Search

Observed genome equivalents, wild-type DNA fragments, mutant DNA fragments, and mutant allele frequency in K2EDTA tubes at 4{degree sign}C and Streck{trade mark, serif} BCT tubes at room temperature.

Average DNA Yield and Range of Plasma Isolated in Healthy Donors vs. Diseased Donors

Schematic of sample collection and analyses for the comparative studies between K2EDTA (EDTA) and Streck{trade mark, serif} BCT (Streck{trade mark, serif}) tubes.

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.

Summary of clinical characteristics, plasma volume and DNA yield in 15 cancer patients.

Next-Generation Sequencing Performance of Plasma Isolated in Different Blood Collection Tubes

Science attempts to be a meritocracy; however, in recent years, there has been increasing evidence for systematic gender bias against women. This bias is present in many metrics commonly used to evaluate scientific productivity, which in turn influences hiring and career success. Here we explore a new metric, the Altmetric Attention Score, and find no evidence of bias across many major journals (Nature, PNAS, PLOS One, New England Journal of Medicine, Cell, and BioRxiv), with equal attention afforded to articles authored by men and women alike. The exception to this rule is the journal Science, which has marked gender bias against women in 2018, equivalent to a mean of 88 more tweets or 11 more news articles and a median of 20 more tweets or 3 more news articles for male than female first authors. Our findings qualify Altmetric, for many types and disciplines of journals, as a potentially unbiased measure of science communication in academia and suggest that new technologies, such as those on which Altmetric is based, might help to democratize academic evaluation.

Background Several predictive biomarkers are currently approved or are under investigation for the selection of patients for checkpoint blockade. Tumor PD-L1 expression is used for stratification of non-small cell lung (NSCLC) patients, with tumor mutational burden (TMB) also being explored with promising results, and mismatch-repair deficiency is approved for tumor site-agnostic disease. While tumors with high PD-L1 expression, high TMB, or mismatch repair deficiency respond well to checkpoint blockade, tumors with lower PD-L1 expression, lower mutational burdens, or mismatch repair proficiency respond much less frequently. Case presentation We studied two patients with unexpected responses to checkpoint blockade monotherapy: a patient with PD-L1-negative and low mutational burden NSCLC and one with mismatch repair proficient colorectal cancer (CRC), both of whom lack the biomarkers associated with response to checkpoint blockade, yet achieved durable clinical benefit. Both maintained T-cell responses in peripheral blood to oncogenic driver mutations – BRAF-N581I in the NSCLC and AKT1-E17K in the CRC – years after treatment initiation. Mutation-specific T cells were also found in the primary tumor and underwent dynamic perturbations in the periphery upon treatment. Conclusions These findings suggest that T cell responses to oncogenic driver mutations may be more prevalent than previously appreciated and could be harnessed in immunotherapeutic treatment, particularly for patients who lack the traditional biomarkers associated with response. Comprehensive studies are warranted to further delineate additional predictive biomarkers and populations of patients who may benefit from checkpoint blockade. Electronic supplementary material The online version of this article (10.1186/s40425-018-0492-x) contains supplementary material, which is available to authorized users.

Purpose: Approximately 10% of patients with mismatch repair–proficient (MMRp) colorectal cancer showed clinical benefit to anti-PD-1 monotherapy (NCT01876511). We sought to identify biomarkers that delineate patients with immunoreactive colorectal cancer and to explore new combinatorial immunotherapy strategies that can impact MMRp colorectal cancer. Experimental Design: We compared the expression of 44 selected immune-related genes in the primary colon tumor of 19 patients with metastatic colorectal cancer (mCRC) who responded (n = 13) versus those who did not (n = 6) to anti-PD-1 therapy (NCT01876511). We define a 10 gene–based immune signature that could distinguish responder from nonresponder. Resected colon specimens (n = 14) were used to validate the association of the predicted status (responder and nonresponder) with the immune-related gene expression, the phenotype, and the function of tumor-infiltrating lymphocytes freshly isolated from the same tumors. Results: Although both IL17Low and IL17High immunoreactive MMRp colorectal cancers are associated with intratumor correlates of adaptive immunosuppression (CD8/IFNγ and PD-L1/IDO1 colocalization), only IL17Low MMRp tumors (3/14) have a tumor immune microenvironment (TiME) that resembles the TiME in primary colon tumors of patients with mCRC responsive to anti-PD-1 treatment. Conclusions: The detection of a preexisting antitumor immune response in MMRp colorectal cancer (immunoreactive MMRp colorectal cancer) is not sufficient to predict a clinical benefit to T-cell checkpoint inhibitors. Intratumoral IL17-mediated signaling may preclude responses to immunotherapy. Drugs targeting the IL17 signaling pathway are available in clinic, and their combination with T-cell checkpoint inhibitors could improve colorectal cancer immunotherapy. See related commentary by Willis et al., p. 5185

