Mutations Of Unknown Significance Research Articles

Mutations of Unknown Significance in the FOXO and EGFR Signaling Pathways in Patients With CML

Mutations of Unknown Significance in the FOXO and EGFR Signaling Pathways in Patients With CML

Genomic Surveillance and Evolutionary Dynamics of Influenza A Virus in Sri Lanka.

Influenza A has been named as a priority pathogen by the WHO due to the potential to cause pandemics. Genomic sequencing of influenza strains is important to understand the evolution of the influenza strains and also to select the appropriate influenza vaccines to be used in the different influenza seasons in Sri Lanka. Therefore, we sought to understand the molecular epidemiology of the influenza viruses in the Western Province of Sri Lanka, including mutational analysis to investigate the evolutionary dynamics. A total of 349 individuals presenting with fever and respiratory symptoms were enrolled in this study from November 2022 to May 2024. Nasopharyngeal and oropharyngeal specimens were collected and screened using quantitative PCR to detect Influenza A, Influenza B, and SARS-CoV-2. Subtyping and genomic sequencing was carried out on influenza A strains using Oxford Nanopore Technology. Influenza A was detected in 49 (14 %) patients, influenza B in 20 (5.7%) and SARS-CoV-2 in 41 (11.7%). Co-infections were observed in five participants. The phylogenetic analysis assigned the H1N1 HA gene sequences within the 6B.1A.5a.2a clade. The HA gene of the H1N1 sequences in 2023 were assigned as belonging to the subclades C.1, C.1.2, and C.1.8, while the 2024 sequences were assigned to subclades C.1.8 and C.1.9. The H3N2 sequences from 2023 were assigned to the 3C.2a1b.2a.2a.1b clade and subclade G.1.1.2, while the 2024 sequences were assigned to the 3C.2a1b.2a.2a.3a.1 clade and subclade J.2. The K54Q, A186T, Q189E, E224A, R259K, K308R, I418V, and X215A amino acid substitutions were seen in the H1N1 in the 2023 and 2024 sequences. The 2024 H1N1 sequences additionally exhibited further substitutions, such as V47I, I96T, T120A, A139D, G339X, K156X, and T278S. In this first study using genomic sequencing to characterize the influenza A strains in Sri Lanka, which showed different influenza A viruses circulating in an 18-month period. As the Sri Lankan strains also had certain mutations of unknown significance, it would be important to continue detailed surveillance of the influenza strains in Sri Lanka to choose the most suitable vaccines for the population and the timing of vaccine administration.

Brugada syndrome in Portugal: a cohort characterization

Abstract Introduction Brugada syndrome (BrS) is an inherited cardiac arrhythmic disorder that can lead to sudden death. Although SCN5A was the first pathogenic associated gene, other potential genes have been described. The relationship between SCN5A, spontaneous type 1 pattern and the predisposition to ventricular arrhythmias is not totally understood. Purpose To characterize mutations in a Portuguese cohort with BrS and explore the genotype-phenotype association in terms of electrocardiographic pattern and arrhythmic risk. Methods Prospective single-center study of patients (pts) with BrS. Genetic test included SCN5A direct sequencing from 2003 a broad panel of 120 genes associated with cardiomyopathies and arrhythmic disorders from 2018. Genetic test results were classified according to pathogenicity: mutations of unknown significance (MUS), mutations potentially pathogenic (MPP) and known pathogenic mutations (KPM). Kaplan-Meyer survival analysis was used to explore the association between genetic test results and the risk of arrhythmic events. Results A total of 94 pts [46±12 years, 67% male,64.9% with type 1 spontaneous pattern] were submitted to genetic testing. No relevant mutations were identified in 68pts and suspicious or pathogenic mutations were recognized in 26pts. The most frequent mutations occurred in SCN5A (N=20,76.9%), and included 8 KPM: 4 in exon 23 (3 of them had the same mutation,c.4018G>A), 2 in exon 26 (c.4534C>T), 1 in exon 28 and 1 in exon 16. All MPP in SCN5A occurred either in exon 23 or 28. Considering MUS, different mutations were described in 6 different exons (8,15,18,23,25 and two in 28). Additionally, 6 pts presented MUS in other genes: SCN10A (N=2), ANK2 (N=2), SFM13A, CAV2 (associated with long QT), CACNA1D and PXDNL. We found no differences in the prevalence of spontaneous type 1 pattern considering gene mutation or mutation pathogenicity. A non-significant trend to higher arrhythmic risk was observed in pts presenting genetic mutations (either KPM, MPP or MUS), Long-Rank: 1.743, p=0.187. Conclusion Gene mutations are identified in a minority of BrS pts, mostly at SCN5A gene. No association was noticed between genetic test results and the ECG pattern. However, pts with identified mutation presented a tendency to higher arrhythmic risk. At present time, genetic tests in BrS are only relevant for familial screening, but long-term collection of data is crucial to elucidate the genotype-phenotype relation and arrhythmic risk. Funding Acknowledgement Type of funding sources: None.

Long-term risk of arch complications in Loeys Dietz syndrome patients undergoing proximal ascending aortic replacement.

