Additional Mutations Research Articles

Phenotype switching of the mutation rate facilitates adaptive evolution.

The mutation rate plays an important role in adaptive evolution. It can be modified by mutator and anti-mutator alleles. Recent empirical evidence hints that the mutation rate may vary among genetically identical individuals: evidence from bacteria suggests that the mutation rate can be affected by expression noise of a DNA repair protein and potentially also by translation errors in various proteins. Importantly, this non-genetic variation may be heritable via a transgenerational epigenetic mode of inheritance, giving rise to a mutator phenotype that is independent from mutator alleles. Here, we investigate mathematically how the rate of adaptive evolution is affected by the rate of mutation rate phenotype switching. We model an asexual population with two mutation rate phenotypes, non-mutator and mutator. An offspring may switch from its parental phenotype to the other phenotype. We find that switching rates that correspond to so-far empirically described non-genetic systems of inheritance of the mutation rate lead to higher rates of adaptation on both artificial and natural fitness landscapes. These switching rates can maintain within the same individuals both a mutator phenotype and intermediary mutations, a combination that facilitates adaptation. Moreover, non-genetic inheritance increases the proportion of mutators in the population, which in turn increases the probability of hitchhiking of the mutator phenotype with adaptive mutations. This in turns facilitates the acquisition of additional adaptive mutations. Our results rationalize recently observed noise in the expression of proteins that affect the mutation rate and suggest that non-genetic inheritance of this phenotype may facilitate evolutionary adaptive processes.

The Histologic and Molecular Spectrum of Highly Differentiated HPV-independent Cervical Intraepithelial Neoplasia.

Recently, the World Health Organization (WHO) recognized the existence of human papillomavirus (HPV)-independent invasive cervical squamous cell carcinoma (SCC), but HPV-independent precursor lesions were not included due to the lack of description of this rare entity. We present the histologic spectrum of highly differentiated squamous HPV-negative and p16 ink4a -negative precursor lesions adjacent to and/or preceding invasive HPV-negative cervical SCC in 3 patients. The histologic features resembled those described for vulvar HPV-negative precursor lesions. One precursor featured a proliferation of atypical basal keratinocytes with mitotic activity, premature squamatization in elongated epithelial rete, and mostly regular superficial squamous differentiation with TP53 mutation and immunohistochemical p53 overexpression termed differentiated cervical intraepithelial neoplasia (d-CIN). The other 2 precursors included verruciform acanthosis with plump rete, minimal atypia, and an EGFR mutation that resembled vulvar acanthosis with altered differentiation, and an exophytic papillary proliferation with a PIK3CA mutation resembling the differentiated exophytic vulvar intraepithelial lesion. Two precursors that preceded the invasive SCC harbored an additional pathogenic SMARCB1 mutation. The cytologic smears of d-CIN revealed 3-dimensional branched basaloid tubular structures and eosinophilic squamous cell clusters mimicking the histologic features. In conclusion, highly differentiated cervical HPV-negative precursors are characteristic intraepithelial squamous lesions with somatic mutations that resemble those described in vulvar HPV-independent carcinogenesis. For optimal reproducibility, we propose a simplistic classification of these HPV-negative cervical precursors in TP53 -mutated d-CIN and p53 wild-type verruciform intraepithelial neoplasia.

PfCRT mutations conferring piperaquine resistance in falciparum malaria shape the kinetics of quinoline drug binding and transport.

The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ.

Characterization of Mechanisms of Resistance in Previously Treated Chronic Lymphocytic Leukemia (CLL) From a Head‐to‐Head Trial of Acalabrutinib Versus Ibrutinib

