Mutations In Individual Genes Research Articles

The Spiral Model of Evolution: Stable Life Forms of Organisms and Unstable Life Forms of Cancers.

If one must prioritize among the vast array of contributing factors to cancer evolution, environmental-stress-mediated chromosome instability (CIN) should easily surpass individual gene mutations. CIN leads to the emergence of genomically unstable life forms, enabling them to grow dominantly within the stable life form of the host. In contrast, stochastic gene mutations play a role in aiding the growth of the cancer population, with their importance depending on the initial emergence of the new system. Furthermore, many specific gene mutations among the many available can perform this function, decreasing the clinical value of any specific gene mutation. Since these unstable life forms can respond to treatment differently than stable ones, cancer often escapes from drug treatment by forming new systems, which leads to problems during the treatment for patients. To understand how diverse factors impact CIN-mediated macroevolution and genome integrity-ensured microevolution, the concept of two-phased cancer evolution is used to reconcile some major characteristics of cancer, such as bioenergetic, unicellular, and multicellular evolution. Specifically, the spiral of life function model is proposed, which integrates major historical evolutionary innovations and conservation with information management. Unlike normal organismal evolution in the microevolutionary phase, where a given species occupies a specific location within the spiral, cancer populations are highly heterogenous at multiple levels, including epigenetic levels. Individual cells occupy different levels and positions within the spiral, leading to supersystems of mixed cellular populations that exhibit both macro and microevolution. This analysis, utilizing karyotype to define the genetic networks of the cellular system and CIN to determine the instability of the system, as well as considering gene mutation and epigenetics as modifiers of the system for information amplification and usage, explores the high evolutionary potential of cancer. It provides a new, unified understanding of cancer as a supersystem, encouraging efforts to leverage the dynamics of CIN to develop improved treatment options. Moreover, it offers a historically contingent model for organismal evolution that reconciles the roles of both evolutionary innovation and conservation through macroevolution and microevolution, respectively.

The performance of A rpob gene mutation linked to A resistant to rifampicin mycobacterium tuberculosis isolate from an Indonesian referral hospital

IntroductionIndonesia has the world's second-highest tuberculosis and is one of the 30 countries with an increased MDR/RR TB burden. Even though the number of people with tuberculosis is rising every year, several cases of latent or active tuberculosis remain unreported. Microscopy has its limitations, and factors such as quality sample, quality smear, bacterial load, and experience of reading AFB influence the sensitivity of the test. Utilizing a quick, point-of-care testing system based on molecular techniques can increase the sensitivity of tuberculosis early diagnosis. Cepheid Xpert ®MTB/RIF is Indonesia's most widely utilized molecular technology. It is a nested real-time PCR diagnostic device that is cartridge-based and semiquantitative. In the TB complex of Mycobacterium tuberculosis, it identifies alterations in the RNA polymerase (rpoB) gene beta-subunit that result in rifampicin resistance (RR). By utilizing the Xpert ® MTB/RIF assay at Soetomo General Hospital, one of Indonesia's primary tertiary referral hospitals, the current study seeks to improve our knowledge of the prevalence of rpoB gene mutations in individuals with tuberculosis. MethodsBetween January 2021 and December 2021, Soetomo General Hospital used the Xpert®MTB/RIF system to screen sputum and extrapulmonary specimens from 3584 for tuberculosis. ResultsOf the 665 samples, 18,5% tested positive for M. tuberculosis, with 7.6% (51 out of 665) showing resistance to Rifampicin Rifampicin compounds at codons 529–533 (probe E) of the rpoB gene were detected in 56.8% (29/51) of rifampicin-resistant tuberculosis (RR-TB) samples. And then Probe D (19.6%). 7.8% for probes A and B, respectively (5.8%). Probe C did not exhibit any mutations. On the other hand, two samples revealed two mutations in probes D and E, while among 13.7% (7/51) of RR TB samples, no mutations in the probe type were observed. ConclusionThe Xpert®MTB/RIF system is a compassionate, fast, accurate, and efficient diagnostic tool for RR-TB, tuberculosis, and the molecular epidemiology of mutations associated with rifampin resistance.

Comparative genomic profiling of circulating tumor DNA and paired formalin-fixed paraffin-embedded (FFPE) DNA from Indian patients with breast cancer.

e14554 Background: Breast cancer (BC) is the most frequent malignancy and a leading cause of mortality in women worldwide. Although most BCs are treated with curative intent, 30-40 % patients experience disease progression or metastatic relapse. Considering high heterogeneity of BC, mutational profiling of solid biopsy may not be adequate for informed treatment decisions in all cases. Liquid biopsy based on circulating tumor DNA (ctDNA) analysis is emerging as an equivalent and alternative to tissue biopsy in clinical settings. Though extremely critical for management of BC, liquid biopsy is not a standard of care in India. Here we compared comprehensive genomic profiles of ctDNA and paired biopsy samples to determine the efficacy of ctDNA assay in detecting actionable mutational targets. Methods: Retrospectively, genomic DNA and matched ctDNA was isolated from both FFPE and blood samples of14 BC patients. The patients were confirmative with pathological findings, having metastatic infiltrating ductal carcinoma, or triple negative breast cancers. Genome-wide HRD, TMB and MSI scores based on the OncoIndx comprehensive gene panel (CGP) was determined. OncoIndx CGP targets 1080 cancer related genes along with 47 HRR genes and 1600 MSI signature sites. Libraries were prepared using a hybridization-capture method based on custom-designed OncoIndx 1080 CGP and sequenced (depth 5500 X) on Illumina Nextseq 2000 in a pair-end mode (150 x2). Variant calling was performed using a proprietary bioinformatics pipeline iCare. Results: All patients were diagnosed with metastatic infiltrating ductal carcinoma, or triple negative breast cancers. From the patient cohort, microsatellite instability (MSI) from ctDNA showed 100% concordance with FFPE DNA while tumor mutation burden (TMB) and homologous recombination deficiency (HRD) scores showed 71.4 % and 64.2 % concordance with FFPE DNA respectively. At the individual gene mutation level, the concordance TP53, BRCA1/2, PI3K family of genes, ESR1, PTEN, ERBB2, PALB2, and BRAF genes between ctDNA and FFPE DNA was 54.7%, 92.8%, 54.7%, 78.5%, 92.8%, 100%, 100%, and 100% respectively. Many important genomic alterations including PIK3CA:p.L334H, PIK3CA:p.E81K, ESR1:p.Q375L, BRCA2:p.X3040_splice, PTEN:p.D19H were exclusive to ctDNA thereby denoting potential tumor heterogeneities which could be missed from FFPE DNA. Conclusions: Our results suggest a high correlation between ctDNA and FFPE DNA detected by OncoIndx test in metastatic or triple negative breast cancer patients of Indian ethnicity. Although not a part of standard of care, liquid biopsy could be of significant potential for management of BC in the standard clinical settings. However, a thorough investigation of cancer stage, and clinical presentations are necessary to carefully evaluate the selection of ctDNA or FFPE DNA-based genomic profiling.

