Targeted Exome Sequencing Research Articles

Abstract 4546: Comprehensive analysis using transcriptional factor based molecular subtypes and correlation to clinical outcomes in small-cell lung cancer

Abstract Background: Several studies have reported the predictive and prognostic value of novel transcriptional factor-based molecular subtypes in small-cell lung cancer (SCLC). We conducted an in-depth analysis using multi-omics data to elucidate the underlying characteristics that lead to differences in clinical outcomes between subtypes. Patients and Methods: Immunohistochemistry (IHC, n=252), target exome sequencing (n=422), and whole transcriptome sequencing (WTS, n=189) data generated from 427 patients with SCLC patients were comprehensively analyzed. The differences in the mutation profile, gene expression profile were analyzed according to the IHC-based molecular subtype. Clinical implication was evaluated based on treatment outcomes of individual patients. Results: IHC based molecular subtyping revealed a high incidence of ASCL1 subtype (SCLC-A, 56.3%) followed by ASCL1/NEUROD1 co-expressed (SCLC-trans, 17.9%), NEUROD1 (SCLC-N, 12.3%), POU2F3 (SCLC-P, 9.1%), triple-negative (SCLC-TN, 4.4%) subtypes showing high concordance with WTS-based subtyping. We delineated the SCLC-trans subtype resembling SCLC-A rather than SCLC-N in terms of both gene expression profiles and clinical outcomes. SCLC-TN type was defined as non-significant expression of A, N, P. Favorable overall survival (OS) was observed in SCLC-A compared to SCLC-N (adjusted HR 2.4, 95% CI 1.5-3.9, p < 0.001) and SCLC-P (adjusted HR 1.7, 95% CI 0.9-2.9, p = 0.087). SCLC-TN showed a similar OS with SCLC-A (adjusted HR 1.2, 95% CI 0.6 -2.7, p = 0598). The clinical outcome based on inflamed phenotype, clustered by effector cell gene expression profile which are only found in 5% of SCLC-N but 60% of SCLC-P, was more likely to benefit from first-line immunotherapy treatment than non-inflamed phenotype (p = 0.013). Inflammed phenotype demonstrated longer progression-free survival to the first line immunotherapy compared to the non-inflammed phenotype. (10.5 vs 4.3, p = 0.013) Conclusions: This study provides fundamental data, including the incidence and basic demographics of molecular subtypes of SCLC using both IHC and WTS from a comparably large cohort including potential differences in the distribution of subtypes based on ethnicity. Additionally, our results reveal differences in the underlying biological pathway activities and immunogenicity based on molecular subtype, possibly related to the difference in clinical outcomes, including immunotherapy response. Citation Format: Sehhoon Park, Tae Hee Hong, Soohyun Hwang, Hyun-Ae Jung, Jong-Mu Sun, Jin Seok Ahn, Myung-Ju Ahn, Jong Ho Cho, Yong Soo Choi, Jhingook Kim, Young Mog Shim, Hong Kwan Kim, Yoon-La Choi, Se-hoon Lee, Keunchil Park. Comprehensive analysis using transcriptional factor based molecular subtypes and correlation to clinical outcomes in small-cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4546.

Abstract 1906: MSH3: An underestimated DNA MMR gene in colorectal carcinogenesis and its potential role in disparity

Abstract Background: Unique mutations in cancer genes have been found in different races including African Americans (AA) population. Maintenance of proficient mismatch repair genes is important in colorectal cancer. Among them, MSH3 is found to be more important in colorectal cancer in AA particularly as its alterations associate with EMAST phenotype that has poor prognosis. However, there are regional variations in the classification and criteria used to determine the pathogenicity of these mutations particularly in AA due to their high genomic diversity. Aim: To analyze unique and novel MSH3 mutations using specific in-silico prediction tools and to validate their biological significances in AA-CRC. Results: In this study, we validated our targeted exome sequencing data of MSH3 mutations in AA and Caucasians (CA) CRC samples. A cost effective in-silico approach was employed to narrow down most significant MSH3 mutations. Interestingly, we and found six non-synonymous novel, pathogenic mutations in MSH3 gene (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, c.G3241A) correspond to MSH3 protein (E413K; S466N; S920F; E976K; H1010Y; E1081K), in AA than CA samples. Also, we observed loss or gain of different chemical bonding patterns (hydrogen, ionic, hydrophobic, and di-sulfide bonding) between wild type and mutant MSH3 protein. Interestingly, some of these mutations were within the ATPase site while others are in MSH3-MSH2 interacting domains. Furthermore, Molecular Dynamics Simulation (MDS) of these six mutants of MSH3 proteins showed an overall effect on MSH3 protein stability especially in MSH3-MSH2 interaction compared to wild type MSH3 protein.Conclusion: Our data showed that, six MSH3 non-synonymous mutations are deleterious as they affect important sites within MSH3-MSH2 interactive and ATPase domain. Assessing the real frequency of these mutations in large cohort and their specific effect on function may shed light on EMAST phenotype, high prevalence, and overall poor prognosis in the AA population. In vitro functional assays using CRISPR-Cas9 knock-in and in vivo studies are underway to mimic these mutations. Citation Format: Mudasir Rashid, Rumaisa Rashid, Minoru Koi, John M. Carethers, Hassan Brim, Hassan Ashktorab. MSH3: An underestimated DNA MMR gene in colorectal carcinogenesis and its potential role in disparity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1906.

