Downstream Mutations Research Articles

Afatinib plus bevacizumab combination after osimertinib resistance in advanced EGFR-mutant non-small cell lung cancer: Phase II ABCD-study

IntroductionMany clinical studies showed a synergy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) and vascular endothelial growth factor inhibitors. We hypothesized afatinib plus bevacizumab exerts clinical potency after developing various osimertinib resistant mechanisms. MethodsEGFR-mutant non-small cell lung cancer patients were enrolled after osimertinib resistance. Afatinib at 30–40 mg/day and bevacizumab at 15 mg/kg tri-weekly were administered until progression. Plasma/histologic rebiopsied samples after osimertinib failure were analyzed to examine resistant mechanisms: gene alterations/copy-number gain using cancer personalized profiling by deep sequencing. ResultsBetween January 2018 and October 2020, 28 patients were enrolled. Response and disease control rates were 17.9 % and 78.6 %, respectively. Median duration of response was 9.0 (range, 4.2–22.3) months. Median progression-free and overall survivals were 2.7 and 9.3 months, respectively. Twenty-eight (100 %) plasma and/or 21 (75 %) histologic rebiopsies identified: 17 (61 %) TP53; 15 (54 %) T790M; 9 (32 %) uncommon EGFR; 9 (32 %) MET; 6 (21 %) C797S; 3 (11 %) BRAF; 2 (7 %) HER2; 2 (7 %) KRAS; and 2 (7 %) PI3K mutations. One (17 %) of 6 C797S patients showed complete response. Three (33 %) of 9 uncommon EGFR-mutated patients achieved radiographic response. Neither 15 T790M-positive nor 6 EGFR downstream signaling mutations: BRAF; KRAS; or PI3K-positive patients responded, but 5 (38 %) of 13 T790M-negative patients responded. Adverse events ≥ grade 3 and incidence ≥ 5 % were: hypertension (29 %); proteinuria (7 %); and diarrhea (7 %). There were neither treatment-related death nor interstitial lung disease. ConclusionsSelected population could obtain clinical benefit from afatinib plus bevacizumab, based on rebiopsy results after osimertinib resistance.

The Roles of RAC1 and RAC1B in Colorectal Cancer and Their Potential Contribution to Cetuximab Resistance.

Colorectal cancer (CRC) is one of the most diagnosed cancers and a leading contributor to cancer-related deaths in the United States. Clinically, standard treatment regimens include surgery, radiation, and chemotherapy; however, there has been increasing development and clinical use of targeted therapies for CRC. Unfortunately, many patients develop resistance to these treatments. Cetuximab, the first targeted therapy approved to treat advanced CRC, is a monoclonal antibody that targets the epidermal growth factor receptor and inhibits downstream pathway activation to restrict tumor cell growth and proliferation. CRC resistance to cetuximab has been well studied, and common resistance mechanisms include constitutive signal transduction through downstream protein mutations and promotion of the epithelial-to-mesenchymal transition. While the most common resistance mechanisms are known, a proportion of patients develop resistance through unknown mechanisms. One protein predicted to contribute to therapy resistance is RAC1, a small GTPase that is involved in cytoskeleton rearrangement, cell migration, motility, and proliferation. RAC1 has also been shown to be overexpressed in CRC. Despite evidence that RAC1 and its alternative splice isoform RAC1B play important roles in CRC and the pathways known to contribute to cetuximab resistance, there is a need to directly study the relationship between RAC1 and RAC1B and cetuximab resistance. This review highlights the recent studies investigating RAC1 and RAC1B in the context of CRC and suggests that these proteins could play a role in resistance to cetuximab.

MDB-79. THE DEUBIQUITINATING ENZYME USP7 COOPERATES WITH SONIC HEDGEHOG SIGNALING TO PROMOTE MEDULLOBLASTOMA

Abstract BACKGROUND Constitutive activation of the Sonic Hedgehog (SHH) signaling pathway during cerebellar development may lead to medulloblastoma (MB), one of the most common malignant pediatric brain tumor. Clinically, SHH-MB with mutations downstream of SMO or TP53-mutated are a real challenge and alternative targeted therapies are sorely needed for this group of patients. In SHH-MB, the transcription factor Atoh1 is constitutively overexpressed and essential for tumor initiation and progression. Previous work demonstrated that proteasomal-mediated degradation of Atoh1 induces tumor regression in vitro and in vivo, therefore deciphering mechanisms controlling Atoh1 stabilization should provide new avenues for drug development in SHH-MB. METHODS Using a proteomic approach, we identified the deubiquitinating enzyme Usp7 as a positive regulator of Atoh1 in MB cells. We then applied a SILAC-based quantitative ubiquitinomics to investigate the Usp7 target landscape in SHH-MB. We also employed several biochemical approaches to demonstrate the functional interaction between Atoh1 and Usp7 in SHH-MB. In parallel, we validated the impact of Usp7 depletion in vitro and in vivo using relevant SHH-MB models. Last, we investigated large transcriptomic and proteomic datasets to unveil the prognostic value of Usp7 in SHH-MB human tumors. RESULTS We demonstrate that Usp7 binds and accumulates Atoh1 protein by counteracting its ubiquitylation. Post-natal electroporation in vivo directly into the developing cerebella also indicates that overexpression of Usp7 promotes proliferation, whereas in the absence of Atoh1 Usp7 was no longer able to spread cell growth, demonstrating a functional link between the Usp7-Atoh1 axis. In vivo, disruption of Usp7 expression in SHH-MB blocks tumor proliferation. Pharmacological inhibition of Usp7 triggers tumor growth in a syngeneic SHH MB mouse model. Lastly, SHH-MB cells with either TP53 or SMO downstream mutations are sensitive to Usp7 inhibition. CONCLUSIONS Overall, our data pinpoint Usp7 as a promising target against Atoh1, a master regulator of SHH-MB.

