Mutational Analysis Research Articles

Subset of retinoblastoma tumours is associated with BRCA1/2 mutations

BackgroundWe investigated the potential association between pathogenic BRCA1/2 variants and retinoblastoma pathogenicity.MethodsIn this single-centre, retrospective case series, we performed hereditary cancer panel tests using blood samples for patients with retinoblastoma...

Analysis of rare mutations associated with Thalassemia and their hematological characteristics in Chenzhou region of Hunan Province

To explore the distribution and hematological characteristics of rare thalassemia-associated mutations in Chenzhou region of Hunan Province with an aim to provide a basis for genetic counseling and effective prevention. A total of 37 370 individuals enrolled from January 2015 to December 2021 were screened by routine blood test and hemoglobin electrophoresis. The genotypes were determined with high-throughput sequencing. A total of 8 455 thalassemia mutations (including 185 rare ones) were detected, which had involved 27 mutational types. Rare type α-Thalassemia --THAI and CD31 (AGG>AAG) have the typical microcytic hypochromic hematological features, whilst SEA-HPFH, CD14 (CTG>-TG), CD37 (TGG>TAG), -90(C>T), Codon 15 (G>A), IVS-I-128 (T>G), CD86 (GCC>GC-) and Chinese Gγ+(Aγδβ)0 had typical microcytic hypochromic and β-thalassemia-associated hematological features of elevated HbA2 or HbF. In addition, the -50（G>A）heterozygotes of β-thalassemia had normal or slightly decreased MCV and MCH without an increase in HbA2. Various forms of thalassemia-associated mutations have been identified in the Chenzhou region of Hunan Province. Above finding has facilitated development of preventive and control strategies for thalassemia as well as birth health programs.

An analysis of PIK3CA hotspot mutations and response to neoadjuvant therapy in breast cancer patients from four prospective clinical trials.

The PI3K signaling pathway is frequently dysregulated in breast cancer (BC), and mutations in PIK3CA, are relevant for therapy resistance in HER2pos BC. Mutations in exons 9 or exon 20 may have different impact on response to neoadjuvant chemotherapy-based treatment regimens. We investigated PIK3CA mutations in 1691 early BC patients, randomized in four neoadjuvant multicenter trials: GeparQuattro (NCT00288002), GeparQuinto (NCT00567554), GeparSixto (NCT01426880) and GeparSepto (NCT01583426). The role of different PIK3CA exons and hotspots for pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) and patient survival was evaluated for distinct molecular subgroups and anti-HER2 treatment procedures. A total of 302 patients (17.9%) of the full cohort of 1691 patients had a tumor with a PIK3CA mutation, with a different prevalence in molecular subgroups: luminal/HER2neg 95 of 404 patients (23.5%), HER2pos 170 of 819 patients (20.8%) and TNBC 37 of 431 patients (7.9%). We identified mutations in PIK3CA exon 20 to be linked with worse response to anti-HER2 treatment (OR=0.507, 95%CI 0.320-0.802, p=0.004), especially in HR positive HER2 positive BC (OR=0.445, 95%CI 0.237-0.837, p=0.012). In contrast, exon 9 hotspot mutations p.E452K and p.E545K revealed no noteworthy differences in response therapy response. Luminal/HER2neg patients show a trend to have worse treatment response when PIK3CA was mutated. Interestingly, patients with residual disease after neoadjuvant treatment, have better survival when PIK3CA was mutated. PIK3CA hotspot mutation p.H1047R are associated with worse pCR rates after NACT in HER2pos BC, while hotspot mutations in exon 9 seems to have less impact.

Genetic insights and therapeutic potential for colorectal cancer: mutation analysis of KRASgene and efficacy of Oleuropein-conjugated iron oxide nanoparticles.

This study aimed to address the challenges of treating advanced stages of colon cancer (CRC) by exploring potential therapeutic options. The research focused on the genetic aspects of CRC, specifically the mutation rate of the KRAS gene, along with other genes like TTN, APC, MUC16, and TP53, using the TCGA dataset. Additionally, the study investigated the efficacy of Oleuropein, a polyphenolic compound found in olives, in combating CRC by using iron oxide nanoparticles coated with glucose and conjugated with Oleuropein. The study characterized the physicochemical properties of the nanoparticles, and the cytotoxic effects of the nanoparticles were evaluated on CRC and normal fibroblast cell lines, demonstrating significantly higher cytotoxicity against CRC cells compared to normal cells. Furthermore, the study analyzed gene expression changes using the GSE124627 dataset to understand the influence of KRAS alterations. It identified numerous upregulated and downregulated genes in KRAS-overexpressing samples, suggesting their involvement in critical cancer-related pathways. These findings suggest that KRAS-influenced genes could serve as potential therapeutic targets for CRC treatment. The study also examined the expression levels of identified genes in CRC samples compared to normal samples. Among the upregulated genes, 22 showed significant increases in cancer samples, while 14 downregulated genes exhibited decreased expression in both KRAS-influenced and cancer samples. Cox regression analysis identified specific upregulated genes, including ANKZF1, SNAI1, PPFIA4, SIX4, and NOTUM, associated with poor prognosis. Kaplan-Meier analysis further confirmed the correlation between increased expression of these genes and higher patient mortality rates. In conclusion, this study provided valuable insights into the genetic aspects of CRC and potential therapeutic strategies. The use of Oleuropein-conjugated iron oxide nanoparticles showed promising cytotoxic effects on colon cancer cells. These findings contribute to advancing our understanding of CRC and offer potential targets for further investigation and the development of novel therapeutic approaches.