Abstract 133: Rewinding the tape of life: somatic profiling in Von Hippel-Lindau (VHL) disease reveals evolutionary contingency and constraints

Abstract Whether cancer evolution follows a deterministic route prescribed by host factors (germline genetics and/or environment) or proceeds along multiple alternative trajectories due to the stochastic nature of evolution is unknown. Patients with VHL disease have a pathogenic germline mutation in VHL (3p25) and tumorigenesis occurs when the wild-type copy of VHL is lost. Patients develop multiple clonally independent tumors including clear cell renal cell carcinoma (ccRCC), pheochromocytoma or paraganglioma (PPGL) and pancreatic neuroendocrine tumors (pNET), offering an opportunity to test repeatability of evolutionary trajectories between and within patients. We performed deep targeted exome sequencing, in 1520 tumors (993 solid ccRCC; 156 cystic ccRCC; 135 mixed solid/cystic ccRCC; 236 extra-renal) from 130 patients with VHL disease to evaluate somatic variants and copy number alterations, ploidy and chromosomal complexity. Tumor phenotype was evaluated from pathology reports and longitudinal growth kinetics from diagnostic MRI data. Analysis of 889 tumor samples is complete and profiling of the full cohort will be presented at the 2023 AACR meeting. Within clonally independent tumors, we performed two tests to assess for evidence of convergence between tumors from the same patient. First, we tested for parallel events in canonical ccRCC drivers. We found significant enrichment of somatic VHL variants occurring in one patient with a germline whole VHL gene deletion encompassing neighboring BRK1. Contiguous BRK1 loss is associated with less aggressive ccRCC phenotype and our findings suggest a specific constraint in this patient. We next assessed for evidence of convergence by measuring the genetic distance of copy number and mutational profiles between and within patients with resampling and permutation. Similar somatic profiles between tumors were only detected in 1/76 patients whose tumors were characterized by infrequent somatic alterations beyond 3p loss indicating little evidence of convergence. Cystic ccRCCs represent an indolent phenotype not associated with metastasis that may progress to solid tumors over time; we found mixed tumors closely resemble somatic profiles of solid tumors whereas cysts were characterized by infrequent copy number gains and low chromosomal complexity. We also identified cysts with specific losses (chr16, chr17, chr19p) mutually exclusive with 3p LOH suggestive of VHL-loss independent cyst formation. In solid ccRCCs, we found chromosomal complexity and loss of specific chromosomal loci (9p and/or 14q) are associated with diameter potentially reconciling the observation linking risk of metastasis to tumor diameter in VHL-related ccRCC. In summary, within the constrained setting of VHL-related ccRCC, evolution is dominated by chance although specific patient and phenotype specific constraints are observed. Citation Format: Scott T. Shepherd, Alex Coulton, Cathy D. Vocke, Chris J. Ricketts, Mark Ball, Fiona Byrne, Yuliia Dovga, Annika Fendler, Barber Taja, Olga O'Neill, Lina Gerontogianni, Gavin Kelly, Laura S. Schmidt, Daniel R. Crooks, Kirstin Choo, Rabindra Gautam, Zoe Blake, Krista Reynolds, Kevin Litchfield, Maria Merino, Marston W. Linehan, Samra Turajlic. Rewinding the tape of life: somatic profiling in Von Hippel-Lindau (VHL) disease reveals evolutionary contingency and constraints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 133.

MP63-08 CLINICAL AND GENOMIC RISK FEATURES OF HIGH GRADE T1 NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP63-08 CLINICAL AND GENOMIC RISK FEATURES OF HIGH GRADE T1 NON-MUSCLE INVASIVE BLADDER CANCER Carissa Chu, Jessica Chen, Manuel DE Jesus Escano, Karissa Whiting, Irina Ostrovnaya, Song Jiang, Wesley Yip, Sari Khaleel, Andrew Lenis, Timothy Clinton, Goh Alvin, CHA Eugene, Timothy Donahue, Guido Dalbagni, Gopa Iyer, David Solit, Judy Sarungbam, Hikmat AL-Ahmadie, Bernard Bochner, and Eugene Pietzak Carissa ChuCarissa Chu More articles by this author , Jessica ChenJessica Chen More articles by this author , Manuel DE Jesus EscanoManuel DE Jesus Escano More articles by this author , Karissa WhitingKarissa Whiting More articles by this author , Irina OstrovnayaIrina Ostrovnaya More articles by this author , Song JiangSong Jiang More articles by this author , Wesley YipWesley Yip More articles by this author , Sari KhaleelSari Khaleel More articles by this author , Andrew LenisAndrew Lenis More articles by this author , Timothy ClintonTimothy Clinton More articles by this author , Goh AlvinGoh Alvin More articles by this author , CHA EugeneCHA Eugene More articles by this author , Timothy DonahueTimothy Donahue More articles by this author , Guido DalbagniGuido Dalbagni More articles by this author , Gopa IyerGopa Iyer More articles by this author , David SolitDavid Solit More articles by this author , Judy SarungbamJudy Sarungbam More articles by this author , Hikmat AL-AhmadieHikmat AL-Ahmadie More articles by this author , Bernard BochnerBernard Bochner More articles by this author , and Eugene PietzakEugene Pietzak More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003321.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: High grade non-muscle invasive bladder cancer invading the lamina propria (HG T1) exhibits variable clinical behavior. Existing risk stratification approaches by clinicopathologic alone are limited in determining who will benefit most from immediate radical cystectomy (RC) versus BCG. We report contemporary outcomes of patients managed with BCG or immediate RC and comparative mutational landscape across the spectrum of HG T1 disease. METHODS: This was a single-center retrospective cohort study of patients newly diagnosed with HG T1 non-muscle invasive bladder cancer between 2014 and 2020>18 months of follow up. All included patients underwent restaging TURBT with confirmed T1 by TURBT/EUA/imaging. Targeted exome sequencing with MSK-IMPACT was performed on 139 HG T1 tumors and 191 T2 tumors. RESULTS: 390 patients were included with median follow up time of 35 months (IQR 19-59 months). 308 were managed with intravesical BCG and 82 immediate RC. Patients selected for immediate RC were younger (median age 66 vs 69, p<0.001) and had higher rates of concurrent CIS (78 vs 53%, p<0.001), deep lamina propria invasion (37 vs 17%, p<0.001), lymphovascular invasion (7 vs 0.3%, p<0.001), and larger tumor size (4.3 cm vs 3.3 cm, p=0.007). Of the 82 patients who immediate radical cystectomy (BCG naïve), 32 (40%) were upstaged to pT2 and/or N+disease at RC. Persistent pT1 disease at re-resection and the presence of variant histology were associated with pathologic upstaging (p<0.01), while tumor size, concurrent CIS, bladder neck involvement, or prostatic urethra involvement were not. Compared to patients without additional risk factors, patients with CIS and at least one additional risk (e.g . “very high risk” T1 disease) harbored more TP53, ERBB2/Her2, and RB1 mutations and fewer FGFR3 mutations and a genomic profile more similar to T2 tumors than NMIBC (Figure 1). CONCLUSIONS: Very high risk T1 tumors are genomically similar to muscle-invasive tumors and are enriched in targetable alterations including ERBB2/Her2. Integration of genomic sequencing with clinical and pathologic features of HG T1 disease may improve risk stratification and treatment selection. Source of Funding: Ruth L. Kirschstein National Research Service Award T32CA082088 American Society of Clinical Oncology Young Investigator Award (YIA) Award © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e874 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Carissa Chu More articles by this author Jessica Chen More articles by this author Manuel DE Jesus Escano More articles by this author Karissa Whiting More articles by this author Irina Ostrovnaya More articles by this author Song Jiang More articles by this author Wesley Yip More articles by this author Sari Khaleel More articles by this author Andrew Lenis More articles by this author Timothy Clinton More articles by this author Goh Alvin More articles by this author CHA Eugene More articles by this author Timothy Donahue More articles by this author Guido Dalbagni More articles by this author Gopa Iyer More articles by this author David Solit More articles by this author Judy Sarungbam More articles by this author Hikmat AL-Ahmadie More articles by this author Bernard Bochner More articles by this author Eugene Pietzak More articles by this author Expand All Advertisement PDF downloadLoading ...