Abstract 944: Pre-clinical activity of the Wnt pathway inhibitor RXC004 in combination with MAPK pathway inhibitors in GI cancer models

Abstract Background: Gastrointestinal (GI) cancers cause more cancer deaths than any other body system, with 3.4 million related deaths in 2018. Wnt pathway activation is common in GI cancers, with the pathogenic upstream Wnt pathway variant sub-population (RNF43 loss of function/RSPO gain of function) showing a combined prevalence of 4.7% in GI cancer patients; pre-clinically this sub-population shows exquisite sensitivity to RXC004, a small molecule Porcupine inhibitor. A striking co-occurrence of upstream Wnt pathway variants with MAPK pathway driver mutations (KRAS, BRAF, NRAS) was detected in 77% of Microsatellite stable (MSS) GI cancers, suggesting co-inhibiting these pathways could enhance clinical activity in these patients. Methods: In vitro proliferation assays were performed in pre-clinical models of upstream Wnt pathway mutated MSS GI cancer (SNU-1411, HPAF-II and AsPC-1) using Wnt (RXC004) or MAPK (Trametinib) pathway inhibitors at multiple concentrations as single agents or in combination. Control cells with downstream Wnt pathway mutations (WiDr and HCT116) were also tested. Synergistic combination effects were detected by BLISS scores. RXC004 (1.5mg/kg QD) and Trametinib (0.3mg/kg QD) were evaluated in an RSPO-fusion xenograft model (SNU-1411) as single agents or in combination. Efficacy was measured by tumour volume and weight; PD markers were assessed in end of study tumour samples. Relative AXIN2 and DUSP6 gene expression changes were determined upon inhibitor treatment by quantitative Polymerase Chain Reaction (qPCR). Results: RXC004 in combination with Trametinib demonstrated strong anti-proliferative synergy in SNU-1411 cells and moderate synergy/additivity in HPAF-II and AsPC-1 cells; no synergy was observed in control cells. In vivo, RXC004 and Trametinib monotherapy led to significant moderate tumour growth control (p<0.05) at the doses tested, whereas their combination led to significantly enhanced efficacy (p<0.05). Gene expression analysis demonstrated a reciprocal Wnt/MAPK signaling mechanism; RXC004 strongly suppressed Wnt signaling (AXIN2) but increased MAPK signaling (DUSP6), whilst Trametinib strongly suppressed MAPK signaling (DUSP6) but increased Wnt signaling (AXIN2). Combination of RXC004 and Trametinib sustained inhibition of both Wnt and MAPK signalling. Similar effects were observed with inhibitors of other MAPK pathway components. Conclusion: Wnt and MAPK pathways are frequently co-activated in GI cancers, particularly in the upstream Wnt pathway mutated sub-population. Pre-clinically we show that Wnt and MAPK pathways act as potential reciprocal resistance mechanisms following single agent inhibition of either pathway; co-inhibition of these pathways leads to synergistic effects in vitro and enhanced efficacy in vivo. This provides a clear rationale to assess this combination approach in the clinic. Citation Format: Simon A. Woodcock, Dorottya Keppel, Catherine Eagle, James Kelly, Eimear Flanagan, Emma Bishop, Inder Bhamra, Clifford D. Jones, Richard Armer, Jane Robertson, Caroline Phillips. Pre-clinical activity of the Wnt pathway inhibitor RXC004 in combination with MAPK pathway inhibitors in GI cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 944.

Extreme restructuring of cis-regulatory regions controlling a deeply conserved plant stem cell regulator.

A striking paradox is that genes with conserved protein sequence, function and expression pattern over deep time often exhibit extremely divergent cis-regulatory sequences. It remains unclear how such drastic cis-regulatory evolution across species allows preservation of gene function, and to what extent these differences influence how cis-regulatory variation arising within species impacts phenotypic change. Here, we investigated these questions using a plant stem cell regulator conserved in expression pattern and function over ~125 million years. Using in-vivo genome editing in two distantly related models, Arabidopsis thaliana (Arabidopsis) and Solanum lycopersicum (tomato), we generated over 70 deletion alleles in the upstream and downstream regions of the stem cell repressor gene CLAVATA3 (CLV3) and compared their individual and combined effects on a shared phenotype, the number of carpels that make fruits. We found that sequences upstream of tomato CLV3 are highly sensitive to even small perturbations compared to its downstream region. In contrast, Arabidopsis CLV3 function is tolerant to severe disruptions both upstream and downstream of the coding sequence. Combining upstream and downstream deletions also revealed a different regulatory outcome. Whereas phenotypic enhancement from adding downstream mutations was predominantly weak and additive in tomato, mutating both regions of Arabidopsis CLV3 caused substantial and synergistic effects, demonstrating distinct distribution and redundancy of functional cis-regulatory sequences. Our results demonstrate remarkable malleability in cis-regulatory structural organization of a deeply conserved plant stem cell regulator and suggest that major reconfiguration of cis-regulatory sequence space is a common yet cryptic evolutionary force altering genotype-to-phenotype relationships from regulatory variation in conserved genes. Finally, our findings underscore the need for lineage-specific dissection of the spatial architecture of cis-regulation to effectively engineer trait variation from conserved productivity genes in crops.