Loeys-Dietz syndrome (LDS) is a rare connective tissue disorder. In LDS patients with normal arch morphology, whether the arch should be prophylactically replaced at the time of proximal aortic replacement remains unknown. We evaluated the risk of long-term arch complications in genetically confirmed LDS patients who underwent proximal ascending aortic replacement. We retrospectively reviewed the records of patients with LDS who have been followed at our institution between 1994 and 2020. Patients were only included if whole exome genetic testing confirmed a mutation in an LDS-causing gene (TGFBR1, TGFBR2, SMAD3, TGFB2, or TGFB3). Mutations were categorized as pathogenic, benign, or of unknown significance. We collected demographic information, aortic dimensions, comorbidities, mortality, and operative course from patients' charts. Descriptive statistics and freedom from reoperation plots were generated. Of the 18 patients with a mutation in an LDS-causing gene, 15 had known pathogenic variants, two had mutations of unknown significance, and one had a benign genetic variant. For the 15 patients with confirmed pathogenic variants of LDS the median follow-up duration was 5 years (interquartile range [IQR]: 4-8). Eleven patients underwent ascending aortic replacements (AAR) ± aortic valve replacement. Two patients required an additional operation; one required arch and staged elephant trunk for a dissection 18 years post-AAR and the other patient required an isolated descending aortic replacement for dissection 5 years post-AAR. Among patients who underwent surgery, the median ascending aortic diameter at intervention was 5.0 cm (IQR: 4.3-5.3). There was no surgical or late follow-up mortality observed for any of the 18 patients in the study. LDS patients who underwent proximal aortic replacement appeared to have low long-term risk of arch complications. While our study is somewhat limited by its sample size and follow-up duration, it suggests that routine prophylactic total arch replacement may not be warranted in LDS patients with nonaneurysmal aortic arches.

Abstract 2975: RAS precision medicine transatlantic partnership: Exploration of RAS and NF1 co-mutations in NSCLC

Abstract Background: RAS is the most commonly mutated oncogene in cancer, with KRAS mutated in ~30% of non-small cell lung cancer (NSCLC). RAS is a small GTPase cycling between GTP-bound ‘ON’ state and GDP-bound ‘OFF’ state. KRAS oncoproteins cycle between these states via hydrolysis and nucleotide exchange with the aid of GAPs, including NF1, and GEFs. Given the ’inactive state’ inhibition of recently developed KRAS G12C inhibitors on the GDP-bound state, this has highlighted the continued reliance of KRAS-mutants upon cycling, and the potential for mutants to retain dependence upon upstream influences, including NF1, for pathogenicity. Methods: 474 patients with advanced RAS- and/or NF1-mutant NSCLC were retrospectively identified from four tertiary cancer centers between 2008 -2021. DNA from archival FFPE samples, serum or combination underwent targeted NGS panels to identify mutations. Molecular, clinical, pathological and treatment outcome data were collected. Online resources including cBioPortal and Project Achilles were used to assess the functional role of any findings. Results: KRAS mutations were identified in 416/474 patients and NF1-mutations in 63/474 patients, eight of whom harbored two NF1-mutations. 24/63 (38%) of NF1-mutant cancers had a concomitant KRAS-mutation. We identified that KRAS G13D was more prevalent in NF1 mutant cancers vs. NF1 wildtype (NF1 MT: 6/24, 25%; vs. NF1 WT: 4/281, 1.4%; p<0.0001). KRAS G12C was identified in 11/24 (45.8%) of the double mutants vs. 109/282 (38.7%) of the NF1 WT patients (p=0.52). Those with G13D/NF1 co-mutation had a predicted pathogenic NF1-mutation in 5/6 (83%) of cases. Functional analysis of NF1 KO in G13D mutant lung cancer cell lines identified that NF1 was more essential in G13D mutant cell lines, with median Chronos score -0.26 G13D vs. -0.04 G12C (p=0.02). mRNA expression data identified a range of mRNA expression Z-scores relative to all samples for NF1-mutant cancers, ranging from lowest expression at -4.54 to +1.63, median -0.51. A higher mRNA expression was identified for NF1 missense mutations of unknown significance compared to truncating mutations with likely pathogenic, loss of function. Conclusions: These results highlight the co-mutational landscape of KRAS G13D with NF1 in NSCLC, suggesting functional importance conferred by NF1 loss, and highlight NF1 mutations as an additional pathogenic even when combined with KRAS mutation. The genomic landscape of KRAS and NF1 mutant NSCLC will be explored further through Whole Genome Sequencing data from the 100,000 Genomes Project (Genomics England). Citation Format: Helen M. Adderley, Mihaela Aldea, Jaqueline Aredo, Mathew Carter, Matthew Church, Pantelis Nicola, Jamie Weaver, Aisha Ghaus, Damien Vasseur, Matthew Krebs, Nicola Steele, Fiona Blackhall, Heather Wakelee, Benjamin Besse, Colin Lindsay. RAS precision medicine transatlantic partnership: Exploration of RAS and NF1 co-mutations in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2975.