PD09-07 ASSESSING THE UTILITY OF A CELL-FREE TUMOR (ct)DNA ASSAY (MSK-ACCESS) IN NODE POSITIVE (N+) MUSCLE INVASIVE BLADDER CANCER (MIBC) PATIENTS (pts) UNDERGOING NEOADJUVANT CHEMOTHERAPY (NAC)

You have accessJournal of UrologyCME1 Apr 2023PD09-07 ASSESSING THE UTILITY OF A CELL-FREE TUMOR (ct)DNA ASSAY (MSK-ACCESS) IN NODE POSITIVE (N+) MUSCLE INVASIVE BLADDER CANCER (MIBC) PATIENTS (pts) UNDERGOING NEOADJUVANT CHEMOTHERAPY (NAC) Andrew B. Katims, Andrew T. Lenis, Ronak H. Shah, Carissa E. Chu, Neha Ratna, Ashely M. Regazzi, Eugene J. Pietzak, David H. Aggen, Samuel A. Funt, Min Yuen Teo, Jonathan E. Rosenberg, Dean F. Bajorin, Timothy F. Donahue, Bernard H. Bochner, Michael F. Berger, David B. Solit, and Gopa Iyer Andrew B. KatimsAndrew B. Katims More articles by this author , Andrew T. LenisAndrew T. Lenis More articles by this author , Ronak H. ShahRonak H. Shah More articles by this author , Carissa E. ChuCarissa E. Chu More articles by this author , Neha RatnaNeha Ratna More articles by this author , Ashely M. RegazziAshely M. Regazzi More articles by this author , Eugene J. PietzakEugene J. Pietzak More articles by this author , David H. AggenDavid H. Aggen More articles by this author , Samuel A. FuntSamuel A. Funt More articles by this author , Min Yuen TeoMin Yuen Teo More articles by this author , Jonathan E. RosenbergJonathan E. Rosenberg More articles by this author , Dean F. BajorinDean F. Bajorin More articles by this author , Timothy F. DonahueTimothy F. Donahue More articles by this author , Bernard H. BochnerBernard H. Bochner More articles by this author , Michael F. BergerMichael F. Berger More articles by this author , David B. SolitDavid B. Solit More articles by this author , and Gopa IyerGopa Iyer More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003240.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Circulating ctDNA is associated with disease progression, worse overall survival, and recurrence in pts with bladder cancer. This study correlated targeted exome ctDNA sequencing with pathologic response to NAC and metastatic recurrence in pts with N+ MIBC undergoing radical cystectomy (RC). METHODS: We prospectively identified pts with cT2-3N1-2M0 bladder cancer who underwent NAC prior to RC. Node positivity was determined radiographically and/or by node biopsy. Plasma samples were collected pre-NAC, mid-treatment, after NAC completion, and 3 months after RC. Samples were analyzed using MSK-ACCESS, an ultrasensitive ctDNA platform designed to identify somatic mutations in 129 cancer associated genes. Primary bladder tumors were sequenced using targeted exome sequencing. RESULTS: 31 samples from 9 pts (6 men) were analyzed. Median age was 63 years (IQR 58-69). NAC regimens included gemcitabine and cisplatin alone (78%) or with paclitaxel (22%). Seven (78%) pts had >1 detectable mutation pre-NAC. The most altered genes detected in both tissue and ctDNA included: TERT (88% vs 28%), TP53 (76% vs 40%), ARID1A (43% vs 17%), RB1 (43% vs 15%), KDM6A (43% vs 15%), and ATM (17% vs 17%). Four (45%) pts had complete response (ypT0N0), 1 (11%) had a partial response (ypT1N0), and 4 (33.3%) were non-responders (ypT2N0-3). All non-responders had disease recurrence after RC (median 3 months, range 2-11 months). Of 7 pts with on-treatment MSK-ACCESS, 2 (29%) had detectable ctDNA (1 ypT2N0, 1 ypT0N0). The pt with a PR had detectable ctDNA post-NAC with 1 mutation identified that was not detected at 3 months. All pts with >ypT2N0 had detectable ctDNA post-NAC. Pts with recurrence/non-responders had a significantly higher mutation count 3 months after RC compared to responders, with a median of 7 vs. 0 mutations, respectively. Two pts with CR had detectable ctDNA at 3 months post-RC but have not recurred (Table 1). CONCLUSIONS: Clearance of ctDNA post-NAC correlated with pathologic complete response. Approximately 70% of pts had ctDNA clearance on NAC. All pts with residual disease at RC had detectable ctDNA post-NAC. N+ disease correlated with persistent ctDNA detected at 3 months post-RC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e242 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Andrew B. Katims More articles by this author Andrew T. Lenis More articles by this author Ronak H. Shah More articles by this author Carissa E. Chu More articles by this author Neha Ratna More articles by this author Ashely M. Regazzi More articles by this author Eugene J. Pietzak More articles by this author David H. Aggen More articles by this author Samuel A. Funt More articles by this author Min Yuen Teo More articles by this author Jonathan E. Rosenberg More articles by this author Dean F. Bajorin More articles by this author Timothy F. Donahue More articles by this author Bernard H. Bochner More articles by this author Michael F. Berger More articles by this author David B. Solit More articles by this author Gopa Iyer More articles by this author Expand All Advertisement PDF downloadLoading ...