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

Mismatch-repair deficiency in solid tumors predicts their response to PD-1 blockade. Based on this principle, pembrolizumab is approved as standard of care for patients with unresectable or metastatic microsatellite instability–high (MSI-H) cancer. Despite this success, a large majority of metastatic colorectal cancer patients are not MSI-H and do not benefit from checkpoint blockade treatment. Predictive biomarkers to develop personalized medicines and guide clinical trials are needed for these patients. We, therefore, asked whether immunohistologic stratification of metastatic colorectal cancer based on primary tumor PD-L1 expression associated with the presence or absence of extracellular mucin defines a subset of metastatic colorectal cancer patients who exhibit a preexisting antitumor immune response and who could potentially benefit from the checkpoint blockade. To address this, we studied 26 advanced metastatic colorectal cancer patients treated with pembrolizumab (NCT01876511). To stratify patients, incorporation of histopathologic characteristics (percentage of extracellular mucin) and PD-L1 expression at the invasive front were used to generate a composite score, the CPM (composite PD-L1 and mucin) score, which discriminated patients who exhibited clinical benefit (complete, partial, or stable disease) from those patients with progressive disease. When validated in larger cohorts, the CPM score in combination with MSI testing may guide immunotherapy interventions for colorectal cancer patient treatment.

RNA-Seq data has recently been used to successfully develop prognostic signatures to predict cancer patients who will have a worse prognosis. We designed a study to ascertain whether a prognostic model, based on RNA-Seq data, would have clinical utility for predicting survival in patients with colon adenocarcinomas (COAD). Of particular interest are early stage COAD patients, for whom the benefits of adjuvant therapy are unclear. High-risk patients predicted by our 5-gene, RNA-Seq signature had significantly shorter survival in both the training (p = 0.00) and test (p = 0.003) cohorts. Here we present an RNA-Seq prognostic signature that can identify high-risk COAD patients with shorter survival. This signature would have clinical utility as part of an RNA-Seq screening program, particularly for identifying early stage COAD patients who could be recommended adjuvant therapy after resection based on the results of this prognostic signature.

Predicting responses to immunotherapy Colon cancers with loss-of-function mutations in the mismatch repair (MMR) pathway have favorable responses to PD-1 blockade immunotherapy. In a phase 2 clinical trial, Le et al. showed that treatment success is not just limited to colon cancer (see the Perspective by Goswami and Sharma). They found that a wide range of different cancer types with MMR deficiency also responded to PD-1 blockade. The trial included some patients with pancreatic cancer, which is one of the deadliest forms of cancer. The clinical trial is still ongoing, and around 20% of patients have so far achieved a complete response. MMR deficiency appears to be a biomarker for predicting successful treatment outcomes for several solid tumors and indicates a new therapeutic option for patients harboring MMR-deficient cancers. Science , this issue p. 409 ; see also p. 358