ERBB2-Mutant Gastrointestinal Tumors Represent Heterogeneous Molecular Biology, Particularly in Microsatellite Instability, Tumor Mutation Burden, and Co-Mutated Genes: An In Silico Study.

During recent years, activating mutations in ERBB2 have been reported in solid tumors of various organs, and clinical trials targeting ERBB2-mutant tumors have been conducted. However, no effective treatment has been established for gastrointestinal tumors targeting ERBB2 mutations. ERBB2-mutant tumors have a higher tumor mutation burden (TMB) and microsatellite instability (MSI) than ERBB2 non-mutant tumors, but not all ERBB2-mutant tumors are TMB- and MSI-high. Thus, a more detailed classification of ERBB2-mutant tumors based on the underlying molecular mechanisms is required. Herein, we classified ERBB2 mutations into three groups-group 1: both ERBB2 mutations and amplifications; group 2: ERBB2 mutations annotated as putative driver mutations but without amplifications; group 3: ERBB2 mutations annotated as non-driver mutations (passenger mutations or unknown significance) and those that were not amplified in gastrointestinal tumors. Esophageal adenocarcinoma, gastric cancer, and colorectal cancer presented significantly higher MSI and TMB in the ERBB2-mutant group than in the ERBB2-wild-type group. The proportions of TMB- and MSI-high tumors and frequency of co-mutated downstream genes differed among the groups. We identified TMB- and MSI-high groups; this classification is considered important for guiding the selection of drugs for ERBB2-mutant tumors with downstream genetic mutations.

The Effect of BSCL2 Gene on Fat Deposition Traits in Pigs.

BSCL2 encodes seipin, a transmembrane endoplasmic reticulum protein associated with lipodystrophy and severe metabolic complications, including diabetes and hepatic steatosis. In pigs, BSCL2 expression increases during adipocyte differentiation. In the present study, we identified significant gene variants associated with fat deposition (FD)-related processes based on subcutaneous fat tissue RNA-seq data. In the association study, to prove our hypothesis, three Polish pig breeds were included: Złotnicka White (ZW, n = 72), Polish Landrace (PL, n = 201), and Polish Large White (PLW, n = 169). Based on variant calling analysis and χ2 tests, BSCL2 mutations showing significantly different genotype/allele distribution between high- and low-fat pigs were selected for a comprehensive association study. Four interesting BSCL2 variants (rs346079334, rs341493267, rs330154033, and rs81333153) belonging to downstream and missense mutations were investigated. Our study showed a significant decrease in minor allele frequency for two BSCL2 variants (rs346079334 and rs341493267) in PL pigs in 2020-2021. In ZW, BSCL2 mutations significantly affected loin and ham fats, meat redness, and growth performance traits, such as feed conversion and daily feed intake. Similar observations were noted for PLW and PL, where BSCL2 mutations influenced fat depositions and meat traits, such as loin eye area, loin mass and fat, carcass yield, and growth performance traits. Based on the observation in pigs, our study supports the theory that BSCL2 expressed in subcutaneous fat is involved in the FD process.

Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet-Induced Adiposity.

Recent genetic evidence has linked WNT downstream mutations to fat distribution. However, the roles of WNTs in human obesity remain unclear. Here, theauthors screenall Wnt-related paracrine factors in 1994 obese cases and 2161 controls using whole-exome sequencing (WES) and identify that 12 obese patients harbor the same mutations in RSPO1 (p.R219W/Q) predisposing to human obesity. RSPO1 is predominantly expressed in visceral fat, primarily in the fibroblast cluster, and is increased with adiposity. Mice overexpressing human RSPO1 in adipose tissues develop obesity under a high-fat diet (HFD) due to reduced brown/beige fat thermogenesis. In contrast, Rspo1ablation resists HFD-induced adiposity by increasing thermogenesis. Mechanistically, RSPO1 overexpression or administration significantly inhibits adipocyte mitochondrial respiration and thermogenesis via LGR4-Wnt/β-catenin signaling pathway. Importantly, humanized knockin mice carrying the hotspot mutation (p.R219W) display suppressed thermogenesis and recapitulate the adiposity feature of obese carriers. The mutation disrupts RSPO1's electrostatic interaction with the extracellular matrix, leading to excessive RSPO1 release that activates LGR4-Wnt/β-catenin signaling and attenuates thermogenic capacity in differentiated beige adipocytes. Therefore, these findings identify that gain-of-function mutations and excessive expression of RSPO1, acting as a paracrine Wnt activator,suppress fat thermogenesis and contribute to obesity in humans.

Loss of mismatch repair promotes a direct selective advantage in human stem cells.