KRAS Mutations Are Associated with Shortened Survival in Patients with Epithelioid Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy of the pleural surface that includes three major histologic subtypes, epitheliod, sarcomatoid and biphasic. Epithelioid mesothelioma is usually associated with better prognosis. The genetic mechanisms driving MPM, the possible target mutations and the correlation with overall survival remain largely unsettled. We performed target exome sequencing in 29 cases of MPM aimed at identifying somatic mutations and, eventually, their correlation with phenotypic traits and prognostic significance. We found that KRAS mutations, occurring in 13.7% of cases, were associated with shortened median survival (7.6 versus 32.6 months in KRAS wild-type; p = 0.005), as it was the occurrence of any ≥3 mutations (7.6 versus 37.6 months; p = 0.049). Conversely, the presence of KDR single nucleotide polymorphism p.V297I (rs2305948) resulted in a favorable variable for survival (NR versus 23.4 months; p = 0.026). With the intrinsic limitations of a small number of cases and patient heterogeneity, results of this study contribute to the characterization of the mutation profile of MPM and the impact of selected somatic mutations, and possibly KDR polymorphism, on prognosis.

Clinical features and outcomes of patients with diacylglycerol kinase epsilon nephropathy: a nationwide experience.

Thrombotic microangiopathy (TMA) is usually caused due to dysregulation of the alternative complement pathway. Rarely, thrombotic microangiopathy is caused by non-complement mediated mutations in diacylglycerol kinase epsilon (DGKE); information about therapy and outcome of these patients is limited. Medical records of patients, younger than 18years, diagnosed with TMA and variants in DGKE were reviewed to include 12 patients from seven centers. Genetic studies included targeted exome sequencing and multiplex-ligation dependent probe amplification of CFH-CFHR5. Patients presented at a median age of 11 (7.5, 12.3) months; all were younger than 2years. All patients had an infectious prodrome; enteroinvasive, enteropathogenic, and enterotoxigenic Escherichia coli were detected in two patients with diarrhea. Chief features included those of microangiopathic hemolysis (n = 11), microscopic hematuria (n = 10), nephrotic range proteinuria (n = 10), hypoalbuminemia (n = 6), elevated total cholesterol (n = 6), and hypocomplementemia (n = 4). Histopathology showed thrombotic microangiopathy (n = 4), overlapping with membranoproliferative pattern of injury (n = 1). At median 3.3years of follow-up, significant hypertension and/or proteinuria (40%), relapses (66.7%), and death or progression to CKD (60%) were common. Genetic sequencing showed 13 homozygous and compound heterozygous variants(7 pathogenic, 3 likely pathogenic) located throughout DGKE; 11 variants were novel. This case series highlights the need to suspect DGKE nephropathy in young patients with TMA, especially those with severe proteinuria. Medium-term outcomes are unsatisfactory with risk of relapses, progressive kidney failure, and death. A higher resolution version of the Graphical abstract is available as Supplementary information.

Loss of CDKN2A/B is a Molecular Marker of High-grade Histology and is Associated with Aggressive Behavior in Acinic Cell Carcinoma

Acinic cell carcinoma (AciCC) is a rare salivary gland cancer with excellent prognosis in most cases. However, a subset of patients will develop distant metastasis and die of disease. Recently, a 2-tiered grading scheme in AciCC was proposed to recognize patients at risk of poor outcome. We performed a genetic analysis of AciCC to explore the underlying molecular correlates of the tumor grade and examined programmed death ligand 1 (PD-L1) expression to identify potential candidates for immunotherapy. A retrospective cohort of 55 patients included 34 high-grade (HG) and 21 low-grade AciCCs. Forty-three cases were subjected to targeted exome sequencing by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets. PD-L1 immunohistochemistry was performed in 33 cases. Tumor mutation burden was low with a median of 1 and 2 mutations in low-grade and HG AciCCs, respectively. CDKN2A/B was the most frequently altered gene, and loss-of-function mutations were found only in HG but not in low-grade AciCCs (18/31 [58.1%] vs 0/12 [0%], P < .001). CDKN2A/B alterations were significantly associated with distant metastasis, which occurred in 16/18 (88.9%) CDKN2A/B mutants versus 11/25 (44%) wild-type cases (P = .004, Fisher exact test). Sequential profiling of multiple temporally distant samples from the same patient demonstrated intratumor heterogeneity, including the detection of CDKN2A/B deletion in the second, in HG metastasis only. ATM and PTEN mutations were detected in 6/31 (19.4%) and 5/31 (16.1%); ARID2, BIRC3, and FBXW7 mutations each in 4/31 (12.9%); and TP53, MTAP, and FAT1 each in 3/31 (9.7%) HG AciCC. PD-L1–positive labeling was more common in HG AciCC (9/17, 52.9% vs 3/16, 18.9%, P = .071). CDKN2A/B mutations in AciCC represent a molecular marker of HG histology and disease progression, providing a rationale for further studies to determine their prognostic and therapeutic significance in this salivary gland cancer. AciCC with ATM mutations may be amenable to targeted therapy. Immunotherapy can be considered to be a treatment option for a subset of patients with AciCC.