30years of prophylactic thyroidectomy for hereditary medullary thyroid cancer : Amilestone in translational medicine

Medullary thyroid cancer (MTC) is the most frequent manifestation of multiple endocrine neoplasia type 2 (MEN2) that determines the oncological outcome. Germline mutations in the rearranged during transfection (RET) protooncogene, atumor suppressor gene on chromosome 10q11.2, were identified 30 years ago as the genetic basis of MEN2 and published in 1993 and 1994. These seminal findings gave rise to the concept of prophylactic thyroidectomy for asymptomatic gene mutation carriers based on apositive RET gene test, which has become the standard of care ever since. Clinical genetic investigations showed genotype-phenotype correlations with respect to the individual gene mutation regarding the penetrance and onset of MTC and to alesser extent also with respect to the other components of MEN2, pheochromocytoma and primary hyperparathyroidism. From this a clinically relevant risk stratification could be derived. Initially, the optimal timing of prophylactic thyroidectomy was primarily based on the RET genotype alone, which was not sufficient for a precise age recommendation and subsequently required additional consideration of calcitonin serum levels for fine tuning. Calcitonin levels first show the risk of lymph node metastasis when they exceed the upper normal limit of the assay independent of carrier age and RET mutation. Routine calcitonin screening of patients with nodular thyroid disease, screening of families on identification of MEN2 index patients, and pre-emptive thyroidectomy in carriers of gene mutations with normal calcitonin levels have led to the fact that nowadays, 30years after the first description of the gene mutations causing the disease, the life-threatening hereditary MTC has become curable: ashining example for the success of translational transnational medical research for the benefit of patients.

Plasmacytoid urothelial carcinoma of the urinary bladder–A clinicopathological and molecular analysis of 52 cases

Plasmacytoid urothelial carcinoma (UC) is a rare histologic subtype of bladder cancer that is associated with an aggressive clinical behavior. We analyzed the clinicopathologic and molecular features of plasmacytoid UC in 52 patients from a single institute. The patients included 44 men and 8 women, with a mean age of 64 years (range, 41–91 years). All bladder cancers were high-grade UC, and plasmacytoid component accounted for a mean of 47% of bladder tumors (range, 5–100%). Distinct gene mutations were found in most plasmacytoid UCs (n = 49); the most common mutations were TP53 (n = 30), followed by TERT (n = 20), and CDH1 (n = 18). Copy number analysis was performed in 34 patients, and 13 of them showed copy number variations. Expression of HER2 was analyzed in 18 patients by immunohistochemistry, and 3 of them showed HER2 overexpression, which was confirmed by fluorescence in situ hybridization analysis. Thirty-two patients died of disease in a median of 15 months (range, 1–45 months). No individual gene mutations were significantly associated with clinical outcome, but mutations in the mammalian target of rapamycin (mTOR) pathway, including PICK3CA and PIK3R1 mutations, were associated with a significantly shorter survival duration (p < 0.05). Plasmacytoid UC is an aggressive histologic subtype that demonstrates frequent somatic gene mutations and CNVs, which may underlie its oncogenesis and progression. Gene mutations of the mTOR pathway are associated with poor outcome in a subset of patients with plasmacytoid UC.

TP53 and/or BRCA1 Mutations Based on CtDNA Analysis as Prognostic Biomarkers for Primary Triple-Negative Breast Cancer.

Precise biomarkers for predicting the therapeutic efficacy of molecularly targeted drugs are limited at the protein level; thus, it has been important to broadly scrutinize individual cancer driver gene mutations for effective cancer treatments. Multiplex cancer genome profiling can comprehensively identify gene mutations that are therapeutic targets using next-generation sequencing (NGS). In addition, circulating tumor DNA (ctDNA) is a DNA fragment released into the blood by tumor cell-derived cell death or apoptosis. Liquid biopsy with ctDNA is a novel clinical test for identifying genetic mutations in an entire population noninvasively, in real-time, and heterogeneously. Although there are several reports on ctDNA, fewer have evaluated ctDNA with NGS before an initial treatment for breast cancer patients. Therefore, we examined whether analyzing tumor-associated gene mutations in primary breast cancer based on ctDNA could serve as a biomarker for prognosis and optimal treatment selection. Ninety-five primary breast cancer patients treated at our department from January 2017 to October 2020 were included. Pretreatment plasma samples were subjected to NGS analysis of ctDNA, and correlations with patients' clinicopathological characteristics were evaluated. Fifty-nine (62.1%) patients were positive for ctDNA. ctDNA tended to be positive in hormone receptor-negative, and TP53 (34%), BRCA1 (20%), and BRCA2 (17%) gene mutations were more frequent. Regarding recurrence-free survival, the prognosis was poor in the TP53 and/or BRCA1 mutation-positive groups, especially in triple-negative breast cancer (TNBC) patients. In conclusion, the results of this study indicate that ctDNA with liquid biopsy could identify the poor prognosis group before treatment among TNBC patients and for those for whom optimal treatment selection is desirable; additionally, optimal treatment could be selected according to the ctDNA analysis results.

Assessing the Phenotype of a Homologous Recombination Deficiency Using High Resolution Array-Based Comparative Genome Hybridization in Ovarian Cancer.