Introduction: Acalabrutinib (Acala) is a highly selective, next-generation covalent Bruton tyrosine kinase inhibitor (BTKi) approved for CLL. In ELEVATE-RR (NCT02477696) at a median follow-up of 41 mo, Acala demonstrated noninferior progression-free survival with fewer cardiovascular adverse events versus ibrutinib (Ibr) in patients (pts) with relapsed/refractory (R/R) CLL. Disease progression on covalent BTKis is often characterized by acquisition of B-cell receptor pathway mutations, but no data have compared mutational profiles of Acala versus Ibr. We report clonal evolution data in pts with CLL progressing on Acala versus Ibr in ELEVATE-RR. Methods: Peripheral blood samples at baseline and relapse from pts in ELEVATE-RR were used. DNA was extracted from enriched CD19+ cells (RoboSep) and subjected to a 50-gene sequencing assay panel with a sensitivity cutoff for BTK and PLCG2 resistance–associated mutations at 0.5% variant allele fraction (VAF). Forty-eight other CLL-associated genes were assessed at 1%–2% VAF. Results: Paired (baseline and progression) samples were available for 47 (excluding 1 Richter) and 30 (excluding 6 Richter) pts in the Acala and Ibr groups, respectively. At progression, emergent BTK mutations were seen in 31 (66%) Acala versus 11 (37%) Ibr pts (P = 0.02) (Figure 1; median VAF: 5.7 vs. 5.8). Emergent PLCG2 mutations occurred in 3 (6%) Acala vs. 6 (20%) Ibr pts (P = 0.14). Only 1 Acala pt had co-occurrence of BTK and PLCG2 mutations versus 4 Ibr pts. BTK C481S, C481Y, and C481R mutations occurred at similar frequency in both groups; a novel E41V mutation within the pleckstrin homology domain of BTK (median VAF: 16%) was seen in 1 Acala pt. L528W and A428D co-mutations were observed in 1 Ibr pt. Six Acala pts had TP53 and BTK co-mutations. Emergent TP53 mutations were seen in both groups (13% [Acala] vs. 7% [Ibr], P = 0.47; median VAF: 5% [Acala] versus 37% [Ibr]). Only 2 Ibr pts had TP53 mutations (1 had TP53/BTK co-mutation). No statistical difference was seen in the proportions of Acala versus Ibr pts who acquired BTK mutations among pts with del(17p) (39% vs. 64%; P = 0.18), del(11q) (77% vs. 46%; P = 0.07), complex karyotype (58% vs. 73%; P = 0.48), unmutated IGHV (90% vs. 100%; P = 0.55), or trisomy 12 positivity (3% vs. 18%; P = 0.16). Additional mutations (Acala vs. Ibr) included DNMT3A (5 vs. 1 pts), TET2 (1 pt for each), and NRAS (1 pt; Acala only). The research was funded by: AstraZeneca Keywords: Chronic Lymphocytic Leukemia (CLL), Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. J. A. Woyach Consultant or advisory role: Abbvie, AstraZeneca, BeiGene, Genentech, Janssen, Merck, Loxo/Lilly, Newave, Pharmacyclics Research funding: Abbvie, Janssen, Karyopharm Therapeutics, Loxo/Lilly, Pharmacyclics, Schrodinger D. Jones Research funding: Abbvie, Acerta/AstraZeneca, Pharmacyclics, Novartis, MingSight, Other remuneration: The Ohio State University: High sensitivity BTK mutation profiling W. Jurczak Consultant or advisory role: Abbvie, AstraZeneca, BeiGene, Lilly, Roche, Takeda Research funding: Abbvie, AstraZeneca, BeiGene, Janssen, Lilly, Roche, Takeda T. Robak Consultant or advisory role: AstraZeneca, BeiGene, Janssen Oncology Honoraria: AstraZeneca, BeiGene, Janssen Research funding: AstraZeneca, BeiGene, Janssen A. Illés Employment or leadership position: University of Debrecen Honoraria: Janssen, Celgene, Novartis, Pfizer, Takeda, Roche Research funding: Takeda, Seattle Genetics A. P. Kater Consultant or advisory role: AstraZeneca, BMS, Roche/Genentech, Janssen, AbbVie, LAVA Research funding: AstraZeneca, BMS, Roche/Genentech, Janssen, AbbVie Other remuneration: Janssen, LAVA, AbbVie, AstraZeneca P. Ghia Honoraria: AbbVie, AstraZeneca, Janssen, BMS, MSD, Loxo Oncology/Lilly, and Roche Research funding: AbbVie, AstraZeneca, Janssen, and BMS J. C. Byrd Consultant or advisory role: Janssen, Novartis, Syndax, Newave, AstraZeneca, Kura, Vincerx, Trilrom, Abbvie Stock ownership: Vincerx Research funding: Zencor, Pharmacyclics Educational grants: Janssen, Novartis Other remuneration: Ohio State University J. F. Seymour Consultant or advisory role: AbbVie, AstraZeneca, Celgene, Genentech, Genor Bio, Gilead, Janssen, Morphosys, Roche, Sunesis, TG Therapeutics Research funding: Abbvie, Celgene, Janssen, Roche Other remuneration: Abbvie, Celgene, Roche, TG Therapetics G. De Jesus Employment or leadership position: AstraZeneca Stock ownership: AstraZeneca R. Lai Employment or leadership position: AstraZeneca G. de Bruin Employment or leadership position: Acerta Pharma B.V. A. Butturini Employment or leadership position: AstraZeneca Stock ownership: AstraZeneca, Amgen, Roche S. Rule Employment or leadership position: AstraZeneca V. Munugalavadla Employment or leadership position: AstraZeneca Stock ownership: AstraZeneca; Gilead Sciences