Lynch syndrome (LS) is the most common hereditary form of colon cancer, resulting from a germline mutation in a DNA mismatch repair (MMR) gene. Loss of MMR in cells establishes a mutator phenotype, which may underlie its link to cancer. Acquired downstream mutations that provide the cell a selective advantage would contribute to tumorigenesis. It is unclear, however, whether loss of MMR has other consequences that would directly result in a selective advantage. We found that knockout of the MMR gene MSH2 results in an immediate survival advantage in human stem cells grown under standard cell culture conditions. This advantage results, in part, from an MMR-dependent response to oxidative stress. We also found that loss of MMR gives rise to enhanced formation and growth of human colonic organoids. These results suggest that loss of MMR may affect cells in ways beyond just increasing mutation frequency that could influence tumorigenesis.

Oncogenic JAK2/STAT Signaling Molecules Activation Is Associated with PD-L1 Upregulation in Acute Myeloid Leukemia

Background: Increased evidence suggests the PD-1/PD-L1 axis is involved in the oncogenesis of myeloid neoplasms through promoting cancer immune escape, which suggests potential benefits by blockade of this axis in these neoplasms by immune checkpoint inhibitors. However, the ongoing clinical trials on PD-1/PD-L1 blocker in MDS/AML have reported controversial results, unlike the promising therapeutic efficacy shown in solid tumors. Identifying reliable biomarkers and scoring of PD-L1 expression and targeting the subgroup of patients who might benefit the most from PD-1/PD-L1 blockade are promising directions. Janus kinase 2 (JAK2) pathway activation has been shown to enhance PD-L1 expression in malignancies by in vitro studies. Here, we investigated PD-L1 expression associated with JAK2/STAT oncogenic pathway in AML patients using the combined positive score (CPS) algorithm. Methods: Immunohistochemical staining (IHC) for PD-L1 and pSTAT3 was performed on the marrow biopsies of 31 AML patients (39 cases), including 11 patients (17 cases) with JAK2/STAT mutation and 20 patients (22 cases) with wild-type JAK2/STAT. For PD-L1 IHC, the cases were evaluated for variable staining intensity (weak, moderate, strong) and membrane staining pattern (partial, complete) in tumor and mononuclear immune cells. The PD-L1 CPS was calculated by summing the number of all PD-L1-stained cells and dividing by the total number of tumor cells, multiplied by 100. The pSTAT3 was evaluated by percentage of positive tumor cells among the total tumor cells. The status of JAK2 / STAT mutations and other molecular abnormalities was evaluated by next generation sequencing (NGS) study. Results: Among patients of JAK2/STAT mutations, nine with JAK2 V617F exon 14 mutations, one with JAK2 I540-E543delinsRG exon 12 mutation and one with STAT5B N642H mutation. Evaluation of PD-L1 expression by IHC revealed four major cell staining patterns in our study cohort: 1. Negative (Figure 1A); 2. Partial weak membrane staining (Figure 1B, CPS<10); 3. Partial-complete, weak-moderate membrane staining (Figure 1C, CPS≥10); 4. Complete, moderate-strong membrane staining (Figure 1D, CPS≥10). Patterns 3-4 were classified as positive PD-L1 staining with a CPS cut-off of 10 (CPS≥10). Comparison of the PD-L1 CPS score in the JAK2/STAT wild-type versus the JAK2/STAT mutant groups revealed significantly higher PD-L1 CPS score in the JAK2/STAT mutant group (Figure 1K, P<0.0001, CPS≥10). One patient showed wild-type JAK2 at initial AML diagnosis. At two subsequent relapses, gain of JAK2 exon 12 (I540-E543delinsRG) mutation with increased VAF from 17% to 41% was detected. Interestingly, PD-L1 expression was correspondingly increased with CPS from 25 to 75 in these subsequent biopsies (Figure 1E, F). In another case with wild-type JAK2, STAT5B N642H mutation was detected and strong PD-L1 expression with CPS of 80 was obtained (Figure 1G). To address if JAK2/STAT3 axis is involved in PD-L1 upregulation, we assessed STAT3 phosphorylation by IHC in JAK2 wild-type and mutated groups. The results revealed significantly increased p-STAT3 expression in the JAK2 mutant compared with the wild-type cases, supporting the idea that oncogenic JAK2 mutation leads to STAT3 phosphorylation (Figure 1H-J). Furthermore, p-STAT3 expression showed a significant positive correlation with the PD-L1 CPS score (Figure 1H, r=0.8663, P<0.0001). Conclusion: In the AML patients with oncogenic JAK2 or its downstream signaling molecule STAT5B mutations, there is significantly enhanced PD-L1 expression. In addition, we proposed that the PD-L1 CPS positivity cut-off score of 10 being used as a quantitative measure of PD-L1 expression in this heterogeneous myeloid neoplasm. In the patient with relapsing AML, PD-L1 expression showed a positive correlation with increased JAK2 mutant VAF. Independent of the oncogenic JAK2 mutation, PD-L1 expression upregulation was also noted with STAT5B N642H mutation. Lastly, we demonstrated enhanced p-STAT3 expression in JAK2 mutant cases and a positive correlation between p-STAT3 and PD-L1 expression. Taken together, these data support the model that JAK2/STAT pathway activation is associated with PD-L1 upregulation in AML patients (Figure 1I), together with the proposed PD-L1 CPS cut-off of 10, can serve as scanning biomarkers to select relevant subgroups of AML patients for PD-1/PD-L1 immunotherapeutic study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