Background: Extrinsic factors such as expression of PD-L1 (programmed death-ligand 1) in the tumor microenvironment (TME) have been shown to correlate with responses to checkpoint blockade therapy. More recently two intrinsic factors related to tumor genetics, microsatellite instability (MSI) and tumor mutation burden (TMB), have been linked to high response rates to checkpoint blockade drugs. These response rates led to the first tissue-agnostic approval of any cancer therapy by the FDA for the treatment of metastatic, MSI-H tumors with anti-PD-1 immunotherapy. Our team sought to explore the biology of such tumors further and suggest potential companion therapeutics to current checkpoint inhibitors. Methods: A CRC patient cohort (n=549) from TCGA was used for analysis. MSI was assessed with the MicrOSAtellite Instability Classifier (MOSAIC). TMB was assessed using Mutect2 and a 5% cutoff for allele frequency. Tumors with >20 somatic mutations per megabase were classified as high mutation. Expression of PD-L1 was assessed by quantifying gene expression – FPKM values from TCGA were used for this. Immune cell populations were analyzed using 3 algorithms: CIBERSORT, TIMER, and xCell. Pathway analysis was conducted with miRbase, an online tool for analyzing gene targets for miRNAs. Results: Analysis by Pearson Correlation revealed 41 miRNAs correlated with mutation burden, 62 miRNAs correlated with microsatellite instability, and 17 miRNAs correlated with PD-L1 expression. Fifteen of these miRNAs were correlated with all 3 tumor features and chosen for further analysis. let-7i, mir-1266, mir-132, mir-146b, mir-155, mir-212, mir-22, mir-223, mir-511, mir-625, and mir-629 were associated positively associated with the 3 tumor features while mir-335, mir-552, and mir-92a-2 were negatively associated with all 3. None of these miRNAs were associated with overall survival. Two cellular pathways known to influence immunological function were predicted by miRbase to be targeted by the miRNAs: PI3K-Akt (p Conclusions: Exploring miRNA targets as companions to treatment by immune checkpoint blockade revealed 15 potential miRNA targets predicted to impact 2 important cellular pathways: PI3K-Akt and TGF-beta. M1 polarization of macrophages was also associated with tumors predicted to respond to therapy by immune checkpoint blockade. Citation Format: Bjarne Bartlett, Vedbar Khadka, Mark Menor, Youping Deng. The miRNA expression profile of inflammatory tumors reveals a unique immune cell profile and potential companion targets for checkpoint blockade immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4058.

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.

Analyses of genomic alterations in cell-free DNA (cfDNA) shed from tumors into the blood stream is rapidly advancing as a method to detect and genotype cancer. Liquid biopsy analyses are a favorable alternative to invasive tissue approaches, particularly for cancers in organs where tissue is difficult to obtain. As these methods become a clinical standard, the method for blood collection and plasma isolation will be an important consideration to ensure optimal assay performance with limited degradation of circulating tumor DNA (ctDNA). We compared blood collection and plasma isolation using both K2EDTA and cell-free DNA Streck™ BCT tubes to determine the stability of cfDNA and ctDNA in blood at room temperature or at 4°C. Blood was collected from nine cancer patients with KRAS mutations using K2EDTA and Streck™ BCT tubes. Over the course of seven days, cell lysis occurred in K2EDTA tubes at room temperature, releasing germline DNA into the blood (1300% increase over seven days), which significantly increased the number of total genomic equivalents. Additionally, we found that the volume of plasma collected from both tube types during blood fractionation decreased over time when tubes were at room temperature (an average of 3.5 mL vs. 1.0 mL, respectively). To evaluate the effect of storage conditions on performance of each tube type, blood was also collected from six cancer patients with KRAS or EGFR mutations and either maintained at room temperature or stored at 4°C for up to three days. Taken together, K2EDTA tubes stored at 4°C prevented cell lysis and preserved the ctDNA in a manner equivalent to Streck™ BCT tubes. These data demonstrate that liquid biopsies can be collected using either tube type with similar performance and appropriate consideration of storage conditions. Citation Format: Sonya T. Parpart-Li, Bjarne Bartlett, Maria Popoli, Vilmos Adleff, Julie Brahmer, Nilo Azad, Sarah Bonerigo, Ilene Browner, Amy Ryan, Victor Velculescu, Mark Sausen, Luis A. Diaz. Optimized plasma collection procedures for liquid biopsy analyses in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3957.

103Background: Mismatch repair deficient (dMMR) colon cancers are densely infiltrated with CD8+T cells and regress when treated with anti-programmed death-1 (PD-1) antibodies. This anti-tumor response is thought to be potentiated by the thousands of somatic mutations that when expressed as proteins result in hundreds of potentially immunogenic neo-antigens that can be recognized by the patient’s immune system. Methods: We previously reported a phase 2 study to evaluate the activity of pembrolizumab (pembro), a PD-1 antibody in treatment refractory dMMR colon cancers (NEJM 2015). We are reporting the expanded trial and updated data for the mismatch repair deficient CRC (dMMR, cohort A) and mismatch repair proficient CRC (pMMR, cohort B) cohorts. Pembro was administered at 10 mg/kg every 14 days in patients with 2 or more prior therapies. The co-primary endpoints were response and progression-free survival rate at 20 weeks. Secondary endpoints included disease control rate (DCR = CR + PR + SD), progression ...