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Post allograft transplant patients are prone to develop a continuum of lympho-proliferative diseases (PTLD) that exhibit distinct clinicopathological forms that range from benign to malignant. The molecular pathogenesis of PTLD is not well established. Methods: We analyzed an unusual case of a 15 year old child who received a lung transplant at age 7 and developed PTLD first in the right tonsil (polymorphic PTLD) 4 months post transplant and then in the left shin and associated lymph node (PTLD-Burkitt Lymphoma) 4 years later. Histopathology and immunohistopathology (IHC) features of the left shin mass were typical for Burkitt Lymphoma with diagnostic features of CD20+, CD10+ve, TdT-ve, EBER+ve, IGH clonal for B cells and Ig-cMyc fusion positve by FISH analysis. The child received chemotherapy and was in remission for 3 years. At 8 years post transplant, he developed an enlargement of the left tonsil. Histopathology and IHC lack evidence of recurrent Burkitt Lymphoma as the B cells are polyclonal, CD10-ve and EBV-ve. FISH was negative for Ig-cMyc fusion. A diagnosis of a polymorphic PTLD was made. With the recently available TruSight RNA Pan-Cancer assay, RNA was extracted from the two most recent tissue samples and NGS libraries were enriched using the 1385 gene panel. Results show the PTLD- Burkitt Lymphoma has additional mutations in the cMyc locus and TP53. The distinctly common mutations notable for Pediatric Burkitt Lymphoma in immunocompetent patients such as ID3, DDX3X, ARID1A and SMARCA4 that are noted in our database and described in a recent publication by B. Burkhardt et al (Nature communication 13:3881;2022) are absent. cMyc, TP53 and all of these mutations are absent in the post chemotherapy polymorphic PTLD in the right tonsil. However, a mutation of Linker Histone 1 (H1-4) gene is detected in the Burkitt PTLD mass and this recurrent polymorphic PTLD tonsil. Conclusions: This result shows there is a persistence of non clonal B cells that are chemotherapy resistant. The patient is currently stable under clinical surveillance. The pathological consequence of H1ST-1(1-4) mutation in the B cells of this patient appears to be supported by a study in a preclinical mouse model that Histone H1 loss drives lymphoma by disrupting 3D chromatin structure (N. Yusufova et. al. Nature 589:299-305; 2021). In this study, knock-in mice carying this mutation alone have prolonged survival as compared with knock-in mice with genetic constructs that contain additional oncogenic driver genes. Based on our findings of the persistence of lymphoid cells with a mutation of gene encoding for a epigenomic gene[HIST-1 (1-4) ], we suspect this mutation plays a role that drives the development of PTLD in this patient. Keywords: Diagnostic and Prognostic Biomarkers, Non-Hodgkin (Pediatric, Adolescent, and Young Adult), Pathology and Classification of Lymphomas No conflicts of interests pertinent to the abstract.

Clinical implications of recurring mutations in myxoid liposarcoma (MLS).

11577 Background: MLS is a subset of liposarcoma characterized by a translocation, (t(12;16), FUS-DDIT3 or less frequently t(12;22), EWSR1-DDIT3). Additional mutations have been reported in TERT promotor, PIK3CA, and PTEN and some were associated with the more aggressive round cell phenotype. Our study aimed at analyzing the clinical significance of these recurring mutations. Methods: MLS patients (pt) with available next-generation sequencing (NGS) data (any clinical grade) between Jan 2014 to Oct 2022 were included. Baseline characteristics, chemotherapy (CMT) response (partial response [PR] and stable disease [SD]), and survival were collected for doxorubicin-ifosfamide (AI), doxorubicin-dacarbazine (ADIC), trabectedin (T), and eribulin (E). Binary logistic regression was used to evaluate mutations associated with response. Cox proportional-hazard regression was used to investigate associations of variables with progression-free survival (PFS) and overall survival (OS). The Kaplan-Meier method was used to estimate survival and log-rank tests were used to compare survival between groups. Results: Of 18 MLS pt included, median age at diagnosis was 42 years (R 30-65). Majority were male (12/18, 67%), had localized disease at diagnosis (13/18, 72%), and had lower extremities as primary location (10/18, 56%). Eight (44%) had round-cell > 5% and 15 (83%) had primary tumor > 5 cm. Somatic mutations were reported in 15 pt (83%); the most common were TERT promotor (61%), PTEN (28%), PIK3CA (22%), and TP53 (17%). None showed significant association with clinical factors (age, sex, size, stage, round cell > 5%, primary location). Forty-eight records (18 AI, 6 ADIC, 19 T, and 5 E; 3 pt received AI and T twice for different recurrences), were evaluated for PFS and 47 records had response data available. For AI, ADIC, T, and E, PR rate was 39% (7/18), 17% (1/6), 32% (6/19), and 40% (2/5), while clinical benefit (CBR: PR + SD≥ 3 mo) was 67% (12/18), 50% (3/6), 63% (12/19), and 80% (4/5), respectively. PR or CBR were not associated with the common mutations for any regimen. Median follow up (FU) from start of CMT was 4 mo (R 0.5-31). PFS (95%CI) was 11 (6.7- Not Reached [NR]), 1.2 (1.1-NR), 23 (6.3-NR), and 8 (1.3-NR) mo for AI, ADIC, T, and E, respectively. PIK3CAm (n = 15) was associated with improved PFS with CMT (PFS [95%CI]: PIK3CAwt 7.8 [6.3-11.2] vs. PIK3CAm 23 mo [1.3-NR]; multivariate hazard ratio [HR] 0.23, p= 0.036, adjusted by gender and ADIC regimen). This appeared to be mainly driven by the PIK3CAm/ TERTwt (n = 8, HR 0.07, p= 0.006). For T, PIK3CAm/ TERTm (n = 3) showed a trend for lower PFS (1.3 mo) in contrast to other groups (23 mo). Conclusions: TERT promoter mutation was the most frequent mutation but was not associated with outcome in our small series. PIK3CAm suggested PFS benefit from CMT while, on the contrary, dual PIK3CAm/ TERTm showed a trend towards shorter PFS with T. The predictive and prognostic role of these mutations in MLS warrant further study.

A systematic comparison of whole genome sequencing and targeted panel sequencing for precision oncology.