Abstract 2143: Clinicopathologic, genomic and immunophenotypic landscape of ATM mutations in non-small cell lung cancer

Abstract Introduction: Defective DNA damage repair machinery is a hallmark of cancer, resulting in increased mutation rates and genomic instability. In non-small cell lung cancer (NSCLC), ATM is mutated in ~10% of cases, representing the most commonly mutated DNA damage and repair gene. However, the clinicopathologic, genomic, and immunophenotypic correlates of ATM mutations in NSCLC are unknown. The impact of ATM mutation on clinical outcomes to PD-(L)1 blockade is also unclear. Methods: Clinicopathologic and genomic data were collected from 3592 patients (pts) with NSCLC who had consented to correlative studies at the Dana-Farber Cancer Institute (DFCI), and whose tumors underwent genomic profiling (OncoPanel). Multiplexed immunofluorescence (mIF) for CD8, PD1, PD-L1, FOXP3, and CK AE1/AE3 was performed on a subset of 416 NSCLC samples to examine tumor-infiltrating immune cells. ATM immunohistochemistry (IHC) was also performed on 184 ATM mutated NSCLCs with available tissue. ATM mutated (ATMMUT) tumors were defined as harboring loss-of-function mutations (nonsense, frameshift, splice site, known deleterious missense mutations). Missense mutation of unknown significance were excluded, unless deemed to affect protein function in silico. Tumors lacking ATM mutations or harboring benign ATM alterations were defined as ATM wild type (ATMWT). Results: A total of 399 deleterious ATM mutations were identified in 10.2% (365/3592) of samples; 138 (34.6%) mutations were truncating (nonsense, frameshift, and splice site mutations); the remaining 261 (65.4%) were missense mutations. Truncating mutations were significantly more likely to result in ATM loss by IHC compared to missense mutations (71.4% vs 28.9%, P<0.01) When we examined the genomic profiles of tumors with versus without deleterious ATM mutations, we found that ATMMUT NSCLCs were significantly enriched with KRAS, STK11, RBM10, and KDM5C co-mutations (P<0.01), while co-mutations in EGFR, CDKN2A and TP53 were nearly mutually exclusive (P<0.01). Among ATMMUT NSCLCs, those with ATM loss by IHC were significantly enriched with KRAS and STK11 co-mutations, while those with retained ATM expression were enriched with TP53 co-mutations (P<0.01). Pts with ATMMUT NSCLCs had similar outcomes to PD-(L)1 inhibition +/- chemotherapy, compared to ATMWT cases, and similar immune cell subsets infiltration (P>0.05). Pts with deleterious mutations in ATM and TP53 (ATMMUT/TP53MUT) had increased response rates to chemo-immunotherapy compared to those with ATMMUT/TP53WT, ATMWT/TP53MUT, or ATMWT/TP53WT genotypes (70% vs 56.2% vs 35.7% vs 27.4%, respectively, P=0.01), as well as increased tumor-stroma interface CD8+ T cells (P<0.01) and higher PD-L1 expression by mIF on tumor (P<0.01) and immune cells (P<0.01). Conclusion: Deleterious ATM mutations defined a subset of NSCLC with unique clinicopathologic, genomic, and immunophenotypic features. Citation Format: Biagio Ricciuti, Joao Victor Alessi, Xinan Wang, Arrien A. Bertram, Victor R. Vaz, Mizuki Nishino, James Lindsay, Kristen D. Felt, Bijaya Sharma, Lynette M. Sholl, Rodig Scott, Mark M. Awad, Michael L. Cheng. Clinicopathologic, genomic and immunophenotypic landscape of ATM mutations in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2143.

Abstract P5-03-09: Multigene panel analysis in breast and ovarian cancer

Abstract Background: Genetic testing is of prognostic and predictive importance in patients with ovarian cancer and breast cancer. The best explored mutations are those of the BRCA 1 and BRCA2 genes, and the result of BRCA testing impacts treatment and prophylactic options. Data on other breast-cancer-associated genes are accumulating. Methods: All patients who underwent genetic counseling and testing for BRCA 1 and BRCA 2 between 2007 and 2019 at the Medical University of Graz were reviewed. If BRCA testing was negative patients were offered panel testing for other breast and ovarian cancer associated genes including ATM, PETEN, RAD50, RAD51D, RAD51C, TP53, STK11, ATM, NBN, CDH, MSH6, MSH2, MLH1, CHEK2, PALB2, BRIP1, SMO, PETEN, SMO. Results: In total 1905 patients were counseled for genetic testing between 2007 to 2019. In 292 patients (mean age 53yrs) panel gene testing was performed. 114 (39%) of these 292 BRCA-negative patients (55 with breast cancer, 24 with ovarian cancer) had a pathogenic variant (n=32) or a mutation of unknown significance (n=82). The most common pathogenic variant occurred in the CHEK2 gene (n=15). Interpretation: In this series approximately one third of high-risk patients who tested negative for a BRCA mutation had a mutation of unknown significance and/or pathogenic mutation in gene panel testing. This indicates that genetic panel testing should be considered more frequently. Citation Format: Elisabeth Katharina Trapp, Michael Speicher, Jochen B Geigl, Vassiliki Kolovetsiou-Kreiner, Julia Reisinger, Josef Haas, Edgar Petru, Karl Tamussino, Gunda Pristauz. Multigene panel analysis in breast and ovarian cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-03-09.