3003Background: Mismatch repair deficiency (MRD) is feature of many cancers at a frequency of approximately 1 in 30 patients independent of tumor histology. Tumors with MRD are deficient in the repair of specific DNA replication errors and as a result accumulate hundreds to thousands of mutations per tumor genome. The high number of somatic mutations increase the chances for at least one of these mutations to result in a highly immunogenic neo-antigenic protein that can trigger a potent anti-tumor immune response in the presence of PD-1 blockade. Methods: To further test this hypothesis, we conducted a phase 2 study to evaluate the activity of pembrolizumab (pembro), a programmed death-1 (PD-1) antibody in MRD tumors independent of tumor histology using a basket design. Pembro was administered at 10 mg/kg every 14 days in patients with > 1 prior therapy. The co-primary endpoints were response and progression-free survival rate at 20 weeks. Secondary endpoints included disease control rate (DCR = CR+PR+SD)...

195 Background: Mismatch repair (MMR) deficient tumors harbor hundreds to thousands of mutations that may produce neoantigens that can be recognized and targeted by T cells. We have shown that MMR deficiency can serve as a predictive biomarker for selection of tumors across tumor histologies that may respond to programmed death-1 (PD-1) blockade. MMR deficiency is present in 15% of colorectal (CRC) however is also detected in 2-20% of gastric, small bowel, and hepatobiliary cancers. Methods: We conducted a phase II study to evaluate anti-PD-1, pembrolizumab, in patients with previously-treated, progressive, advanced cancer. Twenty-one patients with MMR deficient tumors were enrolled onto the non-CRC cohort with an additional 50 patient expansion underway. The pembrolizumab dose is 10mg/kg intravenously every 2 weeks. Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) will be reported in at least the first 17 patients with non-CRC gastrointestinal (GI) cancers. Results: As of September 18, 2015, 17 patients with non-CRC GI cancers have been enrolled on the protocol with additional patients identified. The diseases represented are ampullary (N=4), pancreas (N=4), biliary (N=3), small bowel (N=3), and gastric (N=3) cancers. For the 10 evaluable patients at the time of abstract admission, ORR is 50 % (N=5/10), disease control rate is 70% (N=7/10), OS is 21 months and the median duration of response (range 5.5-17+ months) and PFS have not been reached. The median follow up duration is 7.6 months. Conclusions: PD-1 blockade shows promising activity in mismatch repair deficient GI cancers. Clinical trial information: NCT01876511.

11025 Background: Despite aggressive therapeutic interventions with pancreaticoduodenectomy and adjuvant chemo/chemoradiotherapy, recurrence rates remain high for patients with resectable pancreas ...

LBA100 Background: Somatic mutations have the potential to be recognized as “non-self” immunogenic antigens. Tumors with genetic defects in mismatch repair (MMR) harbor many more mutations than tumors of the same type without such repair defects. We hypothesized that tumors with mismatch repair defects would therefore be particularly susceptible to immune checkpoint blockade. Methods: We conducted a phase II study to evaluate the clinical activity of anti-PD-1, pembrolizumab, in 41 patients with previously-treated, progressive metastatic disease with and without MMR-deficiency. Pembrolizumab was administered at 10 mg/kg intravenously every 14 days to three cohorts of patients: those with MMR-deficient colorectal cancers (CRCs) (N = 11); those with MMR-proficient CRCs (N = 21), and those with MMR-deficient cancers of types other than colorectal (N = 9). The co-primary endpoints were immune-related objective response rate (irORR) and immune-related progression-free survival (irPFS) at 20 weeks. Results: The study met its primary endpoints for both MMR-deficient cohorts. The irORR and irPFS at 20 weeks for MMR-deficient CRC were 40% and 78%, respectively, and for MMR-deficient other cancers were 71% and 67%, respectively. In MMR-proficient CRC, irORR and irPFS at 20 weeks were 0% and 11%, respectively. Response rates and Disease Control Rates (CR+PR+SD) by RECIST criteria were 40% and 90% in MMR-deficient CRC, 0% and 11% in MMR-proficient CRC, and 71% and 71% in MMR-deficient other cancers, respectively. Median PFS and overall survival (OS) were not reached in the MMR-deficient CRC group but was 2.2 and 5.0 months in the MMR-proficient CRC cohort (HR for PFS = 0.103; 95% CI, 0.029 to 0.373; p < 0.001 and HR for OS = 0.216; 95% CI, 0.047 to 1.000; p = 0.05). Whole exome sequencing revealed an average of 1,782 somatic mutations per tumor in MMR-deficient compared to 73 in MMR-proficient tumors (p = 0.0015), and high total somatic mutation loads were associated with PFS (p = 0.02). Conclusions: MMR status predicts clinical benefit of immune checkpoint blockade with pembrolizumab. Clinical trial information: NCT01876511.