8546 Background: The analysis of the cancer genome provides clinically meaningful information such as actionable cancer driver mutations. Whole genome sequencing (WGS) provides a complete mutational landscape at an affordable cost thereby having increase clinical utility compared to the usual approach of targeted panel sequencing (TPS). To understand the capabilities of WGS in oncology, we conducted a study in lung adenocarcinomas. Methods: We produced WGS (surgically resected tumor tissues and matched blood; mean coverage 42x) and TPS (tumor only; SNUH panel encompassing 75 cancer genes; mean coverage 904x) through the Illumina sequencing platform and conducted comprehensive bioinformatic analyses. Oncogenic driver events, including single base substitution (SBS), insertion or deletion (INDEL), complex deletion-insertion, and fusion oncogene, were investigated in both datasets. Lastly, we compared the pattern-based mutational features, such as tumor mutational burden (TMB), mutational signatures, and copy number changes which is associated with genomic instability. Results: In 60 lung adenocarcinomas, the TPS method identified 120 driver mutations from the 75 targeted genes. Of these, 114 mutations were also detected by the WGS method, showing 95.0% sensitivity of WGS in detection of core driver mutations. In uncovered genomic regions by TPS, WGS captured 9 additional driver mutations (such as loss-of-function mutations in PIK3R2, ARID2, and APC) and 37 copy number variations (such as amplification of LMO and SMO, deletion of PIK3R1 and APC) in the cancer genes. TMB calculation is straightforward in WGS however, sophisticated considerations were necessary in TPS for TMB, to remove false positives and germline mutations. TPS showed a good capability for identifying hypermutator tumor samples, but a quantitative estimation was not always feasible due to the substantial background fluctuation, particularly for INDELs. Mutational signatures are unique combinations of mutation types in a given sample, which can give important clues to the mechanism underlying mutations. TPS showed adequate mutational signatures (cosine similarity > 0.7), but was not constantly satisfactory in tumors with high TMB ( > 20,000 genome wide SBS and INDELs). In TPS, approximately 30% of high TMB tumors represented marginal cosine similarity (0.5-0.7), and tobacco smoking related signature (SBS 4) found in WGS was captured only in 50% of the associated cases (9 out of 18). For DNA copy number changes and loss-of-heterozygosity analyses, WGS showed a much higher resolution. Conclusions: Our study demonstrates that WGS has comparable capabilities for the detection of driver mutations in core oncogenes and tumor suppressor genes. WGS was superior for pattern-based features, such as TMB, mutational signatures, and copy number changes. WGS is a medically necessary more comprehensive approach to precision oncology.

Association of alterations in multiple tumor suppressor genes with poor outcomes in patients with EGFR-mutant lung cancer.

9104 Background: Co-occurring alterations in tumor suppressor genes (TSG) have been described as determinants of disease heterogeneity in EGFR-mutant non-small cell lung cancer (NSCLC), however, detailed analyses of their impact on patient outcomes are limited. Methods: Patients with EGFR-mutant NSCLC treated with EGFR tyrosine kinase inhibitors (TKIs) at the Yale Cancer Center who had tumor genomic profiling either before EGFR-TKI therapy (pre-TKI) or at disease progression (post-TKI) were included. Whole exome sequencing (WES) of paired pre- and post-TKI tumor specimens were performed for a subset of patients. The cohort was divided into three subgroups based on alterations in TP53 and five additional TSGs ( RB1, NF1, ARID1A, BRCA1 and PTEN): patients with tumors harboring a TP53 mutation plus a mutation in at least 1 additional TSG ( TP53mut/TSGmut), patients with tumors harboring a TP53 mutation without an additional TSG mutation ( TP53mut/TSGwt), and patients with TP53wt tumors. Clinical characteristics including progression-free (PFS) and overall survival (OS) were assessed. Results: One-hundred-one patients were included in this retrospective study. TP53 mutations were identified in 65 (64%) tumors, of which 23 (35%) and 42 (65%) were classified as TP53mut/TSGmut and TP53mut/TSGwt, respectively. Among those cases with paired WES available (n = 34), frequencies of alterations in the six included TSGs did not significantly differ between pre- and post-TKI tumor specimens. In the full study cohort, the presence of a TP53 mutation was associated with numerically worse PFS (HR 1.46, CI 0.97 – 2.21, p = 0.09) and OS (HR 1.68, CI 1.05 – 2.70, p = 0.04) on first-line EGFR-TKI. Strikingly, after dividing the TP53mut cohort into TP53mut /TSGmut and TP53mut /TSGwt cases, alterations in additional TSG were found to drive the poor outcomes: TP53mut /TSGmut cases had significantly worse PFS and OS on first-line EGFR TKI than TP53mut /TSGwt(mPFS 8.0 vs 10.6 months, p = 0.006; mOS 30.0 vs 33.3 months, p = 0.12) or TP53wt cases (mPFS 8.0 vs 12.6 months, p < 0.0001; mOS 30.0 vs 48.8 months, p = 0.001). There was no significant difference in PFS or OS between patients with TP53mut /TSGwt and TP53wt tumors. Similar outcome differences between the three groups were found in patients who received osimertinib in the second-line setting. Conclusions: The inferior outcomes associated with EGFR/TP53-mutant NSCLC tumors may be due to additional TSG alterations rather than TP53 mutational status alone. Alterations in the investigated TSGs did not appear to emerge under EGFR-TKI therapy, which suggests that they are truncal. Our findings may have implications for understanding the biologic underpinnings of differential outcomes to EGFR TKIs.

