung cancer treated with an ICI. Additional tumor types, stages, and drug classes should be included in future analyses to further validate this. CtDNA may serve as an important tool in clinical development and an early indicator of treatment benefit.

BRCA1 E1644X: a deleterious mutation in an African American individual with early onset breast cancer.

An African American individual with early onset breast cancer has a unique BRCA1 germline mutation, E1644X, that truncates the protein's carboxy terminal region. DNA sequencing for E1644X mutation and five BRCA1 exon-11 single nucleotide polymorphisms showed tumor LOH. Clinical history suggests paternal transmission of the deleterious allele, and tumor polymorphisms provide some insight into the ancestral origins of the mutation.

Noninvasive Detection of Microsatellite Instability and High Tumor Mutation Burden in Cancer Patients Treated with PD-1 Blockade.

PURPOSE: Microsatellite instability (MSI) and high tumor mutation burden (TMB-High) are promising pan-tumor biomarkers used to select patients for treatment with immune checkpoint blockade; however, real-time sequencing of unresectable or metastatic solid tumors is often challenging. We report a noninvasive approach for detection of MSI and TMB-High in the circulation of patients. EXPERIMENTAL DESIGN: We developed an approach that utilized a hybrid-capture-based 98-kb pan-cancer gene panel, including targeted microsatellite regions. A multifactorial error correction method and a novel peak-finding algorithm were established to identify rare MSI frameshift alleles in cell-free DNA (cfDNA). RESULTS: Through analysis of cfDNA derived from a combination of healthy donors and patients with metastatic cancer, the error correction and peak-finding approaches produced a specificity of >99% (n = 163) and sensitivities of 78% (n = 23) and 67% (n = 15), respectively, for MSI and TMB-High. For patients treated with PD-1 blockade, we demonstrated that MSI and TMB-High in pretreatment plasma predicted progression-free survival (hazard ratios: 0.21 and 0.23, P = 0.001 and 0.003, respectively). In addition, we analyzed cfDNA from longitudinally collected plasma samples obtained during therapy to identify patients who achieved durable response to PD-1 blockade. CONCLUSIONS: These analyses demonstrate the feasibility of noninvasive pan-cancer screening and monitoring of patients who exhibit MSI or TMB-High and have a high likelihood of responding to immune checkpoint blockade.See related commentary by Wang and Ajani, p. 6887.

A machine learning approach for somatic mutation discovery.

Variability in the accuracy of somatic mutation detection may affect the discovery of alterations and the therapeutic management of cancer patients. To address this issue, we developed a somatic mutation discovery approach based on machine learning that outperformed existing methods in identifying experimentally validated tumor alterations (sensitivity of 97% versus 90 to 99%; positive predictive value of 98% versus 34 to 92%). Analysis of paired tumor-normal exome data from 1368 TCGA (The Cancer Genome Atlas) samples using this method revealed concordance for 74% of mutation calls but also identified likely false-positive and false-negative changes in TCGA data, including in clinically actionable genes. Determination of high-quality somatic mutation calls improved tumor mutation load-based predictions of clinical outcome for melanoma and lung cancer patients previously treated with immune checkpoint inhibitors. Integration of high-quality machine learning mutation detection in clinical next-generation sequencing (NGS) analyses increased the accuracy of test results compared to other clinical sequencing analyses. These analyses provide an approach for improved identification of tumor-specific mutations and have important implications for research and clinical management of cancer patients.

Whole-Exome Sequencing of Salivary Gland Mucoepidermoid Carcinoma.

PURPOSE: Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. To explore the genetic origins of MEC, we performed systematic genomic analyses of these tumors. EXPERIMENTAL DESIGN: Whole-exome sequencing and gene copy-number analyses were performed for 18 primary cancers with matched normal tissue. FISH was used to determine the presence or absence of the MECT1-MAML2 translocation in 17 tumors. RESULTS: TP53 was the most commonly mutated gene in MEC (28%), and mutations were found only in intermediate- and high-grade tumors. Tumors with TP53 mutations had more mutations overall than tumors without TP53 mutations (P = 0.006). POU6F2 was the second most frequently mutated gene, found in three low-grade MECs with the same in-frame deletion. Somatic alterations in IRAK1, MAP3K9, ITGAL, ERBB4, OTOGL, KMT2C, and OBSCN were identified in at least two of the 18 tumors sequenced. FISH analysis confirmed the presence of the MECT1-MAML2 translocation in 15 of 17 tumors (88%). CONCLUSIONS: Through these integrated genomic analyses, MECT1-MAML2 translocation and somatic TP53 and POU6F2 mutations appear to be the main drivers of MEC. Clin Cancer Res; 23(1); 283-8. ©2016 AACR.