Assessing the utility of a cell-free tumor (ct)DNA assay (MSK-ACCESS) in patients (pts) with node-positive (N+) muscle-invasive bladder cancer (MIBC) undergoing neoadjuvant chemotherapy (NAC).

544 Background: Circulating ctDNA is associated with disease progression, worse overall survival, and recurrence in pts with bladder cancer. This study correlated targeted exome ctDNA sequencing with pathologic response to NAC and metastatic recurrence in pts with N+ MIBC undergoing radical cystectomy (RC). Methods: We prospectively identified pts with cT2-3N1-2M0 bladder cancer who underwent NAC prior to RC. Node positivity was determined radiographically and/or by node biopsy. Plasma samples were collected pre-NAC, mid-treatment, after NAC completion, and 3 months after RC. Samples were analyzed using MSK-ACCESS, an ultrasensitive ctDNA platform designed to identify somatic mutations in 129 cancer associated genes. Primary bladder tumors were sequenced using targeted exome sequencing. Results: Samples (31) from 9 pts (6 men) were analyzed. Median age was 63 years (IQR 58-69). NAC regimens included gemcitabine and cisplatin alone (78%) or with paclitaxel (22%). Seven (78%) pts had &gt;1 detectable mutation pre-NAC. The most altered genes detected in both tissue and ctDNA included: TERT (88% vs 28%), TP53 (76% vs 40%), ARID1A (43% vs 17%), RB1 (43% vs 15%), KDM6A (43% vs 15%), and ATM (17% vs 17%). Four (45%) pts had complete response (ypT0N0), 1 (11%) had a partial response (ypT1N0), and 4 (33.3%) were non-responders (ypT2N0-3). All non-responders had disease recurrence after RC (median 3 months, range 2-11 months). Of 7 pts with on-treatment MSK-ACCESS, 2 (29%) had detectable ctDNA (1 ypT2N0, 1 ypT0N0). The pt with a PR had detectable ctDNA post-NAC with 1 mutation identified that was not detected at 3 months. All pts with &gt;ypT2N0 had detectable ctDNA post-NAC. Pts with recurrence/non-responders had a significantly higher mutation count 3 months after RC compared to responders, with a median of 7 vs. 0 mutations, respectively. Two pts with CR had detectable ctDNA at 3 months post-RC but have not recurred (Table). Conclusions: Clearance of ctDNA post-NAC correlated with pathologic complete response. Approximately 70% of pts had ctDNA clearance on NAC. All pts with residual disease at RC had detectable ctDNA post-NAC. [Table: see text]

Response Evaluation Criteria in Tumors of the Bladder (RECIT-Bladder) for future window of opportunity trials as a platform to accelerate drug development.

571 Background: There are abundant potential permutations of novel or combination therapeutics that could be tested in non-muscle invasive bladder cancer (NMIBC), but the ability to study these therapies is limited. Window of opportunity trials (WOOT) may accelerate otherwise costly and slow drug evaluation by testing response to treatment during the time between the surveillance office cystoscopy that diagnoses a recurrent tumor and removal by transurethral resection (TURBT), but the accuracy of endoscopic tumor measurement is unknown. Through an iterative process modifying the RECIST criteria used for metastatic cancer response assessment; we developed RECIT-Bladder as a short-term endpoint for NMIBC WOOTs. Here, we report the feasibility and reproducibility of paired tumor measurement at office cystoscopy and TURBT in “untreated” patients as the development cohort for RECIT-Bladder. Methods: Bladder tumors were measured at office cystoscopy and again at formal TURBT in a standardized fashion compared to a calibrated object (flexible biopsy forceps). If multiple tumors were present, the two largest, measurable, representative tumors were measured. Patients were excluded if tumor location did not allow for accurate measurement (e.g., bladder neck) or if the lesion was not amenable to measurement (e.g., diffuse CIS or papillary carpeting). Results: We characterized 69 papillary tumors from 34 patients (4 patients with recurrent tumors included ≥2 times). 38% (13/34) had LGTa and the remainder had high grade (HG) bladder cancer (1 CIS, 15 HGTa, 2 HGT1, and 1 HGT2). 98.6% (68/69) were measurable at TURBT. There was a median 23 (16-37 IQR) days between cystoscopy and TURBT during which lesion size increased by a median 0.25 mm (0-0.1mm IQR, p=0.4). Measurements on cystoscopy and at TURBT were highly correlated (correlation coefficient 1.04, p&lt;0.01, r2 = 0.88). There was no correlation between tumor growth and time between cystoscopy and TURBT (correlation coefficient 0.00, p=0.5, r2 = 0.00). MSK-IMPACT targeted exome sequencing was available in 62% of patients (21/34) of whom 71% (15/21) had an FGFR3 mutation (median growth 1 mm over median 23 days). Conclusions: Using a standardized protocol (RECIT-Bladder), tumor measurement at office cystoscopy and follow-up TURBT appears precise and reproducible. RECIT-Bladder may offer a practical short-term clinical trial endpoint for evaluating progression, stable disease, or response to therapy. If validated, RECIT-Bladder offers a pathway to rapidly discover novel therapeutic agents. We have begun a prospective multi-surgeon protocol to validate these results and evaluate potential pharmacodynamic endpoints. RECIT-Bladder is also currently being assessed in the context of an ongoing investigator-initiated WOOT testing oral erdafitinib in FGFR3 altered recurrent NMIBC (NCT04917809).