Liquid and tissue profiling of somatic mutational landscape of non-small cell lung cancer from an Indian cohort.

e21202 Background: Circulating tumor DNA (ctDNA) encompasses spatial and temporal heterogeneity, tumor evolution, and is ideal for repeat sampling and predictive therapeutic course corrections whereas tissue is the first choice for molecular profiling. We report the mutational landscape of an Indian NSCLC cohort, with focus on liquid biopsy and value addition offered by paired tissue and liquid profiling. Methods: The study comprised 507 tumor tissue and 1271 liquid biopsies from NSCLC patients. Evaluation of single nucleotide alterations, copy number alterations and fusions was conducted using targeted next generation sequencing with paired tissue and liquid profiling in a sub cohort (n = 45). Results: In tissue based analysis (n = 507), EGFR-sensitizing mutations were the most common alterations, detected in 41.4% of samples. Among these, Exon 19 deletion was seen in 46.3%, Exon 21 L858R in 23.9%, Exon 20 insertion in 4.6%, Exon 18 G719X in 3.7% and compound mutations in 5%. TP53, KRAS and PIK3CA mutations were detected in 34.5%,13.4% and 6.3% respectively. MYC was commonly amplified (15.4%) gene followed by PIK3CA (4.1%), CCNE1 (3.3%), EGFR (2.9%), and MET (2.9%) amplifications. Tissue fusion analysis (n = 398) detected ALK in 6.3%, ROS1 in 3.3%, and RET in 1.8%. METEx14 was detected in 4.5%. Liquid biopsy (n = 1271) showed comparable mutation spectrum as tissue, with the EGFR gene being the most altered (32.9%). Mutations in TP53 were observed in 26.4%, KRAS in 7.4%, PIK3CA in 4.4% and BRAF in 3%. In EGFR positive individuals, EGFR T790M was detected at higher frequency in liquid (16.7%) than tissue (8.6%), indicating this acquired resistance alteration being better captured in liquid biopsy than tissue due to tumor heterogeneity and tumor evolution. 2 liquid biopsy samples identified additional KRAS driver mutations not seen in the paired tissue analysis, highlighting the utility of simultaneous tissue and liquid analysis. Concurrent actionable alterations were detected in 11% of tissue and 5% of liquid samples. Conclusions: We report the largest liquid biopsy study reporting the genomic landscape from NSCLC cases of Indian origin along with tissue based molecular landscape. Presence of alterations like KRAS and EGFR T790M in liquid but absence in concurrent tissue biopsy shows utility of paired tissue and liquid biopsy profiling. Higher incidence of targetable and co-existing alterations in the Indian population highlight the utility of broad molecular profiling for successful utilization of targeted therapies. [Table: see text]

Clinical characteristics, response to poly (adenosine phosphate-ribose) polymerase inhibitors and platinum chemotherapy in patients with lung cancer harboring BRCA mutations.

e21036 Background: BRCA1/2 are tumor suppressor genes involved in DNA double-strand break repair. Tumors harboring BRCA1/2 pathogenic mutations (BPM) are highly responsive to treatment with poly (adenosine phosphate-ribose) polymerase inhibitors (PARPi) and exhibit increased sensitivity to platinum-based chemotherapy (platinum). Somatic BPM are found in 1-2% of all cases of non-small cell lung cancer (NSCLC), the majority of which are not associated with germline variants. While platinum remains the backbone of first-line treatment of NSCLC, PARPi have not demonstrated efficacy in treatment of NSCLC and testing for BRCA mutations in NSCLC is not routine. We present the clinical characteristics and outcomes of patients with NSCLC harboring BPM treated with platinum and PARPi as part of clinical practice at our center. Methods: We retrospectively evaluated the onco-genetic and pathology data bases for patients with locally advanced or metastatic NSCLC who were treated at Hadassah Medical Center (HMC), underwent somatic molecular or genetic testing, and were found to have somatic BPM (according to ClinVar or Varsom). We compared the clinical outcomes to a matched cohort (age, gender, smoking, and performance status) of non-BPM NSCLC patients treated at HMC. Results: Since 2013 we evaluated 642 patients with locally advanced or metastatic NSCLC using comprehensive somatic or germline molecular panels. Twenty-two patients were found to have somatic BPM of which, 18 (81.8%) were found to be carriers of corresponding germline BRCA1/2 variants. This included 14 (63.6%) carriers of classical Ashkenazi-Jewish variants (10 - BRCA2 617delT, 2- BRCA1 185delAG and 2- BRCA1 5382incC) and representing less than 1% of all BRCA1/2 carriers at HMC. Median age of diagnosis was 67 yeas old (range 40–78). Eleven (50%) patients had a history of smoking, and 8 (36.3%) patients had a history of other malignancies, mainly breast cancer. Pathology for all patients revealed adenocarcinoma and 8 (36.6%) patients had additional actionable mutations ( EGFR, ALK, BRAF or ERBB2). Sixteen (72.7%) patients were treated with platinum, with an objective response rate (ORR) of 75% and a median progression free survival (mPFS) of 14 months (range 4-29), compared to ORR of 45% and mPFS of 8.5 months (range 3-18) in a matched cohort of non-BPM NSCLC patients treated with platinum. Six (27.7%) patients were treated with second or third line PARPi with ORR of 83.3%, mPFS of 21 months (range 6-34). Median overall survival (OS) for the entire cohort was 28 months (range 4-103) with 8 (36.4%) patients surviving for more than 3 years. Conclusions: In this cohort, patients with NSCLC harboring BPM show a durable response to platinum and PARPi with prolonged OS. The results support testing BRCA1/2 in NSCLC patients and treatment with platinum and PARPi for patients with BPM.