HER2 in Uterine Serous Carcinoma: Testing platforms and implications for targeted therapy

ObjectiveHER2 is an important prognostic and therapeutic target in uterine serous carcinoma (USC). Optimal HER2 testing platforms have not been defined and guidelines for testing have changed over time. Our objective is to assess the concordance of HER2 positivity based on chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), and next generation sequencing (NGS) and to determine the rate of downstream mutations that may affect response to HER2 directed therapy. MethodsUtilizing the Caris tumor registry, 2192 USC tumors were identified and analyzed using NGS (NextSeq, 592 Genes and WES, NovaSEQ), IHC, and CISH. PD-L1 expression was tested by IHC. Microsatellite instability was tested by fragment analysis, IHC, and NGS. Tumor mutational burden (TMB) was measured by totaling somatic mutations per tumor. HER2 positivity through IHC and CISH was determined based on 2007 and 2018 ASCO/CAP HER2 breast cancer guidelines. ResultsThere was a higher rate of HER2 positivity by IHC when using the 2018 guidelines compared to the 2007 guidelines (16.3% vs 12.3%). Concordance between IHC and CISH was 98.9%. ERBB2 amplification was identified by NGS in 10.5% of tumors. Compared to CISH results, this corresponds to a concordance rate of 91.6% and a positive predictive value (PPV) of 60.3%. Single gene alterations in HER2 amplified tumors that may implicate HER2 therapy resistance included PI3K (33.1%), KRAS (2.5%), and PTEN (1.3%). ConclusionsThere was high concordance between HER2 positivity based on CISH and IHC. Rate of HER2 positivity is the lowest by NGS. Ultimately these testing platforms need to be validated by response to targeted therapy.

Tumor profiling of KRAS, BRAF, and NRAS gene mutations in patients with colorectal cancer: A Lebanese major center cohort study

BackgroundIn the era of precision medicine, treatment schemes for advanced Colorectal (CRC) disease include monoclonal antibodies which block the epidermal growth factor receptor (EGFR) implicated in tumor proliferation, invasion, migration and neovascularization. Resistance to these agents has been correlated with activating downstream mutations in KRAS, BRAF and NRAS genes, among others, leading to constitutive activation of the EGFR axis bypassing EGFR blockade. The assessment of tumor RASandBRAFmutational status has thus become standard clinical practice. While multiple investigations reported roughly mutations rates of 40% in KRAS, 7% in NRAS and 5–15 % in BRAF, numbers vary across different populations with limited data specifically from the Middle East. MethodsThis is a retrospective observational Laboratory information system (LIS) chart review of all the patients with pathologically confirmed colorectal carcinoma (CRC) or metastatic CRC who underwent KRAS, NRAS and/or BRAF mutational analysis testing at the Molecular Diagnostics Laboratory of the American University of Beirut Medical Center (AUBMC) from January 2012 to December 2018, inclusive. Data retrieved included the results of mutation testing performed for KRAS, NRAS and BRAF genes, the age, gender, and tumor location for each patient. Analysis of the mutations was performed using polymerase chain reaction (PCR) hybridization StripAssay® (ViennaLab, Vienna, Austria). Results130 (47.6%) out of 273 histologically confirmed CRC cases, had positive KRAS mutations, namely in codons 12 (82%), 13 (17%), 146 (1.5%), 117 (0.75%), or 61 (0.75%). Two patients had two concomitant mutations: 12 + 12 (different mutations) and 12 + 146. Of 203 CRC cases tested for NRAS mutations, 16 (7.8%) were found to be positive for a mutation in codon 12 (37.5%), 61 (37.5%), or 13 (12.5%). Two patients had two concomitant mutations: 12 + 13 and 59 + 61. Of 172 CRC cases tested for BRAF mutations, 2 (1.2 %) were positive for the V600E -. ConclusionThis retrospective study is the first to report the frequencies of KRAS, NRAS and BRAF gene mutations in a Lebanese CRC cohort diagnosed and managed at a tertiary care center. The frequencies of the studied somatic gene mutations were similar to previously reported cohorts in other populations however the rate of BRAF mutation was lower in this cohort than expected.

Afatinib (AFA) plus bevacizumab (BEV) combination after osimertinib (OSI) resistance in advanced EGFR-mutant NSCLC: A phase II study (ABCD-study).