Abstract 3166: Evaluation of genetic modulation of ACVR1 (aka ALK2) kinase gene as a clinical biomarker of the ACVR1 inhibitor TP-0184

Abstract The receptor kinase ALK2, the product of the ACVR1 gene, is a member of the bone morphogenic protein (BMP) receptor family and a type I receptor of the greater TGFβ family. Germline mutations in ACVR1, and specifically the R206H manifestation, are a driving factor in the development of fibrodysplasia ossificans progressiva (FOP). Additionally, the childhood brain tumor, diffuse intrinsic pontine glioma (DIPG), is also associated with mutations in the ACVR1 gene including R206H, but accounts for approximately only 25% of DIPG. In adult cancers, mutations in the ACVR1 gene are observed, yet their role is less established. TP-0184 is a small molecule inhibitor of ACVR1 which inhibits the kinase activity of wild-type and mutant forms of ALK2. We examined the genetic make-up of ACVR1 mutations across large data sets to evaluate which mutations may portend benefit with TP-0184 treatment. Using publicly available sequence databases (e.g. cBioPortal, Cosmic, TCGA) we searched for ACVR1 mutations in tissues of cancer patients. Results were compared to the known mutations of ACVR1 in the literature associated with various cancers or demonstrate gain-of-function activity. Multiple mutations were found within ACVR1. The FOP community has done extensive work to identify and understand mutations that can drive aberrant activation of ACVR1. These mutations include L196P, R206H, Q207E, R258S, G328E/R, and G356D to name a few. ACVR1 mutations identified in adult cancers include many of these FOP mutations, but also demonstrate more diversity in their ACVR1 mutational profile with fewer “hotspots” and many mutations of unknown significance. For example, 8.49% (45/530) of endometrial cancer patients in the TCGA database have ACVR1 mutations, yet these are represented by 52 different mutations. The most common ACVR1 mutation in endometrial cancer is the bona fide R206H mutation, but it only makes up 0.9% of the 8.49% of ACVR1 mutations in endometrial cancer. If all ACVR1 mutations of known significance are considered, 2.45% of endometrial cancers have validated gain-of-function mutations in ACVR1. The remaining ACVR1 mutations in endometrial cancer are of unknown significance. Similar results were observed in other types of cancer such as melanoma, prostate and breast cancer and in other publicly available databases. The results from surveying these rare mutations in multiple cancer types will be presented. Since the BMP and TGFβ pathways are frequently dysregulated in cancers, the importance of any mutation that regulates ACVR1 activity in cancer may be an important regulator of tumorigenesis and a target of ACVR1 inhibitors. A Phase I trial with TP-0184 is in progress and will assess the correlation of ACVR1 mutations and compound clinical activity (Clinical trial information: NCT03429218). Further studies are in progress to assess the consequences and causal impact of ACVR1 mutations in cancer. Citation Format: Mark L. Wade, C Lars Mouritsen, Yuta Matsumura, Breeann V. Bryan, David J. Bearss, Steven L. Warner. Evaluation of genetic modulation of ACVR1 (aka ALK2) kinase gene as a clinical biomarker of the ACVR1 inhibitor TP-0184 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3166.

Somatic mutations in non-small cell lung cancer and their correlation with overall survival, tumor mutation burden, and programmed-death cell ligand-1 (PD-L1) status.

e14752 Background: In recent years, treatments for non-small cell lung cancer (NSCLC) have expanded to include targeted therapies and immune checkpoint inhibitors in addition to traditional treatments. These newer modalities of treatment require testing with next-generation sequencing (NGS) testing, which offers testing for hundreds of somatic mutations, both with known and unknown significance. Tumor mutation burden (TMB), which is the number of mutations per coding sequence, is also reported on NGS. Patients with targetable mutations or a high TMB have been found to have improved survival and higher responses to immunotherapy, respectively. We wanted to investigate whether the total number of mutations (both with known and unknown significance) had any association with PD-L1 status, TMB and overall survival. Methods: This was a single institution, retrospective study of 51 patients with stage I-IV NSCLC. Results: In our study, we found that there was a significant correlation between the number of mutations and TMB in all patients with NSCLC (p = 0.001), including adenocarcinoma (p = 0.005). We found that the number of known mutations, mutations of unknown significance and total overall mutations did not have a significant correlation with survival (p = 0.708, p = 0.808, and p = 0.639, respectively) or PD-L1 status (p = 0.214). In a subset analysis of patients with metastatic NSCLC, there was also no correlation between the total number of mutations and overall survival (p = 0.821). Conclusions: In our study, we determined that there was a positive correlation between TMB and higher number of mutations, though the number of mutations did not correlate with PD-L1 status or overall survival. As TMB is associated with higher immunogenic responses, this data may have future therapeutic implications. Additional investigation of the most frequent mutations is ongoing.

Germline mutation landscape of Chinese patients with familial breast/ovarian cancer in a panel of 22 susceptibility genes.

Genetic testing for germline mutations in BRCA1/2 of patients with breast cancer (BC) is part of routine patient care. However, BRCA1/2 mutations account only for a fraction of familial BC. A custom panel of 22 gene sequencing was performed on each patient. Among the 481 female patients, 135 patients were detected to carry pathogenic (P)/likely pathogenic (LP) mutations (28.1%), which corresponded to 12 different cancer predisposition genes [14.6% (70/481) on BRCA1 gene, 5.0% (24/481) on BRCA2 gene, 8.5% (41/481) on non‐BRCA1/2 genes]. Moreover, 24.7% (119/481) of patients had mutation of unknown significance (VUS) in these genes. The most common (8/481) pathogenic mutation is BRCA1 c.5470_5477del, while BRIP1 2392 C > T of patients was detected. All the mutations detected were mainly seen in the homologous recombinant repair pathway. Compared to BRCA2 mutation, BRCA1 mutation is higher in younger female patients (P < 0.01). Some pathogenic mutations were detected in the patients’ familiy members without the past history of tumor and 92 novel mutations were detected (31 on BRCA including 2 P, 16 LP, 13 VUS; 61 on non‐BRCA1/2 including 9 LP, 52 VUS). The detection rate of BRCA1/2 mutations was higher in patients with three or more cancer family members than those with one or two. However, the difference was not statistically different. The results suggest that multigene panel testing can increase mutation detection rate for high‐risk BC patients. Detailed family history can help to categorize new mutations.