Alliance A031902 (CASPAR): A randomized phase (ph) 3 trial of enzalutamide with rucaparib/placebo in first-line metastatic castration-resistant prostate cancer (mCRPC).

TPS277 Background: Despite a growing number of treatment options for first-line mCRPC, approximately 40% of patients (pts) have radiographic progression within the first year. Co-inhibition of androgen receptor (AR) and PARP is a promising therapeutic strategy that leverages synthetic lethality induced by impaired double-strand DNA repair. Two phase III studies have shown improvement in radiographic progression-free survival (rPFS) in HRR-mutant pts with abiraterone + PARPi combinations vs abiraterone alone. However, the results in HRR-wild type pts are conflicting, with only one of the studies demonstrating a benefit with the abiraterone + PARPi combination. ENZ + RUCA has shown an acceptable safety profile &amp; no significant drug-drug interactions (S-DDI) in a phase 1b trial. This allows its evaluation in mCRPC. Methods: CASPAR (A031902) is a phase 3 study in which 984 pts will be randomized on a 1:1 basis to ENZ plus RUCA/PBO. HRR gene aberration is not required for enrollment. All pts will undergo next-generation targeted exome sequencing from archival tumor tissue (new biopsy only required if no archival tissue is available). Treatment will be continued until disease progression and crossover is not allowed. Key eligibility criteria are age ≥ 18 years, ECOG PS 0-2, biopsy-proven prostate adenocarcinoma, progressive (PSA or radiographic) disease per Prostate Cancer Working Group 3 guidelines, measurable or non-measurable disease per RECIST 1.1, no prior treatment for mCRPC (prior docetaxel, abiraterone, darolutamide, or apalutamide in non-mCRPC setting is allowed), no significant uncontrolled comorbidity, and no medications with S-DDI with ENZ/RUCA. Hierarchical co-primary endpoints are overall survival (OS) and rPFS. The OS analysis will be undertaken as a primary endpoint if the rPFS endpoint is met. For a one-sided logrank test with a type 1 error rate equal to 0.025, the study has 90% power to detect a hazard ratio (HR) of 0.71 in rPFS (median rPFS of 15 and 21 months in control and combination arms, respectively), and 80% power to detect an HR of 0.80 in OS (median OS of 32 and 40 months, respectively). Key secondary endpoints are rPFS and OS in pts with vs without pathogenic BRCA1, BRCA2, or PALB2 alterations; and differences in adverse events and quality of life (QOL) outcomes between the treatment arms. QOL assessments include Functional Assessment of Cancer Therapy–Prostate (FACT-P), Brief Pain Inventory Short Form (BPI-SF), and EQ-5D-5L. A key correlative endpoint is the sensitivity of ctDNA testing for HRR gene alterations. Enrollment began in July 2021 &amp; the study is available for participation to all US-NCTN sites. Clinical trial information: NCT04455750 .

Genetic mutation profiles for children with congenital hypothyroidism in Fujian province

Objective: To explore the mutation characteristics of pathogenic genes in children with congenital hypothyroidism (CH) in Fujian. Methods: The clinical data of 116 unrelated CH children diagnosed in Fujian Provincial Maternal and Child Health Hospital from January 2019 to September 2020 were retrospectively analyzed, including 50 females and 66 males, with an average age of (20±10) days at diagnosis. Targeted exome sequencing technology was used to detect the mutation frequency, type and distribution characteristics of 29 genes related to thyroxine synthesis or thyroid development. Results: Three hundred and fifty-one potential functional mutations were detected in 105 of 116 CH patients, with a detection rate of 90.5% (105/116). DUOX2 (66.4%, 77/116) was the most frequent mutated gene, followed by TG (23.3%, 27/116), DUOXA1 (23.3%, 27/116), and TPO (12.1%, 14/116), which were all involved in thyroid hormone synthesis. Among the 105 children with CH, 70 cases carried double allele mutation. Except for 3 cases of thyroid dysplasia related genes (2 cases of TSHR and 1 case of GLIS3), the rest were also related to thyroid hormone synthesis. The gene with the highest carrier rate was DUOX2 (68.8%, 59/70), followed by TG (8.6%, 6/70), TPO (4.3%, 3/70), DUOXA2 (1.4%, 1/70) and DUOXA1 (1.4%, 1/70). Conclusion: The main mutated genes in CH children in Fujian are the key genes involved in thyroid hormone synthesis, such as DUOX2, TG and TPO.

De Novo Mutations Contributes Approximately 7% of Pathogenicity in Inherited Eye Diseases.

The purpose of this study was to describe genotype-phenotype associations and novel insights into genetic characteristics in a trio-based cohort of inherited eye diseases (IEDs). To determine the etiological role of de novo mutations (DNMs) and genetic profile in IEDs, we retrospectively reviewed a large cohort of proband-parent trios of Chinese origin. The patients underwent a detailed examination and was clinically diagnosed by an ophthalmologist. Panel-based targeted exome sequencing was performed on DNA extracted from blood samples, containing coding regions of 792 IED-causative genes and their flanking exons. All participants underwent genetic testing. All proband-parent trios were divided into 22 subgroups, the overall diagnostic yield was 48.67% (605/1243), ranging from 4% to 94.44% for each of the subgroups. A total of 108 IED-causative genes were identified, with the top 24 genes explaining 67% of the 605 genetically solved trios. The genetic etiology of 6.76% (84/1243) of the trio was attributed to disease-causative DNMs, and the top 3 subgroups with the highest incidence of DNM were aniridia (n = 40%), Marfan syndrome/ectopia lentis (n = 38.78%), and retinoblastoma (n = 37.04%). The top 10 genes have a diagnostic yield of DNM greater than 3.5% in their subgroups, including PAX6 (40.00%), FBN1 (38.78%), RB1 (37.04%), CRX (10.34%), CHM (9.09%), WFS1 (8.00%), RP1L1 (5.88%), RS1 (5.26%), PCDH15 (4.00%), and ABCA4 (3.51%). Additionally, the incidence of DNM in offspring showed a trend of correlation with paternal age at reproduction, but not statistically significant with paternal (P = 0.154) and maternal (P = 0.959) age at reproduction. Trios-based genetic analysis has high accuracy and validity. Our study helps to quantify the burden of the full spectrum IED caused by each gene, offers novel potential for elucidating etiology, and plays a crucial role in genetic counseling and patient management.