Ovarian cancer (OC) cells with homologous recombination deficiency (HRD) accumulate genomic scars (LST, TAI, and LOH) over a value of 42 in sum. PARP inhibitors can treat OC with HRD. The detection of HRD can be done directly by imaging these genomic scars, or indirectly by detecting mutations in the genes involved in HR. We show that HRD detection is also possible using high-resolution aCGH. A total of 30 OCs were analyzed retrospectively with high-resolution arrays as a test set and 19 OCs prospectively as a validation set. Mutation analysis was performed by HBOC TruRisk V2 panel to detect HR-relevant mutations. CNVs were clustered with respect to the involved HR genes versus the OC cases. In prospective validation, the HRD status determined by aCGH was compared with external HRD assessments. Two BRCA mutation carriers did not have HRD. OC could approximately differentiate into two groups with characteristic CNV patterns with different survival rates. Mutation frequencies have a linear regression on the HRD score. Mutations in individual HR-relevant genes do not always indicate HRD. This may depend on the mutation frequency in tumor cells. The aCGH shows the genomic scars of an HRD inexpensively and directly.

Diagnostic Utility of Next-Generation Sequencing in Circulating Free DNA and a Comparison With Matched Tissue in Gallbladder Carcinoma

Mutation detection for therapy monitoring in cell-free DNA (cfDNA) is used clinically for some malignancies. Gallbladder carcinoma (GBC) presents a diagnostic challenge and has limited late-stage treatment options. To our knowledge, this novel study examines, for the first time, genomic alterations in cfDNA from GBC to assess diagnostic accuracy and therapeutic options. The concordance of somatic genomic changes in cfDNA and DNA from paired tumor tissue was analyzed. Paired serum and tissue samples from 40 histologically proven GBC, 20 cholecystitis, and 4 normal (noninflamed gallbladder) controls were included. Targeted next-generation sequencing with a 22-gene panel (Colon and Lung Cancer Research Panel v2, Thermo Scientific) in cfDNA and tumor tissue with high depth and uniform coverage on ION Personal Genome Machine (ION, PGM) was performed. A spectrum of 223 mutations in cfDNA and 225 mutations in formalin-fixed paraffin-embedded tissue DNA were identified in 22 genes. Mutations ranged from 1 to 17 per case. In cfDNA frequent alterations were in TP53 (85.0%), EGFR (52.5%), MET (35%) CTNNB1, SMAD4, BRAF (32.5%), PTEN (30%), FGFR3 and PIK3CA (27.5%), NOTCH1 (25.0%), and FBXW7 and ERBB4 (22.5%). At least one clinically actionable mutation was identified in all cfDNA samples. Paired samples shared 149 of 225 genetic abnormalities (66.2%). Individual gene mutation concordance ranged from 44.44% to 82.0% and was highest for EGFR (82.0%), BRAF and NOTCH1 (80.0%), TP53 (73.08%), MET (72.22%), and ERBB4 (71.42%) with a significant level of correlation (Spearman r = 0.91, P ≤ .0001). The sensitivity and specificity of the TP53 gene at the gene level was the highest (94.44% and 100.0%, respectively). Overall survival was higher for ERBB4 and ERBB2 mutant tumors. The adenocarcinoma subtype revealed specific genetic changes in ERBB4, SMAD4, ERBB2, PTEN, KRAS, and NRAS. NGS-based cfDNA mutation profiling can be used to diagnose GBC before surgery to guide treatment decisions. Targeted therapy identified in GBC included SMAD4, ERBB2, ERBB4, EGFR, KRAS, BRAF, PIK3CA, MET, and NRAS.

Abstract B153: Resistance mechanisms to mobocertinib in treating NSCLC with EGFR exon 20 insertion mutations

Abstract Background: Mobocertinib is an oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that received FDA accelerated approval for non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion (ex20ins) mutations, based on data from a pivotal phase 1/2 study of mobocertinib (NCT02716116). In platinum-pretreated patients (pts) with EGFR ex20ins NSCLC (N=114), the overall response rate (ORR) was 28%, median duration of response was 17.5 months, and median progression-free survival was 7.3 months. Despite this encouraging clinical activity, de novo and acquired resistant mechanisms to mobocertinib remain largely unknown. Methods: Tumor samples and circulating tumor DNA (ctDNA) samples were obtained from platinum-pretreated pts with EGFR ex20ins+ NSCLC treated with mobocertinib (NCT02716116) and subjected to next-generation sequencing (NGS) analyses. CtDNA was collected at baseline, cycle 3 day 1 (C3D1) and progression per protocol. Tumor growth assay was performed in BaF3 cells and patient-derived xenograft models to evaluate efficacy of combination therapies to overcome resistance. Results: At baseline, in tumor (n=56), the combined presence of BRAF and PIK3CA mutations alone (n=9) or together with copy number gains (CNV) in EGFR, MYC, CDK4, or CCND1 (n=19) was significantly associated with inferior ORR to mobocertinib with odd ratios of ‘infinity’ (p=0.045) and 13.21 (p=0.0047), respectively. Individual co-occurring gene mutations and CNV were not significantly associated with differential clinical outcomes. Longitudinal analysis of paired tumor-blood samples at baseline and C3D1 showed that 22 out of 55 pts (40%) achieved clearance at C3D1, including 81.2% (13/16) of PR pts, 50% of SD pts, and 20% of PD pts. CtDNA was also used to assess emerging mutations after mobocertinib progression after at least 5 months of treatment (n=18). TP53 (n=11) and acquired secondary EGFR (n=5) were the most common genes with emerging variants, including EGFR T790M (n=4) and D379E (n=1). With this observation, T790M was engineered in Ba/F3 cells in the context of EGFR ex20ins mutations (H773_V774insNPH and 769_D770insASV). T790M did confer resistance to mobocertinib in vitro, and to all EGFR TKIs tested including osimertinib. In patient-derived xenograft models (H773_V774insNPH and 769_D770insASV), the combination of mobocertinib plus the bispecific monoclonal antibody amivantamab demonstrated greater antitumor activity than either agent alone. Conclusions: Our results indicate that bypass pathway alterations are associated with de novo resistance whereas T790M emergence mediates acquired resistance to mobocertinib. Future research is warranted to determine the potential clinical benefits of combination strategies in patients with EGFR ex20ins+ NSCLC. Citation Format: Xiuning Le, Monique Nilsson, Sampurna Chatterjee, Zhenqiang Su, Junqin He, Hibiki Udagawa, Xiaoxing Yu, Alissa Poteete, Qian Huang, John V Heymach, Sylvie Vincent. Resistance mechanisms to mobocertinib in treating NSCLC with EGFR exon 20 insertion mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B153.