9117 Background: After OSI failure, various resistant mechanisms such as C797S and uncommon EGFR mutations, or MET amplification have been reported. AFA is an irreversible EGFR-TKI with a potency as pan-HER inhibitor, including high sensitivity to uncommon EGFR mutations. Many clinical studies have also shown a synergy of EGFR-TKIs and VEGF inhibitors. Methods: ECOG PS 0-1 patients (pt) with EGFR-mutant NSCLC were enrolled after OSI resistance. AFA was prescribed at 30-40 mg QD, and BEV was administered at 15 mg/kg tri-weekly until progression. Plasma/histologic rebiopsied samples taken after OSI failure but before enrollment were analyzed to examine resistant mechanisms including gene alterations/copy-number gain using cancer personalized profiling by deep sequencing. Results: Between January 2018 and October 2020, 28 pts were enrolled. Mutation subtypes were: 9 (32%) Del-19; 5 (18%) Del-19+T790M; 5 (18%) L858R; 7 (25%) L858R+T790M; 1 (4%) Del-19+L858R+T790M; and 1 (4%) G719S. Median line of prior OSI was 2 (range, 1-9). CR/PR was obtained by prior OSI in 24 (86%) pts. Regarding AFA+BEV efficacy, one (4%) CR, 4 (14%) PR, and 17 (61%) SD were confirmed, resulting in response rate of 17.9% and disease control rate of 78.6%. Median DoR was 9.0 (range, 4.2-22.3) months. Median PFS and OS were 2.7 (95% CI, 2.0-4.6) months and not reached, respectively. Twenty-eight (100%) plasma and/or 21 (75%) histologic rebiopsies identified: 17 (61%) TP53; 15 (54%) T790M; 9 (32%) uncommon EGFR; 9 (32%) MET; 6 (21%) C797S; 3 (11%) BRAF; 2 (7%) HER2; 2 (7%) KRAS; and 2 (7%) PI3K mutations; or 14 (50%) EGFR; 6 (21%) MET; and 2 (7%) HER2 amplifications. One (4%) small cell transformation was found. Among 6 C797S pts, 1 CR, 4 SD, and 1 PD were confirmed. Three (33%) of 9 uncommon EGFR and 1 (50%) of 2 HER2 mutation positive pts achieved radiographic response. All 15 T790M-positive pts showed no response, but 5 (38%) of 13 T790M-negative pts responded to AFA+BEV. None (0%) of six pts with EGFR downstream signaling mutations such as BRAF, KRAS, or PI3K responded. Five (50%) of 10 pts without T790M/BRAF/KRAS/PIK3 mutations exhibited confirmed response. FAT1 mutation was found in two (40%) of 5 responded pts. Dose reduction/interruption of AFA was performed in 15 (54%) pts. Median number of BEV administrations was 4 (range, 1-32). There were neither TRD nor ILD. Adverse events ≥grade 3: hypertension (29%); proteinuria (7%); diarrhea (7%); and rash (4%) were observed. One (4%) grade 4 duodenal perforation was reported. Conclusions: AFA+BEV after OSI resistance demonstrated moderate efficacy and favorable safety. A small portion of C797S pts exhibited the sensitivity. Higher potency was suggested in T790M/BRAF/KRAS/PIK3 mutation-negative and uncommon EGFR/HER2 mutation-positive pts. Selected population could obtain clinical benefit from AFA+BEV, based on rebiopsy results after OSI resistance. Clinical trial information: UMIN000030545.

HER2 in uterine carcinosarcoma: Testing platforms and implications for targeted therapy.

5590 Background: Uterine carcinosarcomas (UCS) are rare, aggressive tumors accounting for 5% of all uterine cancers. Recurrence rates are high and 5-year survival is only 18-39%. HER2 is an emerging prognostic and therapeutic target in uterine cancer including UCS. Testing algorithms and platforms in breast and gastric cancers are well studied and validated, but optimal HER2 testing in uterine cancer is not yet established. We aimed to determine HER2 prevalence in UCS and the concordance of chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), and next generation sequencing (NGS) platforms to aid in the development of UCS specific testing guidelines. We also evaluated the rate of downstream mutations and immune biomarkers that may affect response to HER2 directed therapy. Methods: Eight hundred and seventy-five UCS tumor samples (primary 81.4%; metastatic 17.1%; unknown 1.5%) were analyzed using NGS (592, NextSeq; WES, NovaSeq, Caris Life Sciences, Phoenix, AZ). A subset of tumors with HER2 positivity were tested with IHC (4B5, Ventana) and/or CISH (INFORM DUAL HER2 ISH Assay, Ventana) based on 2018 ASCO/CAP HER2 Breast Cancer guidelines. Amplification of ERBB2/HER2 by NGS used a copy number cut-off &gt;6. PD-L1 expression was analyzed by IHC (SP142, positive cut-off ≥1%, Ventana). Tumor mutational burden (TMB) was measured by counting all somatic mutations found per tumor (TMB high cut-off &gt; 10 mutations per Mb). Microsatellite instability (MSI) was tested by fragment analysis (FA), IHC and NGS. Results: Rates of HER2 positivity were 3.4% (28/820) by NGS. Among the IHC/CISH-tested cohort, 7.2% was positive (10/139) by IHC, and 19.5% (27/133) by CISH. In the 105 samples tested with both IHC and CISH, the concordance was 100%. Specifically, 9/105 patients (8.6%) were IHC+/CISH+ and 96 patients (91.4%) were IHC-/CISH-. The concordance between CISH and NGS (N = 127) was 90.6% (sensitivity 100% and specificity 89.3%). Common gene alterations in CISH HER2+ UCS tumors that may implicate resistance to HER2 targeted therapy included mutations in TP53 (100%), FBXW7 (22.2%), PIK3CA (33.3%), PIK3R1 (18.5%), PTEN (3.7%) and KRAS (3.7%) and gene amplification of KRAS (11.1%). CISH+ HER2+ UCS tumors had low immunotherapy biomarker prevalence (0% MSI-H, 0% TMB high, 7.7% PD-L1+). Conclusions: Increased HER2 positivity was detected via CISH testing compared to IHC and NGS, which may reflect the heterogeneity of HER2 amplification due to mixed histology between the sarcoma and carcinoma portion of the tumor. High concordance rates were observed between CISH and IHC. These testing platforms need to be validated by response to HER2 targeted therapies in order to develop UCS specific testing guidelines.