Evidence for diet-gene interaction in early-onset colorectal cancer.

182 Background: About 10 percent of all colorectal cancers are in subjects who are not yet 50 (EO-CRC) and the occurrence of early onset colorectal cancer (EO-CRC) is rising in the US. Patients with CRC are twice as likely to have diabetes or be overweight. Using targeted exome sequencing of germline DNA from EO-CRC subjects, we identified a missense mutation at Ala98Val within the DNA binding domain of Hepatic Nuclear Factor 1 alpha (HNF1A, 12q24.31, Rs1800574). HNF1A is the most frequently mutated gene in diabetic individuals whose onset of diabetes occurs typically before age 25 (MODY3 locus). Our aim was to demonstrate that the HNF1A variant provides a genomic landscape for EO-CRC to develop in the setting of a specific diet type. Methods: HNF1A was identified using targeted exon sequencing of archived leukocyte DNA from subjects with EO-CRC. Flag-tagged WT and HNF1AA98V expressing plasmids were transfected into HCT116 colon cancer cells and nuclear extracts were prepared for Electrophoretic Mobility Shift Assays (EMSAs) to test binding differences. An Hnf1aA98V mutant mouse model was generated using CRISPR/Cas9. WT, Hnf1a A98V/+ and Hnf1a A98V/A98V mice were placed on 3 diets--normal chow, a high fat (~34%) diet (HFD) or a high sugar (~62% fructose) diet (HSD) after weaning at 3 weeks. The mice were followed for 12 months, weighed monthly, and observed for clinical signs of morbidity. After euthanizing, blood, colon and liver were collected for histology and qPCR. Results: HNF1AA98V was identified in 13/145 subjects with EO-CRC. An additional 3 subjects exhibited mutations elsewhere in HNF1A. Flag-tagged WT and mutant HNF1A-expressing plasmids were transfected into HCT116 colon cancer cells and nuclear extracts showed reduced HNF1AA98V binding to its consensus DNA element by EMSA. Only heterozygous or homozygous Hnf1aA98V mice on the HFD showed a significant number of colon polyps (~19%). Only 1/22 heterozygous mice developed a polyp on the HSD. RNA-Seq analysis of colonic mucosa from the homozygous mutant compared to WT mice revealed an increase lipid and b-catenin regulated genes. Specifically, there was an increase in β-catenin, LEF 1 and MYC by immunohistochemistry in the polyp of HNF1AA98V, but not in the adjacent normal tissue. Liver steatosis in the Hnf1aA98V versus WT mice on the HFD was observed. Conclusions: Occurrence of the HNF1AA98V variant is increased among a cohort of EO-CRC patients and is a loss of function mutation that creates a genetic landscape for colon polyps on a HFD.

A Korean boy with a CHD8 mutation who presented with overgrowth, intellectual disability, and autism.

The CHD8 mutation was initially identified as the cause of autism spectrum disorder (ASD) and was previously marked as Autism, susceptibility to, 18 [MIM 615032] on Online Mendelian Inheritance in Man (OMIM). As many patients with CHD8 mutation showed other characteristics along with autism, the phenotype was relabeled as intellectual developmental disorder with autism and macrocephaly (IDDAM) in May 2022. We report a 3-year 10-month-old boy who presented with overgrowth, intellectual disability, dysmorphic facial features, and ASD. Targeted exome sequencing identified a heterozygous nonsense mutation c.2854C>T (p.Arg952Ter) in CHD8, and he was diagnosed with IDDAM. Overgrowth patients with dysmorphic face and/or developmental delay should be investigated for genetic causes. Such causes have been reported in about 50% of patients diagnosed with overgrowth with intellectual disability. Our case suggests that CHD8 genetic testing should be included if overgrowth, intellectual disability, and ASD coexist.

Genetic abnormalities assist in pathological diagnosis and EBV-positive cell density impact survival in Chinese angioimmunoblastic T-cell lymphoma patients.

To explore the application of genetic abnormalities in the diagnosis of angioimmunoblastic T-cell lymphoma (AITL) and the reliable pathological prognostic factors. This study included 53 AITL cases, which were reviewed for morphological patterns, immunophenotypes, presence of Hodgkin and Reed-Sternberg (HRS)-like cells, and co-occurrence of B cell proliferation. The Epstein-Barr virus (EBV)-positive cells in tissues were counted, and cases were classified into "EBV encoded RNA (EBER) high-density" group if >50/HPF. Targeted exome sequencing was performed. Mutation data can assist AITL diagnosis: 1) with considerable HRS-like cells (20 cases): RHOA mutated in 14 cases (IDH2 co-mutated in 3 cases, 4 cases with rare RHOA mutation), TET2 was mutated in 5 cases (1 case co-mutated with DNMT3A), and DNMT3A mutated in 1 case; 2) accompanied with B cell lymphoma (7 cases): RHOA mutated in 4 cases (1 case had IDH2 mutation), TET2 mutated in 2 cases and DNMT3A mutated in 1 case; 3) mimic peripheral T cell lymphoma, not otherwise specified (5 cases): RHOA mutated in 2 cases (IDH2 co-mutated in 1 case), TET2 mutated in 3 cases, and DNMT3A mutated in 1 case; 4) pattern 1 (1 case), RHOA and TET2 co-mutated. Besides RHOAG17V (30/35), rare variant included RHOAK18N, RHOAR68H, RHOAC83Y, RHOAD120G and RHOAG17del, IDH2R172 co-mutated with IDH2M397V in one case. There were recurrent mutations of FAT3, PCLO and PIEZO1 and genes of epigenetic remodeling, T-cell activation, APC and PI3K/AKT pathway. EBER high-density independently indicated adverse overall survival and progression-free survival (P=0.046 and P=0.008, Kaplan-Meier/log-rank). Over half AITL cases might be confused in diagnosis for certain conditions without mutation data. Targeted exome sequencing with a comprehensive panel is crucial to detect both hot-spot and rare mutation variants for RHOA and IDH2 and other recurrent mutated genes in addition to TET2 and DNMT3A. EBER high-density independently indicated adverse survival.