Abstract P4-04-07: High-throughput barcode screening elucidates the functional characteristics and mutual relevance of PIK3CA and PIK3R1 somatic mutations in Chinese patients with breast cancer

Abstract Background Deregulation of the phosphoinositide 3-kinase (PI3K) signaling pathway is essential to malignant cellular processes of breast cancer, including proliferation and drug response. Oncogenic somatic mutations of the PI3K pathway are pervasive in breast cancer. However, identification of impactful mutations and determination of their relevance to major components of this pathway remains difficult. This study was conducted to identify the landscape of somatic mutations in the PI3K pathway in a Chinese population. Notably, we developed a recombination-based mutation barcoding (ReMB) library which enables a high-throughput mutation-phenotype screens for vulnerable mutations that contribute to the cancer development and drug resistance. Methods We collected 149 breast cancer specimens in a Chinese population and performed Ion Torrent Amplicon Sequencing for the key genes in PI3K/AKT pathway: PIK3CA, PIK3R1, AKT1, AKT2, AKT3, PTEN, PDK1 at 1000× coverage. To discriminate between 'driver' and 'passenger' mutations, we developed a recombination-based mutation barcoding (ReMB) library that contained the novel identified mutations and that reported in TCGA and COSMIC databases in PIK3CA and PIK3R1 genes, each mutation tagging with a specific barcode to identify their potential oncogenic and drug-resistant characteristics. The unique barcode representing each mutation was detected using Illumina Miseq sequencing following proliferation and drug response selection (doxorubicin and BKM-120) assays to screen the functional mutations. Results We identified that mutations in PIK3CA (44%), PIK3R1 (17%), AKT3 (15%) and PTEN (12%) were prevalent and diverse in Chinese patients with a high proportion of tumours harboring multiple mutations, especially PIK3CA plus PIK3R1 mutations (9.0%). With ReMB screening, we found 11 non-synonymous impactful mutations in PIK3CA; these included eight proliferation-driving mutations, nine doxorubicin-resistant mutations, and eight BKM120-resistant mutations. The highest-ranking PIK3CA mutations include the deleterious mutations E542K, E545K and H1047R/L as well as mutations of unknown significance, including E39K, N345I, E453K, and G1049R. We also identified six non-synonymous impactful mutations inPIK3R1, including five proliferation-driving mutations, six doxorubicin-resistant mutations, and five BKM120-resistant mutations. The PIK3R1 impactful mutations include E160D, Q329L, N564D and K674R. Most impactful mutations in PIK3CA and PIK3R1 occurred at residues lying at the interfaces between p110α and p85α, or between the functional domains within p110α. These PIK3CA and PIK3R1 impactful mutations exhibit a mutually exclusive pattern, leading to oncogenesis and hyperactivity of PI3K pathway. Additionally, impactful mutations in PIK3CA are tightly associated with hormone receptor positivity. Conclusion This study identified the landscape of somatic mutations in the PI3K pathway in Chinese breast cancer patients. A novel developed ReMB screening platform allows the rapid identification of impactful PIK3CA and PIK3R1 mutations in breast cancer and has important implications for PI3K-targeted therapy. Citation Format: Chen L, Yang L, Hu X, Shao Z. High-throughput barcode screening elucidates the functional characteristics and mutual relevance of PIK3CA and PIK3R1 somatic mutations in Chinese patients with breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-04-07.

Screening for Wiskott-Aldrich syndrome by flow cytometry

Screening for Wiskott-Aldrich syndrome by flow cytometry

Long Term Responses to Rigosertib in Lower-Risk Myelodysplastic Syndromes (MDS) Patients