A Novel and Cost-Efficient Measurable Residual Disease (MRD) Detection Assay Using a Single Molecule Molecular Inversion Probe-Based Next Generation Sequencing (NGS) Assay Predicts Overall Survival in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation

Introduction Relapse remains the main reason for poor outcomes of patients (pts) with acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) is the preferred post-remission treatment in pts at high risk of relapse. The presence of measurable residual disease (MRD) after intensive frontline treatment or before or after HSCT has emerged as an important predictor of relapse. Various methods are used for MRD detection, including flow cytometry and molecular assays detecting individual gene mutations (mut) such as NPM1mut. Next generation sequencing (NGS)-based assays covering multiple genes commonly mutated in AML may allow sensitive MRD detection in a broad range of pts with diverse genetic characteristics. Methods We established a targeted, error corrected NGS MRD assay utilizing single-molecule molecular inversion probes (smMIPs). The smMIP protocol uses a hybridization-capture approach to selectively enrich and amplify both DNA strands of 92 regions in 32 genes in a single reaction. Unique molecular identifiers (UMIs) facilitate computational correction of sequencing errors to enable reliable detection of variants ≥0.5% variant allele frequency (VAF), with the additional advantage of relatively low reagent and sequencing costs of &amp;lt; $100 per sample. The smMIP assay was used to evaluate MRD status on pre-HSCT bone marrow samples from 98 AML pts who received an HSCT in first (n=83) or second (n=15) complete remission with or without count recovery. Median age at HSCT was 59 (range 20-75) years, 42% were female; 70% had de novo AML, 19% had an antecedent myeloid neoplasm and 11% had AML post cytotoxic therapy. European LeukemiaNet (ELN)-2022 risk groups at diagnosis were favorable in 11%, intermediate in 33%, adverse in 20% and unknown in 22%. All pts had received intensive induction chemotherapy. Twenty-nine percent received a myeloablative conditioning regimen (MAC), while 23% received reduced intensity conditioning (RIC) and 48% a non-myeloablative (NMA) conditioning regimen. Donors were matched related (18%), haploidentical (6%), or matched (55%) or mismatched (21%) unrelated. The median follow-up for pts alive was 34.8 months. Results Overall, we detected 190 variants in 71 (72%) of 98 pre-HSCT remission samples. The most commonly affected genes were DNMT3A (in 37% of pts), TET2 (21%), PPM1D (12%), IDH2 (10%) and TP53 (5%). Twenty-nine percent of pts had only variants affecting the clonal hematopoiesis-related genes DNMT3A, TET2 or ASXL1 (‘DTA‘), while 44% of patients had ≥1 variant involving a non-DTA gene. MRD-positive pts, defined as those with ≥1 non-DTA variant, had similar baseline and HSCT-related characteristics (includingage, sex, ELN risk group, de novo vs. secondary AML) compared to pts without non-DTA variants. Overall survival (OS) was significantly inferior in NGS MRD-positive pts compared to those with no non-DTA variant (Figure A, 5-year OS, 49% vs. 85%, P=.011). MRD-positive pts also had a non-significant higher cumulative incidence of relapse at 5 years (46% vs. 24%, P=.14), while non-relapse mortality was similar in both groups (P=.72). We next performed multivariable analyses, considering variables associated with OS at an univariable P&amp;lt;.10 (MRD status, patient sex, therapy-related AML, conditioning intensity), and using stepwise backward variable selection to identify factors significantly associated with OS (Figure B). In the final multivariable model, pre-HSCT MRD detection remained associated with inferior OS (hazard ratio 2.51; 95% confidence interval, 1.07 - 5.91; P=.035). Female sex associated with favorable OS, while myeloablative conditioning (MAC) showed a borderline significant association with superior OS. Of note, we did not detect an interaction between conditioning intensity and detection of MRD with regard to OS, i.e. the prognostic association of MRD detection was not different between pts receiving myeloablative or less-intensive conditioning. However, this analysis was limited by the small patient number. Conclusion Detection of MRD by targeted NGS using the smMIPs approach provides prognostic information in pts undergoing allogeneic HSCT, including those without established molecular markers such as NPM1mut. This method is applicable to a large proportion of AML pts and is cost-efficient for broad clinical use. S.M.K. and T.H., and M.J. and K.H.M. contributed equally to this abstract.

Ibrutinib and Rituximab Versus Fludarabine, Cyclophosphamide, and Rituximab for Patients in NCRI FLAIR Study with Previously Untreated CLL: Assessment of Mutational Landscape of Patients at Baseline and Prognostic Impact