晚发胫前隐性营养不良型大疱性表皮松解症1例及基因检测

晚发胫前隐性营养不良型大疱性表皮松解症1例及基因检测

Epidemiological trends, antifungal drug susceptibility and SQLE point mutations in etiologic species of human dermatophytosis in Al-Diwaneyah, Iraq

Dermatophytes show a wide geographic distribution and are the main causative agents of skin fungal infections in many regions of the world. Recently, their resistance to antifungal drugs has led to an obstacle to effective treatment. To address the lack of dermatophytosis data in Iraq, this study was designed to investigate the distribution and prevalence of dermatophytes in the human population and single point mutations in squalene epoxidase gene (SQLE) of terbinafine resistant isolates. The identification of 102 dermatophytes isolated from clinical human dermatophytosis was performed through morphological and microscopic characteristics followed by molecular analysis based on ITS and TEF-1α sequencing. Phylogeny was achieved through RAxML analysis. CLSI M38-A2 protocol was used to assess antifungal susceptibility of the isolates to four major antifungal drugs. Additionally, the presence of point mutations in SQLE gene, which are responsible for terbinafine resistance was investigated. Tinea corporis was the most prevalent clinical manifestation accounting for 37.24% of examined cases of dermatophytosis. Based on ITS, T. indotineae (50.98%), T. mentagrophytes (19.61%), and M. canis (29.41%) was identified as an etiologic species. T. indotineae and T. mentagrophytes strains were identified as T. interdigitale based on TEF-1α. Terbinafine showed the highest efficacy among the tested antifungal drugs. T. indotineae and T. mentagrophytes showed the highest resistance to antifungal drugs with MICs of 2–4 and 4 μg/mL, while M. canis was the most susceptible species. Three of T. indotineae isolates showed mutations in SQLE gene Phe397Leu substitution. A non-previously described point mutation, Phe311Leu was identified in T. indotineae and mutations Lys276Asn, Phe397Leu and Leu419Phe were diagnosed in T. mentagrophytes XVII. The results of mutation analysis showed that Phe397Leu was a destabilizing mutation; protein stability has decreased with variations in pH, and point mutations affected the interatomic interaction, resulting in bond disruption. These results could help to control the progression of disease effectively and make decisions regarding the selection of appropriate drugs for dermatophyte infections.

High-throughput mutational analysis of a methyltransferase ribozyme

Methyltransferase ribozyme 1 (MTR1) is a catalytic RNA that has been isolated from a random RNA pool by in vitro selection. The ribozyme catalyzes site-specific formation of 1-methyl adenosine (m1A) using 6-methyl guanine (m6G) as a methyl group donor. The ribozyme has been extensively characterized by biochemical and structural analyses. Here, we describe high-throughput screening of single point mutants in the catalytic domain of MTR1 and determine their effect on ribozyme activity. Our mutational profiling method successfully assessed the activity of the 141 MTR1 variants tested in each experiment and revealed that the ribozyme is very sensitive to nucleotide substitutions in the catalytic core domain. Our technique can be applied to methyltransferase ribozymes that catalyze formation of different modifications such as 7-methylguanosine (m7G) or 3-methylcytidine (m3C).

Germline and somatic MUTYH mutations: Clinicopathologic and mutational analysis in a pan-cancer cohort.