Abstract A fraction of lower risk MDS patients benefit from approved therapies but generally for a limited duration, and several require experimental therapies.1 Rigosertib, a small molecule inhibitor of RAS effector pathways, has been tested in phase I and II trails at our center. We present case histories of two patients with lower-risk MDS who experienced a complete response to Rigosertib which persisted for more than five years. Case 1: A 63-year-old Hispanic female with history of aplastic anemia and PNH since 1990 was diagnosed with low grade MDS in 2011. She was previously treated with mycophenolate mofetil, cyclosporine, and prednisone, which improved her counts. Subsequently, the counts deteriorated and she became heavily transfusion dependent for both red cells and platelets; WBC 1.5 × 103/μL, hemoglobin 7.2 g/dL, and platelet count 11,000/μL. A bone marrow biopsy in 4/11 showed a hypo- to normocellular marrow, subtle dysplastic changes. In August 2011, she started a phase 1 clinical trial of Rigosertib (ON01910.Na) taking 570mg twice a day for 14 of 21-day cycles. She responded well to rigosertib, and within three months, she stopped requiring blood and platelet transfusions (Fig 1A). After 18 months of treatment, she came off the study and has continued to have normal blood counts since. The latest CBC on July 10, 2017 showed WBC 3.8, Hb 12.8 and platelets 146,000. BM from the same day showed a mildly hypocellular (20-30%) marrow with patchy trilineage hematopoiesis and relative expansion of erythroid colonies. Myeloid elements and megakaryocytes were reduced. Megakaryocytes were not dysplastic. Cytogenetics were normal. She enjoys a normal quality of life. Case 2: A 78-year-old white male was diagnosed with refractory cytopenia with multilineage dysplasia in January 2009. He had low blood cell counts five years prior to diagnosis, becoming progressively worse over time. A BM showed multi-lineage dysplasia, no increase in blasts and normal cytogenetics. He initially had a good response to 40,000 and then to 60,000 unit erythropoietin (EPO) per week for about a year but became transfusion dependent. In 2010, he started Revlimid with an initial response but treatment was discontinued due to severe rash. In March 2011, he began the same phase 1 clinical trial as patient 1. He was instructed to take investigational drug Rigosertib (ON01910.Na) 280mg caps (60 tablets) 2 caps along with 70 mg caps (60 tablets) twice a day for 14 days of the 21-day cycle. He also started darbepoeitin 500mg every two weeks. Within 8 weeks of starting Rigosertib, he became transfusion independent and remained so for almost six years (Fig 1B). Because of periodic episodes of dysuria he discontinued rigosertib in October 2013 but continued darbepoetin intermittently. Gene sequencing in May 2014 showed pathogenic somatic mutations in ASXL1 and U2AF1 and mutations of unknown significance in SRSF2 and PHF6 . genes. The patient remained transfusion independent until June 2017 when he required a transfusion. He received darbepoetin 500ug on 4/4, 4/18, 5/2, 5/16, 5/30, 6/13, and 7/5/17. In the last 8 weeks, he has required multiple transfusions. While it is possible that the long term response in this case is related to Epo rather than Rigosertib, the patient had clearly become transfusion dependent after losing response to Epo. He only regained transfusion independence when rigosertib was added to the regimen. Conclusion: The precise mechanism of action of Rigosertib in lower risk MDS remains unknown, but it has been proposed that the overall biological effects appear to be mediated by direct binding of the compound to the Ras-binding domain (RBD) found in many Ras effector proteins involved in signal transduction, including Raf kinases and PI3K. Upon treatment with Rigosertib, cells either undergo apoptosis or differentiation, thereby potentially reducing the clone size and producing a clinical response. Despite known point mutations in RAS in solid and liquid malignancies, it has been a difficult target. The allosteric mechanism by which Rigosertib blocks signaling proteins downstream of RAS represents an exciting novel approach for making this oncogene into a “druggable” target. The depth of responses in these two cases as well as the duration, suggest that Rigosertib exerts its effects at a stem cell level Download : Download high-res image (277KB) Download : Download full-size image Disclosures Raza: Janssen RD Celgene Inc.: Research Funding; Genoptix: Speakers Bureau; Novartis: Speakers Bureau; Kura Oncology: Research Funding; Onconova Therapeutics: Research Funding, Speakers Bureau; Syros Pharmaceuticals: Research Funding. Heaney: Onconova Therapeutics: Research Funding; Novartis: Consultancy. Ali: Onconova Therapeutics: Consultancy; Kura Oncology: Consultancy.

LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated ovarian cancers.

Endometrioid (ENOC) and clear cell ovarian carcinoma (CCOC) share a common precursor lesion, endometriosis, hence the designation endometriosis associated ovarian cancers (EAOC). Long interspersed nuclear element 1 (LINE-1 or L1), is a family of mobile genetic elements activated in many cancers capable of moving neighboring DNA through 3' transductions. Here we investigated the involvement of specific L1-mediated transductions in EAOCs. Through whole genome sequencing, we identified active L1-mediated transductions originating within the TTC28 gene in 34% (10/29) of ENOC and 31% (11/35) of CCOC cases. We used PCR and capillary sequencing to assess the presence of specific TTC28-L1 transductions in formalin-fixed paraffin-embedded (FFPE) blocks from six different anatomical sites (five tumors and one normal control) for four ENOC and three CCOC cases, and compared the results to the presence of single nucleotide variations (SNVs)/frame shift (fs) mutations detected using multiplex PCR and next generation sequencing. TTC28-L1 mediated transductions were identified in at least three tumor samplings in all cases, and were present in all five tumor samplings in 5/7 (71%) cases. In these cases, KRAS, PIK3CA, CTNNB1, ARID1A, and PTEN mutations were found across all tumor sites while other selected SNV/fs mutations of unknown significance were present at varying allelic frequencies. The TTC28-L1 transductions along with classical driver mutations were near ubiquitous across the tumors, suggesting that L1 activation likely occurred early in the development of EAOCs. TTC28-L1 transductions could potentially be used to determine clonal relationships and to track ovarian cancer progression.

Next generation sequencing of non-muscle invasive bladder cancer to reveal potential biomarkers and rational therapeutic targets.