Introduction: Recent studies using next generation sequencing have revealed recurrent mutated genes in CLL are associated with clinical outcome. We assessed the baseline mutational profile of 771 patients from FLAIR treated with either fludarabine, cyclophosphamide and rituximab (FCR) or ibrutinib and rituximab (IR). The prognostic impact of individual gene mutations on disease progression was investigated. Method: FLAIR is a phase III, multicentre, randomised, controlled, open, parallel group trial for previously untreated CLL requiring therapy according to IWCLL criteria. Patients with &amp;gt;20% chromosome 17p deletion were excluded. Patients were randomised to FCR or IR. Somatic hypermutation (SHM) status was determined by PCR amplification of IGHV-IGHD-IGHJ gene rearrangements using IGHV leader/FR1 primers. Bidirectional Sanger sequencing was analysed using IMGT V-Quest and the ARResT/AssignSubsets tool. Extracted DNA was sequenced using an Illumina MiSeq and analysed using an in-house pipeline. Amplicon based targeted sequencing of 33 recurrently mutated genes in lymphoid malignancies were performed in parallel. Detected variants were reported down to minimum variant allele fractions of 3-5% and coverage of 100X. Low level variants were confirmed by repeat sequencing. Results: 771 patients were randomly assigned to receive FCR (385) or IR (386). IGHV gene SHM status was available for 728/771 (94.4%): 388 (53.3%) IGHV unmutated (&amp;gt;98% of nucleotide identity to germline), 294 (40.4%) IGHV mutated and 46 (6.3%) stereotyped subset #2 (n=28 IGHV mutated, n=18 IGHV unmutated). Gene mutations were assessed in 767/771 (99.5%) patients at baseline and mutations (&amp;gt;1) were detected in 480/767 (62.6%). Mutation frequencies ranged from 0.1-18.8% with mutations in SF3B1 (18.8%), ATM (14.5%), NOTCH1 (10.0%), MYD88 (6.1%), POT1 (5.7%), TP53 (5.0%), BRAF (4.4%) and RPS15 (4.0%) being the most frequent. No detectable mutations at baseline were found in 287/767 (37.4%) and there was no significant difference in PFS or OS compared to patients with &amp;gt;1 mutation. A significantly shorter PFS and OS was observed for TP53 mutated patients compared to wildtype (HR 2.23 [95% CI 1.31-3.79]; p=0.003 and HR 2.43 [95% CI 1.04-5.66]; p=0.039 respectively). When further subdivided by treatment, a trend for shorter PFS was observed in TP53 mutated patients in both arms compared to wildtype, but significance was only achieved for FCR (HR 2.48 [95% CI 1.25-4.91]; p=0.009) and not IR (HR 2.27 [95% CI 0.97-5.30]; p=0.059). A significantly shorter PFS was observed for FCR compared to IR in patients with ATM mutations at baseline (HR 0.35 [95% CI 0.16-0.80]; p=0.012) and RSP15 (HR 0.19 [95% CI 0.04-0.96]; p=0.044). A trend for shorter PFS was also observed in NOTCH1 mutated patients treated with FCR however, this was not found to be significant when compared to wildtype or NOTCH1 mutated IR patients. CREBBP mutation at baseline (n=19; 2.5%) was associated with shorter PFS (HR 2.63 [95% CI 1.29-5.35]; p=0.008) and OS (HR 4.37 [95% CI 1.75-10.94]; p=0.002) compared to wild type (Fig). When further subdivided by treatment arm, shorter PFS and OS was also observed for CREBBP mutated patients treated with FCR compared to wildtype (n=11; HR 4.37 [95% CI 1.75-10.94]; p=0.002 and HR 10.14 [95% CI 3.83-26.83]; p&amp;lt;0.001 respectively). A similar trend was not observed with IR (n=8; PFS HR 0.79 [CI 0.11-5.74]; p=0.819 and OS HR NE [95% CI NE-NE]; p=0.988). CREBBP mutated patients treated with FCR had a shorter PFS compared to IR (HR 0.1 [95% CI 0.01-0.86]; p=0.036). The PFS and OS for IGHV subset #2 was similar to IGHV mutated patients (Fig). In FCR treated patients there was no difference between mutated (n=13) and unmutated subset #2 (n=7) but with IR there was a trend that IGHV mutated subset #2 patients (n=15) had better PFS and OS than their IGHV unmutated counterparts. Conclusions: Our study confirms that TP53 mutations result in shorter PFS and OS in CLL. We demonstrate improved PFS when patients with ATM or RPS15 mutations are treated with IR compared to FCR. Mutations in the CREBBP gene have recently been described as a novel candidate driver in CLL. We report a significant improvement in disease progression in CREBBP mutated patients treated with IR when compared to FCR. In contrast to previous studies in frontline CLL IGHV subset #2 patients had a similar outcome to good risk IGHV mutated patients.

Malakoplakia with aberrant ALK expression by immunohistochemistry: a case report

BackgroundMalakoplakia is a rare inflammatory disease of the urogenital tract. There have been no reports of malakoplakia expressing anaplastic lymphoma kinase (ALK) to date. Here, we present one case of malakoplakia with aberrant ALK expression by immunohistochemistry and discuss the clinical significance.Case presentationA 65-year-old Chinese woman with a history of diabetes presented with solid masses in the liver and kidney and elevated lesions on the mucosal surface of the colon. Right nephrectomy and partial liver resection were performed. Microscopically, sheets of histiocytes with poor intercellular adhesion were seen, with Michaelis–Gutmann bodies present in both the intracellular and extracellular interstitium. CD10-, CD68-, and CD163-positive cells were present, with Michaelis–Gutmann bodies confirmed by staining with Alcian blue, periodic acid-Schiff (PAS), periodic acid-Schiff with diastase, Von Kossa, and Prussian blue. Aberrant ALK1 and ALK (D5F3) expression was observed in the cytoplasm and nucleus of cells. However, ALK gene mutation was not detected by fluorescence in situ hybridization or whole exome next-generation sequencing. NGS revealed nine individual somatic gene mutations: GOT1L1, GLIS2, SPOUT1, TMEM97, MUC3A, NSD2, SFXN5, ADAD1 and RAD50. The significance of the somatic gene mutations detected in this study is not clear, and the relationship between them and malakoplakia cannot be clarified by existing scientific studies. The pathological diagnosis was malakoplakia with aberrant ALK expression by immunohistochemistry. The antibiotics imipenem and vancomycin were started based on the results of drug sensitivity analysis and the patient was subsequently discharged. She experienced no discomfort during 30 months of follow-up.ConclusionThis is the first reported case of malakoplakia with aberrant ALK expression, it should be differentiated from ALK-positive histiocytosis to avoid misdiagnosis.