Profiling of the most reliable mutations from sequenced SARS-CoV-2 genomes scattered in Uzbekistan

Due to rapid mutations in the coronavirus genome over time and re-emergence of multiple novel variants of concerns (VOC), there is a continuous need for a periodic genome sequencing of SARS-CoV-2 genotypes of particular region. This is for on-time development of diagnostics, monitoring and therapeutic tools against virus in the global pandemics condition. Toward this goal, we have generated 18 high-quality whole-genome sequence data from 32 SARS-CoV-2 genotypes of PCR-positive COVID-19 patients, sampled from the Tashkent region of Uzbekistan. The nucleotide polymorphisms in the sequenced sample genomes were determined, including nonsynonymous (missense) and synonymous mutations in coding regions of coronavirus genome. Phylogenetic analysis grouped fourteen whole genome sample sequences (1, 2, 4, 5, 8, 10–15, 17, 32) into the G clade (or GR sub-clade) and four whole genome sample sequences (3, 6, 25, 27) into the S clade. A total of 128 mutations were identified, consisting of 45 shared and 83 unique mutations. Collectively, nucleotide changes represented one unique frameshift mutation, four upstream region mutations, six downstream region mutations, 50 synonymous mutations, and 67 missense mutations. The sequence data, presented herein, is the first coronavirus genomic sequence data from the Republic of Uzbekistan, which should contribute to enrich the global coronavirus sequence database, helping in future comparative studies. More importantly, the sequenced genomic data of coronavirus genotypes of this study should be useful for comparisons, diagnostics, monitoring, and therapeutics of COVID-19 disease in local and regional levels.

Delivery of Targeted Co(III)-DNA Inhibitors of Gli Proteins to Disrupt Hedgehog Signaling.

The Hedgehog (Hh) signaling pathway is integral for embryonic development and normal cell maintenance. However, aberrant expression of the Hh pathway is recognized as the oncogenic driving force for basal cell carcinoma (BCC). Current chemotherapeutic treatments that inhibit Hh signaling allow treatment of only locally advanced and metastatic BCCs via inhibition of the transmembrane protein, smoothened. It is further recognized that downstream mutations often lead to chemoresistant tumor recurrence. The Gli proteins are the ultimate regulators of Hh signaling and belong to a family of Cys2His2 zinc finger transcription factors (ZnFTFs) that we have shown can be irreversibly inhibited by a series of cobalt(III) Schiff base-DNA (CoSB-DNA) conjugates. However, a significant challenge is the delivery of CoSB-DNA complexes in mammalian tissues. Herein, we report a polyethyleneimine-functionalized graphene oxide nanoconjugate (GOPEI) that delivers CoGli, a CoSB-DNA complex that targets Gli specifically. We describe the characterization of the surface functionalization of GOPEI and accumulation in ASZ murine BCC cells via confocal microscopy and inductively coupled plasma-mass spectrometry (ICP-MS). Lysosomal escape of CoGli is further confirmed by confocal microscopy. We report the successful targeting of Gli by CoGli and a 17-fold improvement in potency over small-molecule Gli inhibitor GANT-61 in inhibiting Hh-driven migration of ASZ murine BCC cells. This study provides a promising starting point for further investigating CoGli inhibitors of Hh signaling in developed mammalian tissues.

Mechanisms of Resistance to Noncovalent Bruton’s Tyrosine Kinase Inhibitors

BackgroundCovalent (irreversible) Bruton’s tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood.MethodsWe performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors.ResultsAmong 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell–receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors.ConclusionsResistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.)

Statistical Approach of the Role of the Conserved CSB-PiggyBac Transposase Fusion Protein (CSB-PGBD3) in Genotype-Phenotype Correlation in Cockayne Syndrome Type B.

Cockayne syndrome is a rare condition that encompasses a very wide spectrum of clinical severity. Mutations upstream of a transposon called PiggyBac Transposable Element Derived 3 in intron 5 of the CSB/ERCC6 gene could bring about less severe forms than mutations located downstream of that transposon insertion. Our aim was to study genotype-phenotype correlation by determining whether the position of each mutation of the CSB/ERCC6 gene has an impact on the phenotype. A hundred and forty-seven Cockayne patients, who had two pathogenic mutations in the CSB/ERCC6 gene and for whom clinical data was available, were retrospectively selected and included in the study. Data analysis was performed under the Bayesian paradigm. Analysis of the proportion of the different subtypes of Cockayne syndrome according to the position of the mutations was done using an ordinal logistic regression model. Using a vague prior, the risk of developing a more severe subtype when exposed to 2 mutations downstream compared to 2 mutations upstream was 2.0 [0.9–4.5]. Estimations varied through the sensitivity analysis. We could reasonably conclude that a relationship between the number of downstream mutations and the Cockayne syndrome clinical expression exists but it is still difficult to give a precise estimate of this relationship. The real effect could be more complex that the one described in the initial model and other genetic factors might be taken into consideration together with the mutation site to better explain clinical variability.