The Effect of Preservative and Temperature on the Analysis of Circulating Tumor DNA.

Purpose: Analysis of genomic alterations in cell-free DNA (cfDNA) is evolving as an approach to detect, monitor, and genotype malignancies. Methods to separate the liquid from the cellular fraction of whole blood for circulating tumor DNA (ctDNA) analyses have been largely unstudied, although these may be a critical consideration for assay performance.Experimental Design: To evaluate the influence of blood processing on cfDNA and ctDNA quality and yield, we compared the cfDNA levels in serum with those in plasma. Given the limitations of serum for ctDNA analyses, we evaluated the effects of two plasma processing approaches, K2EDTA and Cell-Free DNA BCT (BCT) tubes, on cfDNA and ctDNA recovery. A total of 45 samples from nine patients with cancer were collected in both tube types. Once collected, blood was processed into plasma immediately or kept at room temperature and processed into plasma at 1, 3, 5, or 7 days.Results: As early as 24 hours after collection, plasma isolated from blood collected in K2EDTA tubes contained an elevated level of cfDNA that increased over time compared with BCT tubes where no significant increase in cfDNA levels was observed. When samples from an additional six patients with cancer, collected in the same manner, were stored at 4°C in K2EDTA tubes over the course of 3 days, total cfDNA and ctDNA levels were comparable between samples collected in BCT tubes. At day 3, there was a trend toward a decrease in ctDNA levels in both tubes that was more pronounced when measuring the mutant allele fraction for cases stored at 4°C in K2EDTA tubes.Conclusions: In summary, methods of blood processing have a strong influence on cfDNA and ctDNA levels and should be a consideration when evaluating ctDNA in peripheral circulation. Clin Cancer Res; 23(10); 2471-7. ©2016 AACR.

Direct detection of early-stage cancers using circulating tumor DNA.

Early detection and intervention are likely to be the most effective means for reducing morbidity and mortality of human cancer. However, development of methods for noninvasive detection of early-stage tumors has remained a challenge. We have developed an approach called targeted error correction sequencing (TEC-Seq) that allows ultrasensitive direct evaluation of sequence changes in circulating cell-free DNA using massively parallel sequencing. We have used this approach to examine 58 cancer-related genes encompassing 81 kb. Analysis of plasma from 44 healthy individuals identified genomic changes related to clonal hematopoiesis in 16% of asymptomatic individuals but no alterations in driver genes related to solid cancers. Evaluation of 200 patients with colorectal, breast, lung, or ovarian cancer detected somatic mutations in the plasma of 71, 59, 59, and 68%, respectively, of patients with stage I or II disease. Analyses of mutations in the circulation revealed high concordance with alterations in the tumors of these patients. In patients with resectable colorectal cancers, higher amounts of preoperative circulating tumor DNA were associated with disease recurrence and decreased overall survival. These analyses provide a broadly applicable approach for noninvasive detection of early-stage tumors that may be useful for screening and management of patients with cancer.

Genomic and Immunological Tumor Profiling Identifies Targetable Pathways and Extensive CD8+/PDL1+ Immune Infiltration in Inflammatory Breast Cancer Tumors.

Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer that remains poorly understood at the molecular level. Comprehensive tumor profiling was performed to understand clinically actionable alterations in IBC. Targeted next-generation sequencing (NGS) and IHC were performed to identify activated pathways in IBC tumor tissues. siRNA studies examined the impact of IBC genomic variants in cellular models. IBC tumor tissues were further characterized for immune infiltration and immune checkpoint expression by IHC. Genomic analysis identified recurrent alterations in core biologic pathways, including activating and targetable variants in HER/PI3K/mTOR signaling. High rates of activating HER3 point mutations were discovered in IBC tumors. Cell line studies confirmed a role for mutant HER3 in IBC cell proliferation. Immunologic analysis revealed a subset of IBC tumors associated with high CD8(+)/PD-L1(+) lymphocyte infiltration. Immune infiltration positively correlated with an NGS-based estimate of neoantigen exposure derived from the somatic mutation rate and mutant allele frequency, iScore. Additionally, DNA mismatch repair alterations, which may contribute to higher iScores, occurred at greater frequency in tumors with higher immune infiltration. Our study identifies genomic alterations that mechanistically contribute to oncogenic signaling in IBC and provides a genetic basis for the selection of clinically relevant targeted and combination therapeutic strategies. Furthermore, an NGS-based estimate of neoantigen exposure developed in this study (iScore) may be a useful biomarker to predict immune infiltration in IBC and other cancers. The iScore may be associated with greater levels of response to immunotherapies, such as PD-L1/PD-1-targeted therapies. Mol Cancer Ther; 15(7); 1746-56. ©2016 AACR.