The impact of secondary-type mutations in newly diagnosed acute myeloid leukemia treated with venetoclax and decitabine or azacitidine.

e19048 Background: The recently revised risk classification schema in ELN 2022 for acute myeloid leukemia (AML) now includes a more prominent presence of secondary-type mutations (STMs): ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2. However, the risk classification schema of ELN 2022 is based on the outcomes of patients treated with intensive chemotherapy. The impact of STMs in patients treated with lower-intensity venetoclax-based strategies is unknown. Furthermore, IDH and NPM1 mutations are associated with a significant survival benefit in those treated with venetoclax-based strategies, but it is unknown how these mutations impact outcomes when they cooperate with STMs. The purpose of this study was to determine the impact of STMs included in ELN 2022 and cooperating mutations on lower-intensity venetoclax-based strategies. Methods: We retrospectively analyzed 79 patients with AML treated with front-line venetoclax and a hypomethylating agent (HMA; decitabine or azacitidine) at VCU Massey Cancer Center from January 1, 2018 to January 1, 2022. We excluded nine patients without available NGS at diagnosis. We recorded doses and dates of chemotherapy regimens, molecular profiles at diagnosis, and overall survival. We analyzed survival by the Kaplan-Meier method and compared groups with the Mantel-Cox test. The date of death was used to calculate overall survival; patients were censored at the date of the last contact. Results: We identified 32 patients with STMs and compared them to 38 patients without STMs; the median overall survival was nonsignificantly shorter in those with STMs (5.3 m. with STMs versus 8.2 m. without, p = 0.323). The presence of mutated TP53, RAS, or FLT3-ITD appeared to be associated with shorter overall survival in patients with STMs: co-mutated STMs with TP53, RAS, or FLT3-ITD had an overall survival of 2.6 m. compared to 8.6 m. for those with STMs but no additional adverse mutations ( p = 0.093). However, when we excluded patients with mutated TP53, NRAS, KRAS, and FLT3-ITD, there was no significant difference in overall survival in those with and without STMs (8.5 m. with STMs versus 14.7 m. without, p = 0.323). The presence of STMs did not appear to reduce the favorable prognosis associated with IDH mutations without cooperating mutated TP53, RAS, or FLT3-ITD (20.0 m. with STMs versus 23.4 m. without STMs, p = 0.493). Conclusions: While the presence of secondary-type mutations was associated with shorter survival, the association was nonsignificant in those treated with front-line venetoclax + HMA. Additionally, the presence of STMs did not appear to worsen survival in patients that respond favorably to venetoclax-based strategies, such as IDHmut AML. These findings, together with those of larger studies, may contribute to a refined risk classification schema for patients treated with lower-intensity strategies.

P077 Clinical and functional efficacy of elexacaftor/tezacaftor/ivacaftor in people with cystic fibrosis carrying the N1303K mutation

Background: Elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) significantly improves health outcomes in people with cystic fibrosis (pwCF) carrying one or two F508del mutations. According to in vitro assays performed in FRT cells, 178 additional mutations respond to ELX/TEZ/IVA. The N1303K mutation is not included in this list of mutations. Recent in vitro data suggested that ELX/TEZ/IVA increases N1303KCFTR-activity. Based on the in vitro response, seven patients commenced treatment with ELX/TEZ/IVA.

Assessing the utility of liquid biopsy for detecting actionable genetic mutations among indigenous Ghanaian women with metastatic breast cancer.

e13017 Background: Breast cancer is the leading cancer diagnosis in women worldwide, and the leading cause of cancer-related mortality in Ghanaian women. Up to 70% of breast cancer diagnoses in Ghanaian women are advanced-stage, leading to fewer opportunities for early intervention. Late presentation of breast cancer may be attributed to lack of comprehensive screening programs, among other factors. Next-Generation Sequencing (NGS)-based liquid biopsy of circulating free DNA (cfDNA) is non-invasive and been demonstrated to support diagnosis, monitoring and treatment selection in cancer. Unfortunately, people of African ancestry are under-represented in evaluations of this technology and little is known about the feasibility of such approaches in low-resource settings. Methods: The AMBER-01 study recruited 20 newly diagnosed, histologically confirmed, treatment-naive women with metastatic breast cancer at the Cape Coast Teaching Hospital. Testing was conducted at CAP and CLIA accredited laboratories (Lucence, Palo Alto; Singapore). Tissue NGS and cfDNA liquid biopsy analysis was ordered on a total of 20 patients following tissue quality analysis outcomes. All 20/20 (100%) liquid biopsy samples were acceptable for analysis, whereas only 6/20 (30%) passed QC for tissue NGS testing. cfDNA liquid biopsy and tissue NGS analyses were performed using Lucence LiquidHALLMARK and Tissue500 technology, respectively. Results: Liquid biopsy detected 42 cfDNA mutations in 17/20 patients. Of the 17 patients found to have mutations on liquid biopsy, 3 (17.6%) patients had mutations associated with African ancestry, including BRCA K719E, ARAF S262I and GATA3 G125dup. Eight actionable mutations specific to breast cancer were found in 7/17 (41.2%) patients while actionable mutations non-specific to breast cancer were detected in 12/17 (70.6%) patients. Tissue biopsy analysis detected mutations in all 6 patients tested, with 3/6 (50%) patients presenting actionable mutations specific to breast cancer. Of the 20 patients recruited, liquid biopsy was able to identify actionable treatments for 7 patients, while tissue was only able to identify actionable treatment for 3 patients. Of the 6 patients with matched liquid and tissue results, liquid biopsy detected additional mutations in 3 (50%) patients that were not detected by tissue analysis including: BRCA2 Q1379E, GNAQ Q176*, and a low Microsatellite Instability (MSI) reported as stable in tissue. Conclusions: Liquid biopsy detected multiple actionable variants in Ghanaian women with breast cancer that a tissue-only workflow missed. cfDNA analysis featured less variations in sample preparation which is a key consideration in resource-limited settings. Further work will optimize NGS panels to account for novel ancestry specific variants. Liquid biopsy presents a great opportunity to improve cancer care in Africa.