302 Background: We examined a cohort of index pre-treatment NMIBC tumors using Next Generation Sequencing to identify genetic alterations with potential clinical implications. Methods: 105 patients on a prospective IRB-approved protocol had their pre-treatment index NMIBC tumor and matched germline DNA sequenced with a 341 cancer-associated gene panel in a CLIA-certified clinical laboratory. A genitourinary pathologist reviewed representative H&amp;E slides to confirm grade, stage, and urothelial histology. Restaging TUR was performed in all HGT1 tumors. Results: To characterize the genomic landscape of NMIBC, we analyzed 105 tumors across the disease spectrum including LGTa (n = 23), HGTis (n = 12), HGTa (n = 32) and HGT1 (n = 38). The most frequently altered genes in NMIBC were the TERT promoter (74%), FGFR3 (50%), KDM6A (47%), ARID1A (28%), PIK3CA (27%), KMT2D (24%), STAG2 (21%), and CDKN2A (17%). 81% of tumors had inactivating alterations in a chromatin-modifying gene. Alterations in the RTK/RAS/PIK3 pathway occurred in 83% of tumors, including 58% of high-grade NMIBC having alterations in either ERBB2 or FGFR3. Of the 105 patients, 62 were treated uniformly with a 6-week induction course of BCG without maintenance. We investigated all genes altered on the 341-gene panel in at least 5 patients for their association with recurrence after BCG therapy in this 62 patient cohort. On cox-regression analysis, only truncating mutations in the chromatin-modifying gene ARID1A were associated with recurrence after BCG (HR = 3.14 [95%CI = 1.51, 6.51] p = 0.002). This remained significant when adjusting for multiple comparisons (p = 0.04) and when including ARID1A missense mutations of unknown significance (p = 0.002). Conclusions: Next Generation Sequencing of index pre-treatment NMIBC tumors identified an association between ARID1A mutations and recurrence after BCG therapy. Further investigation is needed to determine whether ARID1A mutations are a potential predictive/prognostic biomarker or therapeutic target. Moreover, most NMIBC tumors had at least one potentially “actionable” alteration that could serve as a target in rationally designed trials of intravesical or systemic therapy.

Novel CDKN2A mutations in Austrian melanoma patients.

CDKN2A is the most prominent familial melanoma gene, with mutations occurring in up to 40% of the families. Numerous mutations in the gene are known, several of them representing regional founder mutations. We sought to determine, for the first time, germline mutations in CDKN2A in Austria to identify novel mutations. In total, 700 individuals (136 patients with a positive family history and 164 with at least two primary melanomas as the high-risk groups; 200 with single primary melanomas; and 200 healthy individuals as the control groups) were Sanger sequenced for CDKN2A exon 1α, 1β, and 2. The 136 patients with affected relatives were also sequenced for CDK4 exon 2. We found the disease-associated mutations p.R24P (8×), p.N71T (1×), p.G101W (1×), and p.V126D (1×) in the group with affected relatives and p.R24P (2×) in the group with several primary melanomas. Furthermore, we discovered four mutations of unknown significance, two of which were novel: p.A34V and c.151-4 G>C, respectively. Computational effect prediction suggested p.A34V as conferring a high risk for melanoma, whereas c.151-4 G>C, although being predicted as a splice site mutation by MutationTaster, could not functionally be confirmed to alter splicing. Moreover, computational effect prediction confirmed accumulation of high-penetrance mutations in high-risk groups, whereas mutations of unknown significance were distributed across all groups. p.R24P is the most common high-risk mutation in Austria. In addition, we discovered two new mutations in Austrian melanoma patients, p.A34V and c.151-4 G>C, respectively.

Abstract 2175: A diagnostic gene expression assay for the classification of pheochromocytoma

Abstract Background/Aims: Pheochromocytoma (PCC) is a neoplastic growth of adrenal or extra-adrenal chromaffin cells. PCC is one of the most heritable cancers with up to 35% of cases attributed to a germline mutation in one of 13 or more genes. Previous microarray gene-expression studies have observed that PCC clusters into two or more subtypes corresponding to the underlying mutations that cause either dysregulation of hypoxia inducible factor (HIF) (e.g. VHL, SDHx) or constitutive activation of receptor tyrosine kinase (RTK) signalling (e.g. RET, NF1). The aim of this study was to conduct an unbiased assessment of the number of gene-expression subtypes of PCC using a compendium of published gene-expression data. Furthermore, we aimed to design a gene-expression based diagnostic assay to accurately classify tumours as an adjunct to clinical mutation analysis and for research purposes. Methods: Consensus clustering was applied to a gene-expression compendium of 253 samples to assess the most stable number of subtypes. Differential gene-expression analysis was performed to identify genes specific to each class. The expression of these genes in the microarray data was used to train a cross-platform k-NN classifier. The classifier was tested on an independent RNA-seq dataset to assess accuracy. Forty-seven genes (plus five control genes) were selected for Nanostring gene-expression analysis. The Nanostring assay was performed on RNA isolated from FFPE blocks corresponding to samples in the RNA-seq dataset to assess concordance and accuracy of the cross platform classifier. Results: Consensus clustering identified six robust PCC subtypes. Based on clinical annotation from the published data, four classes belong to the RTK signalling group, three of which (annotated RTK1 to 3) contain a mixture of NF1, RET, RAS, and TMEM127 gene mutations, and a fourth (annotated MAX-like) containing samples with mutations in the MAX gene.The remaining two classes belong to the HIF signalling group, one representing samples with a VHL mutation and the other mutations in one of the SDHx subunits. Applying the classifier we were able to classify RNA-seq data for samples with RET (6), NF1 (6), RAS (2), and TMEM127 (1) as belonging to the RTK 1-3 groups. Accurate classification of nine of eleven VHL samples and two SDHx samples was achieved. Classification of corresponding Nanostring data provided concordant classification for eleven of twelve samples. Conclusion: PCC can be divided into six biological classes indicative of the underlying driver mutation. The ability to accurately classify samples from FFPE material would provide a valuable adjunct to current practices in genetic testing laboratories, providing corroboration for known mutations and guidance on samples with mutations of unknown significance in PCC genes. Furthermore, the classifier can be used for research purposes to subtype tumours with unknown driver mutations for the interpretation of candidate driver genes. Citation Format: Aidan Flynn, Diana Benn, Roderick Clifton-Bligh, Joshy George, Anthony J. Gill, Rodney J. Hicks, Richard W. Tothill. A diagnostic gene expression assay for the classification of pheochromocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2175. doi:10.1158/1538-7445.AM2015-2175