Factors Associated With the Development and Severity of Polycystic Liver in Patients With Autosomal Dominant Polycystic Kidney Disease.

Factors related to the development and severity of polycystic liver disease (PLD) have not been well established. We aimed to evaluate the genetic and epidemiologic risk factors of PLD in patients with autosomal dominant polycystic kidney disease (ADPKD). Adult patients with inherited cystic kidney disease were enrolled from May 2019 to May 2021. Demographic, clinical, and laboratory data were collected at the initial study visit. The severity of PLD was graded based on the height-adjusted total liver volume: < 1,000 mL/m (Gr1), 1,000-1,800 mL/m (Gr2), and > 1,800 mL/m (Gr3). Targeted exome sequencing was done by a gene panel including 89 ciliopathy-related genes. We searched out the relative factors to the presence and the severity of PLD using logistic regression analysis. Of 602 patients with typical ADPKD, 461 (76.6%) patients had PLD. The patients with PLD showed female predominance and a higher frequency of other ADPKD-related complications. The genetic variants with truncating mutation of PKD1 (PKD1-protein-truncating [PT]) or PKD2 commonly affected the development and severity of PLD. An older age, female sex, and higher kidney volume with Mayo classification 1C-1E was significantly associated with the development of PLD, but not with the severity of PLD. On the other hand, higher body mass index, lower hemoglobin, and higher alkaline phosphatase (ALP) were the significant risk factors of severe PLD (≥ Gr2). Hepatic involvement in ADPKD could be related to kidney manifestations and genetic variants including PKD1-PT or PKD2. Monitoring hemoglobin and ALP and evaluating the genetic variants might help predict severe PLD. Clinical Research Information Service Identifier: KCT0005580.

Modeling of a Novel Patient-Based MYO5B Point Mutation Reveals Insights Into MVID Pathogenesis

Modeling of a Novel Patient-Based MYO5B Point Mutation Reveals Insights Into MVID Pathogenesis

Identification of Novel Mutations in Children with Hereditary Spherocytosis by Targeted Exome Sequencing: A Single Center Experience

Aim: Hereditary spherocytosis (HS) is a prevalent cause of congenital hemolytic anemia in Northern&#x0D; Europeans. It is characterized by spherocytes resulting from defects in the erythrocyte structural&#x0D; membrane proteins spectrin and ankyrin. To date, more than five candidate genes, including ANK1,&#x0D; SPTB, SPTA1, SLC4A1, and EPB42 have been linked to HS. Here, we aim to investigate the presence&#x0D; of novel as well as known mutations in eight Turkish children with clinically suspected HS.&#x0D; Material and Methods: We presented the clinical features of the patients and identified the causative&#x0D; gene variants using targeted exome sequencing. Eight children who were clinically suspected of having&#x0D; HS enrolled in this study. A family and medical history, clinical examination, relevant laboratory test&#x0D; results, osmotic fragility test (OFT), and genetic results were evaluated.&#x0D; Results: Six causative variants, including three ANK1 variants, two SPTB variants and one SLC4A1&#x0D; variant were detected. All these mutations were novel variants. ANK1 and SPTB are the most common&#x0D; mutant genes in children with HS.&#x0D; Conclusion: This study expanded the mutation spectrum of ANK1, SPTB and SLC4A1. This is the first&#x0D; study to determine the genetic and clinical characteristics of children with HS in Turkey.

Implications of genetic variations, differential gene expression, and allele-specific expression on metformin response in drug-naïve type 2 diabetes

PurposeMetformin is widely used to treat type 2 diabetes mellitus (T2DM) individuals. Clinically, inter-individual variability of metformin response is of significant concern and is under interrogation. In this study, a targeted exome and whole transcriptome analysis were performed to identify predictive biomarkers of metformin response in drug-naïve T2DM individuals.MethodsThe study followed a prospective study design. Drug-naïve T2DM individuals (n = 192) and controls (n = 223) were enrolled. T2DM individuals were administered with metformin monotherapy and defined as responders and non-responders based on their glycated haemoglobin change over three months. 146 T2DM individuals were used for the final analysis and remaining samples were lost during the follow-up. Target exome sequencing and RNA-seq was performed to analyze genetic and transcriptome profile. The selected SNPs were validated by genotyping and allele specific gene expression using the TaqMan assay. The gene prioritization, enrichment analysis, drug-gene interactions, disease-gene association, and correlation analysis were performed using various tools and databases.Resultsrs1050152 and rs272893 in SLC22A4 were associated with improved response to metformin. The copy number loss was observed in PPARGC1A in the non-responders. The expression analysis highlighted potential differentially expressed targets for predicting metformin response (n = 35) and T2DM (n = 14). The expression of GDF15, TWISTNB, and RPL36A genes showed a maximum correlation with the change in HbA1c levels. The disease-gene association analysis highlighted MAGI2 rs113805659 to be linked with T2DM.ConclusionThe results provide evidence for the genetic variations, perturbed transcriptome, allele-specific gene expression, and pathways associated with metformin drug response in T2DM.