Vimentin single cysteine residue acts as a tunable sensor for network organization and as a key for actin remodeling in response to oxidants and electrophiles

Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a “sensor” transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin.

“Paradoxical” p16 overexpression in cutaneous melanoma: Molecular and immunohistochemical analysis of a rare phenomenon with a focus on cell cycle regulatory molecules

BackgroundOne of the most relevant genetic alterations in cutaneous melanoma (CM) is the biallelic inactivation/loss-of-heterozygosis (LOH) of cyclin-dependent kinase inhibitor 2 A (CDKN2A), which results in the immunohistochemical loss of p16 frequently found in CM. However, we recently described a rare case of dermal/deep-seated melanoma arising in giant congenital nevus (DDM-GCN) with p16 overexpression combined with p53 loss and tumor protein 53 (TP53) mutation. Herein, we reported a case series of CM with p16 overexpression and analyzed their clinicopathologic features, immunohistochemical expression of the cell cycle regulatory molecules (CCRM: p53, p21, Cyclin D1, Rb), and mutational landscape. MethodsWe retrospectively tested for p16 all cases of CM diagnosed at our institution between January 1st 2019-April 1st 2022. In CM with p16 overexpression, we reported clinicopathologic features, immunohistochemical results for melanocytic markers and CCRM, and mutational landscape investigated with a next-generation sequencing (NGS) panel. In cases with zonal p16 overexpression, the immunohistochemical assessment for melanocytic markers and CCRM, as well as the NGS analysis have been performed in both components {with and without p16 overexpression [p16(+)c and p16(-)]}. ResultsOverexpression of p16 was found in 10/2879 (0.35%) CM [5/10 (50%) diffuse and 5/10 (50%) zonal]. We combined the immunohistochemical results for CCRM and molecular data to classify the cases as follows: a) Group 1 with altered expression of at least one CCRM but no TP53 mutations [3/10 (30%), all with Rb altered/lost]; b) Group 2 with altered expression of at least one CCRM and TP53 mutations [4/10 (40%), all with p53 altered]; c) Group 3 with normal expression of CCRM and no TP53 mutations [3/10 (30%), all with mutations in MAPK pathway genes (NRAS and BRAF)]. In CM with zonal p16 overexpression, the histologic appearance of p16(+)c was heterogeneous, whereas combining CCRM profiles and molecular data the cases could be categorized as follows: a) cases with the same CCRM and molecular profiles in both p16(+)c and p16(-)c; b) cases with p16(+)c showing additional genetic mutations and/or modifications of CCRM expression. Conclusionsp16 overexpression is a rare event, occurring in advanced-stage, clinically- and histologically-heterogeneous CM. These lesions may be classified into three different groups based on CCRM expression and mutational profiles (including TP53 mutation). The analysis of CM with zonal p16 overexpression suggests that, at least in a subset of cases, this phenomenon could represent a sign of “molecular progression” due to the acquisition of additional genetic mutations and/or modifications of the CCRM profile.

SARS-CoV-2 omicron sub-lineages differentially modulate interferon response in human lung epithelial cells

Although most of the attention was focused on the characterization of changes in the Spike protein among variants of SARS-CoV-2 virus, mutations outside the Spike region are likely to contribute to virus pathogenesis, virus adaptation and escape to the immune system. Phylogenetic analysis of SARS-CoV-2 Omicron strains reveals that several virus sub-lineages could be distinguished, from BA.1 up to BA.5. Regarding BA.1, BA.2 and BA.5, several mutations concern viral proteins with antagonistic activity to the innate immune system, such as NSP1 (S135R), which is involved in mRNAs translation, exhibiting a general shutdown in cellular protein synthesis. Additionally, mutations and/or deletions in the ORF6 protein (D61L) and in the nucleoprotein N (P13L, D31-33ERS, P151S, R203K, G204R and S413R) have been reported, although the impact of such mutations on protein function has not been further studied. The aim of this study was to better investigate the innate immunity modulation by different Omicron sub-lineages, in the attempt to identify viral proteins that may affect virus fitness and pathogenicity. Our data demonstrated that, in agreement with a reduced Omicron replication in Calu-3 human lung epithelial cells compared to the Wuhan-1 strain, a lower secretion of interferon beta (IFN-β) from cells was observed in all sub-lineages, except for BA.2. This evidence might be correlated with the presence of a mutation within the ORF6 protein (D61L), which is strikingly associated to the antagonistic function of the viral protein, since additional mutations in viral proteins acting as interferon antagonist were not detected or did not show significant influence. Indeed, the recombinant mutated ORF6 protein failed to inhibit IFN-β production in vitro. Furthermore, we found an induction of IFN-β transcription in BA.1 infected cells, that was not correlated with the cytokine release at 72 h post-infection, suggesting that post-transcriptional events can be involved in controlling the innate immunity.