Clinicopathological and Targeted Exome Gene Features of a Patient with Metastatic Acinic Cell Carcinoma of the Parotid Gland Harboring an ARID2 Nonsense Mutation and CDKN2A/B Deletion.

We describe the presentation, treatment, clinical outcome, and targeted genome analysis of a metastatic salivary acinic cell carcinoma (AciCC). A 71-year-old male presented with a 3 cm right tail of a parotid lesion, first detected as a nodule by the patient seven months earlier. He had a right total parotidectomy with cranial nerve VII resection, right facial nerve resection and grafting, resection of the right conchal cartilage, and right modified radical neck dissection. The primary tumor revealed AciCC with two distinct areas: a well-differentiated component with glandular architecture and a dedifferentiated component with infiltrative growth pattern associated with prominent stromal response, necrosis, perineural invasion, and cellular pleomorphism. Tumor staging was pT4 N0 MX. Immunohistochemistry staining showed pankeratin (+), CD56 (−), and a Ki67 proliferation index of 15%. Upon microscopic inspection, 49 local lymph nodes resected during parotidectomy were negative for cancer cells. Targeted sequencing of the primary tumor revealed deletions of CDKN2A and CDKN2B, a nonsense mutation in ARID2, and single missense mutations of unknown significance in nine other genes. Despite postoperative localized radiation treatment, follow-up whole body PET/CT scan showed lung, soft tissue, bone, and liver metastases. The patient expired 9 months after resection of the primary tumor.

Abstract 1303: Genetic cancer incidence

Abstract Objective: Hereditary Breast Ovarian Cancer (HBOC) is genetic malignancy associated with BRCA1/2 genes. Selecting confirmative tests for target population are not clearly defined and expensive. We undertook this study to find the detection rate of HBOC among Korean ovarian carcinoma patients based on family history and immunohistochemistry right after by surgeon. Methods: This study examined 22 patients diagnosed with ovarian carcinomas by single surgeon and counselor. All patients were provided genetic counseling based on immunohistochemistry (IHC) of mismatch repair genes (BRCA1, BRCA2) and 1st, 2nd 3 rd relatives' family history of cancer. Additionally, direct full sequencing to verify germ line mutations was performed to all patients after consent. Results: Median age was 54.Preop CA 125 was 1824(37.71∼16009). There were 3 refusals after counseling. Patients with family cancer history were 3/21(14%). The number of patients with negative IHC results for BRCA1/BRCA2 was 9 and 6.Both negative patients were 3/21(14%). Both normal staining patients were 4/21(19%). There were 3/19 patients (16%) with germ line mutations (two BRCA1 and one BRCA2).Variation of unknown significance mutation patients were 5/19(26%). Conclusion: Our data indicate approximately 17% of patients have a germ line mutation in this study group. Active genetic counseling by surgeon with IHC and family history can help detect HBOC among ovarian carcinoma patients. Citation Format: Min Kyu Kim, Soo Youn Bae, Jee Yeon Lee. Genetic cancer incidence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1303. doi:10.1158/1538-7445.AM2014-1303

Functional Analysis in Mouse Embryonic Stem Cells Reveals Wild-Type Activity for Three Msh6 Variants Found in Suspected Lynch Syndrome Patients

Lynch syndrome confers an increased risk to various types of cancer, in particular early onset colorectal and endometrial cancer. Mutations in mismatch repair (MMR) genes underlie Lynch syndrome, with the majority of mutations found in MLH1 and MSH2. Mutations in MSH6 have also been found but these do not always cause a clear cancer predisposition phenotype and MSH6-defective tumors often do not show the standard characteristics of MMR deficiency, such as microsatellite instability. In particular, the consequences of MSH6 missense mutations are challenging to predict, which further complicates genetic counseling. We have previously developed a method for functional characterization of MSH2 missense mutations of unknown significance. This method is based on endogenous gene modification in mouse embryonic stem cells using oligonucleotide-directed gene targeting, followed by a series of functional assays addressing the MMR functions. Here we have adapted this method for the characterization of MSH6 missense mutations. We recreated three MSH6 variants found in suspected Lynch syndrome families, MSH6-P1087R, MSH6-R1095H and MSH6-L1354Q, and found all three to behave like wild type MSH6. Thus, despite suspicion for pathogenicity from clinical observations, our approach indicates these variants are not disease causing. This has important implications for counseling of mutation carriers.