MDB-25. LARGE CELL/ANAPLASTIC MEDULLOBLASTOMA REPRESENTS MORE THAN ONE DISEASE ENTITY

Abstract Medulloblastoma (MB) with predominant large cell or anaplastic cytological features have been identified to be associated to an aggressive clinical course and poor survival. Because cases can show mixed cytologies, these histologically defined MB types were merged in the recent WHO classification of tumors of the CNS to large cell/anaplastic (LC/A) MB. In this study, we collected FFPE samples of 137 LC/A MB of all age groups (0-39 years) diagnosed at the DGNN Brain Tumor Reference Center in Bonn, Germany, and performed a comprehensive histological, immunophenotypical, genetic and epigenetic analysis by next-generation panel sequencing (NGS), 450k/850k methylation bead-array hybridization and molecular inversion probe (MIP) technology. 73% of cases showed predominant anaplastic histology, 27% predominant large cell histology. Methylation-based subtyping and sequencing showed that the cohort of LC/A MB represented different biological entities: 6% WNT-MB, 7% SHH-MB-TP53-wildtype, 10% SHH-MB-TP53-mutant, and 77% non-WNT/non-SHH MB (with predominant epigenetic subtype II). High-resolution copy number profiling by MIP demonstrated MYC amplification in 40% of non-WNT/non-SHH MB. MYC amplification was highly associated to infant disease and predominant large cell histology. On the contrary, predominant anaplastic histology was highly associated to non-infant disease, MYCN amplification and SHH activation. By NGS we detected recurrent mutations in 37 MB-related genes; the frequency of mutations of individual MB-related genes differed significantly between the histologically and genetically defined MB types. Our data clearly demonstrate that large cell MB and anaplastic MB represent different biological entities. Therefore, and also in respect to the future development of targeted treatment approaches to these highly malignant MB types, they should be precisely diagnosed as separate MB entities.

Evaluation of the influence of single-nucleotide polymorphisms of vitamin D receptor (rs2228570), BDNF (rs6265), and NMDA (rs4880213) genes on gene expression in different tissues

Background. Questions regarding the association of individual and combined gene variations and mutations with thyroid disease and nervous system disorders remain insufficiently researched and require further study to facilitate early diagnosis of nervous system damage on the background of thyroid pathology, disease prognosis, and timely treatment and prevention. An important issue is the identification of the influence of individual polymorphisms in these genes on the functional activity of cells, including gene expression. Currently, gene expression genetics largely depends on the identification of expression quantitative trait loci (eQTL), which are the links between gene expression and genotype at a locus. The purpose of the study was to search for eQTL in single nucleotide polymorphisms (SNPs) of the BDNF gene (rs6265), VDR gene (rs2228570), and NMDA gene (rs4880213). The results were presented as nominal p-values for each SNP of the BDNF, VDR, and NMDA genes. Materials and methods. We use publicly available databases (QTLbase: http://www.mulinlab.org/qtlbase/index.html, GTExPortal: https://gtexportal.org). Results. Using the QTLbase, we identified statistically significant (p≤0.05) associations of rs6265 with the expression of 17 genes (BDNF-AS, BDNF, LDHC, AC104563.1, BBOX1, SPTY2D1OS, YWHABP2, LINC00678, LIN7C, GTF2H1, METTL15, IMMP1L, KIF18A, HPS5, NAV2, LGR4, CCDC34) in various tissues. For rs4880213, we found a significant association with the expression levels of 49 genes (ARRDC1-AS1, TPRN, SSNA1, SAPCD2, UAP1L1, NPDC1, MAN1B1, PTGDS, SNHG7, NDOR1, TRAF2, PHPT1, EGFL7, EHMT1, RNF208, PNPLA7, LCNL1, DPP7, LCN12, STPG3, CCDC183-AS1, ABCA2, RNF224, ENTPD2, PAXX, CLIC3, C9orf163, LCN15, MAN1B1-DT, FAM166A, FAM166A, LRRC26, STPG3-AS1, AGPAT2, ANAPC2, DPH7, ZMYND19, NSMF, MRPL41, EXD3, TUBB4B, NELFB, ARRDC1, EDF1, FBXW5, DIPK1B, MAMDC4, RABL6, TMEM141, TMEM203) in 16 different tissues. Additionally, we identified statistically significant (p ≤ 0.05) associations of rs2228570 with the expression of 29 genes (ASB8, TMEM106C, KANSL2, DDX23, CCNT1, HDAC7, RPAP3, PFKM, SENP1, RND1, PCED1B, AC004466.1, AMIGO2, ZNF641, ENDOU, RAPGEF3, VDR, AC004241.1, AC004801.2, AC121338.1, LINC02354, SNORA2A, LINC02416, AC074029.3, AC004241.5, AC008083.3, COL2A1, CCDC184, SLC48A1) in 17 different tissues. Conclusions. Single nucleotide polymorphisms of the BDNF (rs6265), VDR (rs2228570), and NMDA genes (rs4880213) affect gene expression in various cells and tissues. The use of this extensive eQTL catalog provides an important resource for understanding the molecular basis of common genetic diseases.

Abstract 1183: Mutation type and location in breast cancer susceptibility genes are associated with differential risk in the general population

