Wang, Kai

Importance The rise of novel, more infectious SARS-CoV-2 variants has made clear the need to rapidly deploy large-scale testing for COVID-19 to protect public health. However, testing remains limited due to shortages of personal protective equipment (PPE), naso- and oropharyngeal swabs, and healthcare workers. Simple test methods are needed to enhance COVID-19 screening. Here, we describe a simple, and inexpensive spit-test for COVID-19 screening called Patient Self-Collection of Sample-CoV2 (PSCS-CoV2). Objective To evaluate an affordable and convenient test for COVID-19. Methods The collection method relies on deep throat sputum (DTS) self-collected by the subject without the use of swabs, and was hence termed the Self-Collection of Sample for SARS-CoV-2 (abbreviated PSCS-CoV2). We used a phenol-chloroform extraction method for the viral RNA. We then tested for SARS-CoV-2 using real-time reverse transcription polymerase chain reaction with primers against at least two coding regions of the viral nucleocapsid protein (N1 and N2 or E) of SARS-CoV-2. We evaluted the sensitivity and specificity of our protocol. In addition we assess the limit of detection, and efficacy of our Viral Inactivating Solution. We also evaluated our protocol, and pooling strategy from volunteers on a local college campus. Results We show that the PSCS-CoV2 method accurately identified 42 confirmed COVID-19 positives, which were confirmed through the nasopharyngeal swabbing method of an FDA approved testing facility. For samples negative for COVID-19, we show that the cycle threshold for N1, N2, and RP are similar between the PSCS-CoV2 and nasopharynx swab collection method (n = 30). We found a sensitivity of 100% (95% Confidence Interval [CI], 92-100) and specifity of 100% (95% CI, 89-100) for our PSCS-CoV2 method. We determined our protocol has a limit of detection of 1/10,000 for DTS from a COVID-19 patient. In addition, we show field data of the PSCS-CoV2 method on a college campus. Ten of the twelve volunteers (N1 < 30) that we tested as positive were subsequently tested positive by an independent laboratory. Finally, we show proof of concept of a pooling strategy to test for COVID-19, and recommend pool sizes of four if the positivity rate is less than 15%. Conclusion and Relevance We developed a DTS-based protocol for COVID-19 testing with high sensitivity and specificity. This protocol can be used by non-debilitated adults without the assistance of another adult, or by non-debilitated children with the assistance of a parent or guardian. We also discuss pooling strategies based on estimated positivity rates to help conserve resources, time, and increase throughput. The PSCS-CoV2 method can be a key component of community-wide efforts to slow the spread of COVID-19.

PURPOSE Intrahepatic cholangiocarcinoma (IHCCA), a global health problem, is increasing in incidence and has differing etiologies worldwide. Next-generation sequencing (NGS) is rapidly being incorporated into the clinical management of biliary cancers. IHCCA is enriched with actionable mutations, and there are several promising targeted therapies under development. NGS data from Asia, where IHCCA is most prevalent, are limited. METHODS Comprehensive genomic profiling of formalin-fixed paraffin-embedded tumor tissue from 164 Asian and 283 Western patients with IHCCA was performed using NGS. We measured the distribution of DNA repair genetic aberrations (GAs) in IHCCA, along with actionable mutations. Also, we evaluated the association between DNA repair GAs and tumor mutation burden (TMB). Based on the TMB status, patients were distinguished into 3 levels: low (< 6 mut/Mb), intermediate (6-10 mut/Mb), and high (TMB-H; ≥ 10 mut/Mb). RESULTS Seventy-two percent of Asian patients had ≥ 1 actionable GA, with a significantly higher frequency in KMT2C , BRCA1/2, and DDR2 compared with Western patients ( P = .02, .003, and .003, respectively); 60.9% of Western patients had ≥ 1 actionable GA and higher frequency of CDKN2A/B and IDH1/2 GAs ( P = .0004 and < .001, respectively). GAs in nuclear factor kappa B pathway regulators and DNA repair genes occurred more frequently in Asian patients ( P = .006 and .001, respectively). There was a higher frequency of TMB-H in Asian compared with the Western cohort (12.2% v 5.9%; P = .07). CONCLUSION A higher burden of DNA repair mutations and frequency of patients with TMB-H in the Asian IHCCA cohort compared with the Western patients suggests a potential role for DNA repair and immune checkpoint inhibitors in the Asian population. Future clinical trials should account for this genetic heterogeneity.