Additional Oncogenic Alterations in RAS-Driven Differentiated Thyroid Cancers Associate with Worse Clinicopathologic Outcomes.

RAS mutations occur across the spectrum of thyroid neoplasms, and more tools are needed for better prognostication. The objective of this study was to evaluate how additional genetic events affecting key genes modify prognosis in patients with RAS-mutant thyroid cancers, and specifically differentiated thyroid cancers (DTC). We performed a clinical-genomic analysis of consecutive patients with DTC, poorly differentiated (PDTC), or anaplastic thyroid cancer (ATC) between January 2014 and December 2021, in whom a custom-targeted next-generation sequencing assay was performed. Patients harboring RAS mutations were included, and we compared their clinical features and outcomes based upon the presence of additional oncogenic alterations. Seventy-eight patients were identified, with 22% (17/78) harboring a driver RAS mutation plus an additional oncogenic alteration. All six (100%) ATCs had an additional mutation. Compared with DTCs harboring a solitary RAS mutation, patients with DTC with RAS and additional mutation(s) were more likely to be classified as American Thyroid Association high-risk of recurrence (77% vs. 12%; P < 0.001) and to have larger primary tumors (4.7 vs. 2.5 cm; P = 0.002) and advanced stage (III or IV) at presentation (67% vs. 3%; P < 0.001). Importantly, over an average 65-month follow-up, DTC-specific-mortality was more than 10-fold higher (20% vs. 1.8%; P = 0.011) when additional mutations were identified. Identification of key additional mutations in patients with RAS-mutant thyroid cancers confers a more aggressive phenotype, increases mortality risk in DTC, and can explain the diversity of RAS-mutated thyroid neoplasia. These data support genomic profiling of DTCs to inform prognosis and clinical decision-making.

Temperature-boosted PAM-less activation of CRISPR-Cas12a combined with selective inhibitors enhances detection of SNVs with VAFs below 0.01%

The precise identification of rare single nucleotide variations (SNVs) concomitant with excess wild-type DNA is a valuable method for minimally invasive disease diagnosis and early prediction of drug responsiveness. Selective enrichment of mutant variants via strand displacement reaction offers an ideal approach of SNVs analysis but fails to differentiate wildtype from mutants with variant allele fraction (VAF) < 0.01%. Here, we demonstrate that integration of PAM-less CRISPR-Cas12a and adjacent mutation-enhanced inhibition of wild-type alleles enables highly sensitive measurement of SNVs well below the 0.01% VAF threshold. Raising the reaction temperature to the upper limit of LbaCas12a helps to boost PAM-less activation of collateral DNase activity, which can be further enhanced using PCR additives, leading to ideal discriminative performance for single point mutations. Along with selective inhibitors bearing additional adjacent mutation, it allowed detection of model EGFR L858R mutants down to 0.001% with high sensitivity and specificity. Preliminary investigation on adulterated genomic samples prepared in two different ways also suggests that it can accurately measure ultralow-abundance SNVs extracted directly from clinical samples. We believe that our design, which combines the superior SNV enrichment capability of strand displacement reaction and unparalleled programmability of CRISPR-Cas12a, has the potential to significantly advance current SNV profiling technologies.

Triple-negative Thrombocythemia and Subsequent Acute Lymphoblastic Leukemia with Additional Somatic Mutations.

Triple-negative essential thrombocythemia (ET) is a condition in which mutations in JAK2, CALR and MPL are all negative. Transformation to acute myeloid leukemia may occur during the course of ET, while B-acute lymphoblastic leukemia (B-ALL) is rare. We experienced a case diagnosed as B-ALL during the course of triple-negative ET. Notably, cytoreduction was required for the excessive increase in blood cells during the bone marrow recovery period after chemotherapies. Whole-exome sequencing identified 17 somatic mutations: 9 were identified in both ET and B-ALL samples, while 8 were specific to B-ALL, suggesting that these 8 might have caused the transformation.

Respective roles of Pik3ca mutations and cyproterone acetate impregnation in mouse meningioma tumorigenesis.

Despite their rarity, PIK3CA mutations in meningiomas have raised interest as potentially targetable, ubiquitous mutations owing to their presence in sporadic benign and malignant tumors but also in hormone-related cases. Using new genetically engineered mouse models, we here demonstrate that Pik3ca mutations in postnatal meningeal cells are sufficient to promote meningioma formation but also tumor progression in mice. Conversely, hormone impregnation, whether alone or in association with Pik3ca and Nf2 mutations, fails to induce meningioma tumorigenesis while promoting breast tumor formation. We then confirm in vitro the effect of Pik3ca mutations but not hormone impregnation on the proliferation of primary cultures of mouse meningeal cells. Finally, we show by exome analysis of breast tumors and meninges that hormone impregnation promotes breast tumor formation without additional somatic oncogenic mutation but is associated with an increased mutational burden on Pik3ca-mutant background. Taken together, these results tend to suggest a prominent role of Pik3ca mutations over hormone impregnation in meningioma tumorigenesis, the exact effect of the latter is still to be discovered.