Abstract Two recent studies have defined population-based risk of breast cancer (BC) due to pathogenic variants (PVs) in well-established BC susceptibility genes. Prior studies in high-risk BC cohorts identified variable BC risks based on the position and type of mutation in BRCA1 and BRCA2, and based on the type of mutation in ATM, CHEK2, and PALB2. Whether this variability holds true in the general population has not been evaluated. To determine the BC risks associated with mutations of different types and in different locations in BC susceptibility genes in the general population, we carried out case-control analyses of 32247 cases and 32544 controls in the CARRIERS study, including 12 US population-based studies, and case-control analyses, subset to mutation carriers. We used age-adjusted logistic regression to estimate odds ratios (OR), relative OR (rOR), and 95% confidence intervals (CI) for PVs in BRCA1, BRCA2, ATM, CHEK2, and PALB2. ORs were estimated using the full population of mutation carriers and noncarriers while rORs were estimated within subsets of individual gene mutation carriers. We identified 273 BRCA1, 421 BRCA2, 253 ATM, 351 CHEK2, and 148 PALB2 in women with BC and 35 BRCA1, 84 BRCA2, 139 ATM, 139 CHEK2, and 38 PALB2 variant carriers in those without BC. For BRCA1, we did not find differences in risk by mutation type or location. However, variants leading to nonsense mediated decay (NMD) predicted to have re-initiation at p.M128 had a non-statistical 3-fold higher risk for BC compared to those leading to NMD without re-initiation (rOR 3.1, 0.99-13.52, p = 0.0826). BRCA2 loss of function (LoF) mutations in exons 1-10 had a higher BC risk compared to LoF mutations in the exon 11 (rOR 2.7, 1.1-7.9, p=0.048) ovarian cancer cluster region (OCCR). We did not detect any differences in risk by mutation type or location in ATM, CHEK2, or PALB2. CHEK2 missense variants p.I157T (rOR 0.5, 0.4-0.7, p &amp;lt;0.001) and p.T476M (rOR 0.5, 0.3 - 0.8, p=0.005) were associated with lower BC risk relative to CHEK2 c.1100delC (OR 2.5, 2.0-3.3, p&amp;lt;0.001). The CHEK2 c.444+1G&amp;gt;A splicing variant was associated with highest BC risk among common CHEK2 variants (OR 4.2, 1.9-10.5, p&amp;lt;0.001), but was not statistically significantly different from c.1100delC (rOR 1.7, 0.8-4.3, p=0.230). In summary, we identified a higher BC risk for LoF variants in BRCA2, lower risk for CHEK2 missense mutations, and non-statistical higher risk for NMD/re-initiation BRCA1 variants and CHEK2 c.444+1G&amp;gt;A in this population-based study. Despite the large numbers included in the overall CARRIERS study, the number of PV carriers was relatively small, which may have limited the detection of associations. Nonetheless, we were able to detect genotype-risk associations that have implications for counseling women about their BC risk, and certainly for the development of individualized risks. Citation Format: Mwangala P. Akamandisa, Nicholas Boddicker, Chunling Hu, Siddhartha Yadav, Jeffrey N. Weitzel, Peter Kraft, Susan M. Domchek, Fergus J. Couch, Katherine L. Nathanson, for the CARRIERS Consortium . Mutation type and location in breast cancer susceptibility genes are associated with differential risk in the general population [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 1183.

α- and β-Globin Gene Mutations in Individuals with Hemoglobinopathies in the Chattogram and Sylhet Regions of Bangladesh

Hemoglobinopathies, including α- and β-thalassemias and sickle cell disease, are among the most widely disseminated hereditary blood disorders worldwide. Bangladesh is considered a hotspot for hemoglobinopathies, and these diseases cause a significant health concern in the country. However, the country has a dearth of knowledge on the molecular etiology and carrier frequency of thalassemias, primarily due to a lack of diagnostic facilities, limited access to information, and the absence of efficient screening programs. This study sought to investigate the spectrum of mutations underlying hemoglobinopathies in Bangladesh. We developed a set of polymerase chain reaction (PCR)-based techniques to detect mutations in α- and β-globin genes. We recruited 63 index subjects with previously diagnosed thalassemia. Along with age- and sex-matched control subjects, we assessed several hematological and serum indices and genotyped them using our PCR-based methods. We identified that parental consanguinity was associated with the occurrence of these hemoglobinopathies. Our PCR-based genotyping assays identified 23 HBB genotypes, with the codons 41/42 (–TTCT) (HBB: c.126_129delCTTT) mutation leading the spectrum. We also observed the presence of cooccurring HBA conditions, of which the participants were not aware. All index participants in this study were on iron chelation therapies, yet we found they had very high serum ferritin (SF) levels, indicating inefficient management of the individuals undergoing such treatments. Overall, this study provides essential information on the hemoglobinopathy mutation spectrum in Bangladesh and highlights the need for nationwide screening programs and an integrated policy for diagnosing and managing individuals with hemoglobinopathies.

Abstract P040: Mutational heterogeneity of colorectal cancers across ancestries and its implications for cancer early detection

Abstract Introduction: Early cancer detection with liquid biopsies has the potential to reduce mortality rates by improving screening strategies, adoption, and adherence. Towards this, a deeper understanding of the cancer genomic landscape among patients of different ethnicities is critical to ensure comparable performance across a broad population. In this study, we sought to understand how the mutational landscape of patients with colorectal cancer (CRC) varies across ethnicities. Methods: A total of 20,845 de-identified patients with stage I-IV CRC, who had undergone commercial personalized and tumor-informed ctDNA testing (SignateraTM) as of April 14, 2022, were eligible for inclusion in the study. We performed ancestry inference using Principal Component Analysis on single nucleotide polymorphisms (SNPs) and validated the predictions in a subset of 2,032 patients with whole exome sequencing data available along with self-reported ethnicity. Individuals were assigned to one of four major ethnic groups: African (AFR), European (EUR), East Asian (EAS) and South Asian (SAS). Results: Our cohort consisted of 69.1% EUR, 9.6% AFR, 20% EAS, and 1.3% SAS patients. On comparing the somatic driver mutations across ancestry groups, we observed APC mutations in 71% of EUR, 79% of AFR, 80% of EAS and 69% of SAS (p&amp;lt;0.0001), KRAS mutations in 39% of EUR, 50% of AFR, 40% of EAS and 38% of SAS (p&amp;lt;0.0001), and TP53 mutations in 64% in EUR, 64% in AFR, 74% in EAS and 69% in SAS (p&amp;lt;0.0001) patients, respectively. The microsatellite instability (MSI) rates similarly varied across ancestral groups, with EUR ancestry (12.3%) having a significantly higher rate of MSI than AFR (8.9%) and EAS (7.5%) (p&amp;lt;0.0001). Comparing the mutational patterns in left vs. right colon across 712 patients with available tumor location information, we found 64% of tumors in EAS to be left sided vs. 49% in EUR (p=0.0002). Left-sided tumors were enriched for MSI and RAS mutations and right-sided tumors were enriched for APC and TP53 mutations, confirming known differences in left- vs. right-sided colorectal tumors. Among left sided tumors, we also observed TP53 mutations in 79% of EAS as compared to 64% of EUR (p=0.001), APC mutations in 84% of EAS as compared to 70% of EUR (p=0.002) and RNF43 mutations in 1% of EAS as compared to 7% in EUR (p=0.002) suggesting differences in mechanisms of WNT pathway inactivation across ethnicities. Finally, we looked at the predicted performance of a cancer screening panel which is based on large and heterogeneous data and maximizes recurrent mutation coverage while minimizing panel size. Despite the differences in individual driver gene mutation frequency, we found that the mean number of mutations covered by the panel in AFR, EAS and SAS individuals relative to EUR was 1.07, 1.1 and 1.02, respectively. Conclusions: Our results demonstrate that the expected performance of a theoretical cancer screening panel was similar across the different ancestry groups despite differences in their somatic landscape. Citation Format: Preethi Srinivasan, Boris Gutman, Vasily Aushev, Shruti Sharma, Taly Arbel, Hsin-Ta Wu, Meenakshi Malhotra, Raheleh Salari, Bernhard Zimmermann, Ryan Swenerton, Trupti Kawli, Breeana Mitchell, Matthew Rabinowitz, Alexey Aleshin, Johannes G. Reiter. Mutational heterogeneity of colorectal cancers across ancestries and its implications for cancer early detection. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P040.