Abstract P4-01-01: Resistance to next generation tyrosine kinase inhibitors (TKIs) in HER2-positive breast cancer (BC): Role of HER and PIK3CA mutations and development of new treatment strategies and study models

Abstract Background: We recently reported that acquired resistance to the dual HER1/2 TKI lapatinib (Lap) was mediated by HER2 L755S, while resistance to the pan-HER TKI neratinib (Nrb) was associated with co-acquisition of an additional pathogenic PIK3CA mutation. Though the role of HER2 mutations is gaining attention in HER2-positive (+) BC, less is known about their role and clinical implications in next generation TKI resistance, particularly when co-occurring with PIK3CA mutations in HER2+ BC. Investigating optimal treatment combinations and the development of new clinically relevant 3D models are warranted.Materials and Methods: HER2+ BT474 parental (P) cells and models with acquired resistance to Lap (LapR) and Nrb (NrbR) (SABCS20-PD3-09) were used. Xenografts established in mice using P, LapR, and NrbR cells and 3D organoids derived from these xenografts using the Hans Clevers (HC, PMID 29224780) or Mark Burkard (MB, PMID 31175091) method were characterized by qRT-PCR and western blot. Drug efficacy was assessed by growth changes in 2D and 3D models using the IncuCyte system or by microscopy-based analysis. Results: We previously showed that Lap and Nrb resistance confers cross-resistance to tucatinib (Tuca) and trastuzumab, and that targeting the HER and downstream PI3K pathway, especially using small molecule agents that are key for treatment of CNS lesions, is effective only in combination with Nrb or poziotinib (Pozio), but not Tuca. Our new studies revealed that the MEK inhibitor (i) AZD6244 (selumetinib; Sel), mTORi everolimus (Eve), and selective estrogen receptor degrader fulvestrant (Ful) were not effective as single agents in inhibiting the growth of either LapR or NrbR models. Whilst the LapR cells were highly sensitive to the irreversible HER1/2 TKI afatinib (Afa) and the irreversible dual/pan-HER TKI pyrotinib (Pyro) as single agents, the NrbR models were cross-resistant to both TKIs, highlighting the importance of the co-occurring PIK3CA mutation in resistance. Interestingly, Afa and Pyro were only partly effective when combined with Eve+Ful, Sel+Eve, or Sel+the PIK3CAi alpelisib in inhibiting NrbR growth. Consistent with our previously reported findings for Nrb and Pozio, Pyro was highly effective with TDM1. As opposed to the P xenografts, the LapR and NrbR tumors grew in the presence of the respective TKI, confirming their resistant phenotype in vivo. P and resistant xenograft-derived organoids (XDOs) were successfully established using the HC but not MB method, but the HC-derived XDOs were subsequently grown in MB condition and used for molecular and functional studies. Preliminary characterization showed that the LapR tumors and XDOs harbor HER2 L755S, whereas the NrbR tumors and XDOs also have a concomitant PIK3CA E542V mutation, findings that are in line with our 2D results, suggesting that the xenografts and XDOs retain and recapitulate the molecular profile of their 2D or tumor counterparts. Early drug efficacy studies indicate that, akin to the 2D models, the LapR XDOs are highly sensitive to Nrb, whereas both the LapR and NrbR XDOs exhibit cross-resistance to Tuca but remain sensitive to Pozio.Conclusions: Our data suggest that the potency of next generation irreversible HER TKIs in HER2+ BC may be challenged by the emergence of mutations in HER2, together with other co-occurring downstream mutations, such as PIK3CA. Our findings present a clear roadmap for the development of combinatorial therapies that should be individualized for patients with HER2+ BC. Our newly developed XDO strategy may offer a new platform to confirm and prioritize optimal drug combinations to overcome this resistance and may facilitate the near future development of patient-derived organoids for precision medicine of resistant HER2+ BC. Citation Format: Sreyashree Bose, Ragini Mistry, Chia Chia Liu, Sarmistha Nanda, Lanfang Qin, Pier Selenica, Andrea Gazzo, Yingjie Zhu, Michael A. Mancini, Fabio Stossi, Irmina Diala, Lisa D. Eli, Britta Weigelt, Jorge S. Reis-Filho, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff, Jamunarani Veeraraghavan. Resistance to next generation tyrosine kinase inhibitors (TKIs) in HER2-positive breast cancer (BC): Role of HER and PIK3CA mutations and development of new treatment strategies and study models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-01.

Characterization of the SARS-CoV-2 genomes in Egypt in first and second waves of infection

At Wuhan, in December 2019, the SRAS-CoV-2 outbreak was detected and it has been the pandemic worldwide. This study aims to investigate the mutations in sequence of the SARS-CoV-2 genome and characterize the mutation patterns in Egyptian COVID-19 patients during different waves of infection. The samples were collected from 250 COVID-19 patients and the whole genome sequencing was conducted using Next Generation Sequencing. The viral sequence analysis showed 1115 different genome from all Egyptian samples in the second wave mutations including 613 missense mutations, 431 synonymous mutations, 25 upstream gene mutations, 24 downstream gene mutations, 10 frame-shift deletions, and 6 stop gained mutation. The Egyptian genomic strains sequenced in second wave of infection are different to that of the first wave. We observe a shift of lineage prevalence from the strain B.1 to B.1.1.1. Only one case was of the new English B.1.1.7. Few samples have one or two mutations of interest from the Brazil and South Africa isolates. New clade 20B appear by March 2020 and 20D appear by May 2020 till January 2021.