4076 Background: Gallbladder carcinoma (GBC) is commonly diagnosed at an advanced, unresectable disease stage and has a poor prognosis. Comprehensive genomic profiling (CGP) has a developing role in guiding systemic, precision anti-cancer therapy. Methods: We performed hybrid capture-based CGP on FFPE samples for 491 consecutive advanced GBCs. Mean coverage depth was >550X for up to 315 cancer-related genes plus 37 introns from 28 genes frequently rearranged in cancer. All 4 classes of genomic alterations (GA) were detected. Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA. Results: Median age was 64 (range 25-88) and 69% (337/491) of patients were female. 96% (470), 2.9% (14), and 1.4% (7) of GBC patients had the diagnosis of adenocarcinoma, adenosquamous, or carcinoma not specified (NOS), respectively. Commonly altered genes were TP53 (62%), CDNK2A (31%), ARID1A (18%), and SMAD4 (15%), and most of the genetic aberrations (GA) were short variants. Potentially targetable SCNAs (including ERBB genes, MET, FGFRs, and the CCND1-FGF3/4/19 11q13 amplicon) were identified in 21% of cases (Table). Oncogenic BRAF, ALK, or FGFR2/3 rearrangements were found in 7 cases (1.4%). Moreover, 7.8% of cases had BRCA2 or ATM GA, and 0.8% had INI1loss suggesting benefit from PARP or EZH2 inhibitors, respectively. TMB was low; the 25th, 50th, and 75th percentiles were 2.5, 3.8 and 6.3 mutations/Mb, respectively. Less than 1% of cases had microsatellite instability. Radiological response to TKIs and immunotherapy was noted. Conclusions: In addition to the significant opportunity for anti-HER targeted therapies, other subsets of GBC cases harbored kinase GA (particularly SCNA), the 11q13 amplicon, or BRCA2/ ATM/ INI1mutations that are linked to therapeutic benefit. How the frequency of both driver SCNA and DNA repair alterations in GBC can be linked with inflammation awaits additional investigation. [Table: see text]

570 Background: Vascular endothelial growth factor- (VEGF-) and mammalian target of rapamycin- (mTOR-) directed therapies represent a standard of care in metastatic renal cell carcinoma (mRCC). However, these agents are not employed based on an assessment of predictive biomarkers such as genomic alterations (GA). We sought to determine if an association exists between GA detected by comprehensive genomic profiling (CGP) and the response to VEGFR and mTOR pathway targeted therapies in a cohort of mRCC treated in a clinical practice setting. Methods: The results of CGP performed in the course of clinical care on 31 consecutive mRCC obtained from patients who had received VEGFR- and/or mTOR-inhibitors were reviewed. Duration of treatment (DOT), extent and duration of clinical response was obtained from review of medical records. All classes of genomic alterations - base substitutions, short insertions, deletions, gene fusions, rearrangements and copy number - were assessed via CGP. Descriptive statistics were used to determine the frequency of GAs in groups segregated by the DOT with VEGF-directed agents. Results: The most common GAs detected in this series were in VHL (70%), PBRM1 (48%), SETD2 (32%), TSC1 (29%), MLL (19%), TERT (16%), ARID1B (16%) and KDM5C (16%). Across 61 administrations of VEGF-directed therapy in 27 patients, exceptional responses (DOT > 21 months) were more frequent amongst patients with GAs in KDM5C, PBRM1, and VHL. Conversely, these patients also featured a lower frequency of GA associated with response to mTOR-directed therapy, such as TSC1. Conclusions: Multiple GAs are more prevalent in exceptional responders to VEGF-directed therapy. Prospective validation of these findings may lead to use of CGP to optimize therapeutic selection.