Rare Variants in Systemic Lupus Erythematosus: From Monogenic to Polygenic Disease

The clinical and genotypic characterization of autoimmune diseases, including systemic lupus erythematosus (SLE), has made great strides recently as a result of tremendous advancements in gene sequencing technologies. Systemic lupus erythematosus is a complex multisystem disease characterized by high clinical variability due to abnormalities in both the innate and adaptive immune systems. Several genetic variants as well as environmental and hormonal factors have been identified, but the etiology of lupus is not fully understood yet. The ability of genome-wide association studies to scan thousands of individuals has enabled researchers to associate thousands of common variants to lupus. Common polymorphisms may jointly predispose to lupus, but their individual impact on the disease is minimal. It's becoming progressively more evident that rare mutations have a far higher influence. The role of rare variation in lupus has been the subject of intense research. Several approaches including genotyped-based follow-up of the variants in families, hierarchical screening, and imputation, have been applied to elucidate their functional involvement. Nevertheless, due to their rarity and the absence of standardized methodology, rare variants are still challenging to study. Most lupus patients present a polygenic form of the disease, which is defined by the complex interplay between genetic and environmental factors. Still, certain lupus patients and patients with lupus-like phenotypes might be affected by monogenic lupus, a group of disorders largely caused by individual gene mutation abnormalities. Although monogenic lupus is rare, it has been associated with a sizable number of genes in a range of pathways, mostly resulting in early-onset phenotypes. The study of rare variants causing monogenic lupus has resulted in incredibly useful breakthroughs in our understanding of the function of rare variants in the disease, nonetheless further research is still required.

Laminin-111 mutant studies reveal a hierarchy within laminin-111 genes in their requirement for basal epithelial tissue folding

The process of morphogenesis carefully crafts cells into complex organ structures which allows them to perform their unique functions. During brain development, the neuroepithelial tissue must go through apical and basal folding which is mediated through the instruction of both intrinsic and extrinsic factors. While much is known about apical folding, the mechanisms that regulate basal folding are less understood. Using the highly conserved zebrafish midbrain-hindbrain boundary (MHB) as an epithelial tissue model, we have identified the basement membrane protein laminin-111 as a key extrinsic factor in basal tissue folding. Laminin-111 is a highly conserved, heterotrimeric protein that lines the basal surface of the neuroepithelium. Laminin-111 is comprised of one alpha, one beta, and one gamma chain encoded by the genes lama1, lamb1, and lamc1, respectively. Human mutations in individual laminin-111 genes result in disparate disease phenotypes; therefore, we hypothesized that each laminin gene would have a distinctive role in tissue morphogenesis. Using zebrafish mutants for laminin-111 genes, we found that each laminin chain has a unique impact on basal folding. We found that lamc1 is the most critical gene for MHB morphogenesis, followed by lama1, and finally lamb1a. This hierarchy was discovered via three-dimensional single cell shape analysis, localization of myosin regulatory light chain (MRLC), and with analysis of MHB tissue folding later in development. These findings are essential for development of novel techniques in tissue engineering and to elucidate differences in human diseases due to specific chain mutations.

Construction and Validation of a Prognostic Model Based on mRNAsi-Related Genes in Breast Cancer.

Background Breast cancer is a big threat to the women across the world with substantial morbidity and mortality. The pressing matter of our study is to establish a prognostic gene model for breast cancer based on mRNAsi for predicting patient's prognostic survival. Methods From The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, we downloaded the expression profiles of genes in breast cancer. On the basis of one-class logistic regression (OCLR) machine learning algorithm, mRNAsi of samples was calculated. Kaplan-Meier (K-M) and Kruskal-Wallis (K-W) tests were utilized for the assessment of the connection between mRNAsi and clinicopathological variables of the samples. As for the analysis on the correlation between mRNAsi and immune infiltration, ESTIMATE combined with Spearman test was employed. The weighted gene coexpression network analysis (WGCNA) network was established by utilizing the differentially expressed genes in breast cancer, and the target module with the most significant correlation with mRNAsi was screened. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to figure out the biological functions of the target module. As for the construction of the prognostic model, univariate, least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analyses were performed on genes in the module. The single sample gene set enrichment analysis (ssGSEA) and tumor mutational burden were employed for the analysis on immune infiltration and gene mutations in the high- and low-risk groups. As for the analysis on whether this model had the prognostic value, the nomogram and calibration curves of risk scores and clinical characteristics were drawn. Results Nine mRNAsi-related genes (CFB, MAL2, PSME2, MRPL13, HMGB3, DCTPP1, SHCBP1, SLC35A2, and EVA1B) comprised the prognostic model. According to the results of ssGSEA and gene mutation analysis, differences were shown in immune cell infiltration and gene mutation frequency between the high- and low-risk groups. Conclusion Nine mRNAsi-related genes screened in our research can be considered as the biomarkers to predict breast cancer patients' prognoses, and this model has a potential relationship with individual somatic gene mutations and immune regulation. This study can offer new insight into the development of diagnostic and clinical treatment strategies for breast cancer.