Abstract Background Thyroid carcinoma, which is rare in pediatric patients (age 0–18 years) but more common in adolescent and young adult (AYA) patients (age 15–39 years), carries the potential for morbidity and mortality. Methods Hybrid-capture-based comprehensive genomic profiling (CGP) was performed prospectively on 512 consecutively submitted thyroid carcinomas, including 58 from pediatric and AYA (PAYA) patients, to identify genomic alterations (GAs), including base substitutions, insertions/deletions, copy number alterations, and rearrangements. This PAYA data series includes 41 patients with papillary thyroid carcinoma (PTC), 3 with anaplastic thyroid carcinoma (ATC), and 14 with medullary thyroid carcinoma (MTC). Results GAs were detected in 93% (54/58) of PAYA cases, with a mean of 1.4 GAs per case. In addition to BRAF V600E mutations, detected in 46% (19/41) of PAYA PTC cases and in 1 of 3 AYA ATC cases, oncogenic fusions involving RET, NTRK1, NTRK3, and ALK were detected in 37% (15/41) of PAYA PTC and 33% (1/3) of AYA ATC cases. Ninety-three percent (13/14) of MTC patients harbored RET alterations, including 3 novel insertions/deletions in exons 6 and 11. Two of these MTC patients with novel alterations in RET experienced clinical benefit from vandetanib treatment. Conclusion CGP identified diverse clinically relevant GAs in PAYA patients with thyroid carcinoma, including 83% (34/41) of PTC cases harboring activating kinase mutations or activating kinase rearrangements. These genomic observations and index cases exhibiting clinical benefit from targeted therapy suggest that young patients with advanced thyroid carcinoma can benefit from CGP and rationally matched targeted therapy.

BACKGROUND Activating, nonamplification ERBB2 mutations ( ERBB2 mut) are not detected by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH), but are detected by DNA sequencing and may predict clinical responses to human epidermal growth factor receptor (HER2)‐targeted therapy. The authors queried 5605 advanced/metastatic breast cancers (mBC) to uncover the frequency of ERBB2 mut genomic alterations. Clinical responses to anti‐HER2 therapeutics were identified. METHODS DNA was extracted from 40 µm of formalin‐fixed paraffin‐embedded (FFPE) sections. Comprehensive genomic profiling (CGP) was used to evaluate up to 315 genes (592× mean coverage depth). Results were analyzed for base substitutions, short indels, copy number changes, and selected rearrangements. RESULTS Of 5605 cases, 698 (12.5%) featured ERBB2 alterations, including 596 (10.6%) ERBB2 amplifications ( ERBB2 amp) and 138 (2.4%) ERBB2 mut; 38 cases (0.7%) had co‐occurring ERBB2amp and ERBB2mut. ERBB2mut predominantly affected the kinase (124 cases; 90%) or extracellular (15 cases; 11%) domains. Both primary BC (52 cases; 38%) and metastatic site biopsies (86 cases; 62%) were found to harbor ERBB2 mut, which were distributed across carcinoma not otherwise specified (NOS) (69 cases; 50%), invasive ductal carcinoma (IDC) (40 cases; 29%), invasive lobular carcinoma (ILC) (27 cases; 20%), and mucinous mBC (2 cases; 1%). Genes commonly coaltered with ERBB2 were tumor protein 53 ( TP53 ) (49%); phosphatidylinositol 3‐kinase catalytic subunit alpha ( PIK3CA ) (42%); cadherin 1, type 1 ( CDH1 ) (37%); MYC (17%); and cyclin D1 protein ( CCND1 ) (16%). CDH1 mutations were enriched in ERBB2 mut mBC ( P <0.0006) and associated with recurrent mBC. Selected patients with ERBB2 mut, without ERBB2 amp, who responded to anti‐HER2 targeted therapies are presented herein. CONCLUSIONS Within this large series, 1.8% of cases harbored ERBB2 mut, which are undetectable by standard‐of‐care IHC or FISH tests. Metastatic BC driven by ERBB2 mut respond to anti‐HER2 targeted therapies, and expanding clinical trials designed to detect ERBB2 mut by CGP and optimize targeted treatments are warranted. Cancer 2016 . © 2016 American Cancer Society . Cancer 2016;122:2654–2662. © 2016 American Cancer Society.