10590 Background: MUTYH is a tumor suppressor gene involved in the base-excision DNA repair pathway. Biallelic pathogenic or likely pathogenic germline variants (gPV) in MUTYH are associated with MUTYH-associated polyposis (MAP), inclusive of colorectal carcinoma (CRC) and additional cancer risks. While monoallelic MUTYH gPV carriers do not have the same cancer risk as those with biallelic MAP, further work is needed to fully characterize secondary somatic events, including allelic imbalances. Methods: Using the MSK-IMPACT targeted NGS to detect both MUTYH gPVs and MUTYH somatic variants in patients (pts) with a solid tumor diagnosis, we analyzed clinicopathologic characteristics at cancer diagnosis, ancestry label as defined per cBioPortal, genomic biomarkers, as well as allele-specific copy number estimates using FACETS annotation. Results: Out of 38,907 samples with germline testing available, 587 pts with a solid tumor and presence of MUTYH gPV were identified. Of these, 15 pts had ≥2 MUTYH gPVs, including 4 pts with homozygous MUTYHgPVs (G396D, T179C, p.G480* and c.389-1G>C) and 11 pts with compound heterozygous MUTYHgPVs. In pts of European ancestry (n=403), the MUTYH G396D and T179C variants were predominant, present in 56% and 19% of cases, respectively. All patients with the R241W variant were listed as being of South Asian ancestry (n=6). Concurrent gPV in other genes were observed in 12% (n=69), with high/moderate-penetrance gPVs in 4% (n=24) including BRCA2 (n=6), BRCA1 (n=5), ATM (n=4) and PALB2 (n=3). Among all pts with MUTYH gPVs, 254/587 pts had concurrent tumor samples with FACETS results. Among pts with monoallelic MUTYH gPVs and FACETS annotation available, 24% of evaluable tumors exhibited biallelic MUTYH inactivation due to loss of heterozygosity (LOH). CRC (29% vs 13%; p=0.004) and cholangiocarcinoma (11% vs. 2%; p=0.04) were enriched in the biallelic MUTYH inactivation cohort vs. the cohort of pts with monoallelic MUTYH gPVs. Co-alterations in KRAS G12C and APC were enriched in the biallelic as compared to the monoallelic cohort (9% vs. 3%, p=0.04 and 27% vs. 12%, p=0.005, respectively). In addition, we identified 244 pts for whom FACETS annotation was available without MUTYH gPVs, but with somatic variants at MUTYH. Of these 244 pts, 117 pts had biallelic MUTYH inactivation either from homozygous deletions (n=87), somatic hit + LOH (n=27) or ≥2 somatic hits (n=3). The predominant cancer types with biallelic somatic MUTYH inactivation were breast cancer (16%), non-small cell lung cancer (14%) and CRC (11%). Conclusions: This study represents the largest cohort of pts with MUTYH gPVs and FACETS analysis on concurrent tumor samples, confirming a high prevalence of LOH in pts with monoallelic MUTYH gPVs and enrichment in KRAS G12Cin pts with MUTYH gPVs and biallelic MUTYH inactivation.

Prediction accuracy of biomarkers for response to immune checkpoint inhibitors in advanced non-small cell lung cancer: A systematic review and meta-analysis.

e14652 Background: To compare the predictive accuracy of PD-L1 IHC, tissue or blood tumor mutation burden (tTMB, bTMB), gene expression profiling (GEP), driver gene mutation, and combined marker for immunotherapy response of advanced non-small cell lung cancer (NSCLC). Methods: Systematic searches of PubMed, Cochrane CENTRAL, and Embase were conducted from inception to July 6, 2023. Clinical trials involved with predictive biomarkers exploration for immune checkpoint inhibitors (ICIs) in advanced NSCLC were eligible. The response according to the biomarkers were extracted. The primary outcome was the area under the curve (AUC) of the summary receiver operating characteristic (SROC). Sensitivity, specificity, likelihood ratios and predictive values were evaluated. The subgroup analysis of different PD-L1 cut-off, TMB test and driver gene mutation were performed. Results: 36 articles of 59 biomarker tests were included. The AUC of combined marker (0.75; 95%CI, 0.71-0.78) was higher than PD-L1 (0.64; 95%CI, 0.60-0.68), tTMB (0.64; 95%CI, 0.60-0.68), bTMB (0.68; 95%CI, 0.64-0.72), GEP (0.67; 95%CI, 0.63-0.71), and driver gene mutation (0.51; 95%CI, 0.47-0.55). Combined marker also had higher specificity, positive likelihood ratio and positive predictive value than single biomarkers (Table). In subgroup analysis, PD-L1 with cut-off 50% had higher AUC (0.67; 95%CI, 0.63-0.71) than cut-off 1% (0.62; 95%CI, 0.58-0.66); EGFR mutation had higher AUC (0.69; 95%CI, 0.65-0.73) than KRAS (0.53; 95%CI, 0.49-0.58); tTMB measured by Whole Exosome Sequencing (WES) had approximate AUC to that measure by targeted gene panel. The predictive accuracy of the biomarkers was further compared for patients receiving ICI-based combination therapy. The AUC of combined marker (0.70; 95%CI, 0.66-0.74) remained the highest for combination therapy. Conclusions: Combined marker (PD-L1+tTMB/bTMB/GEP) has superior predictive accuracy than single biomarkers for immunotherapy response of NSCLC. bTMB is a promising liquid biopsy biomarker. The prescription of ICI for patients with EGFR mutation should be cautious. Further investigation is warranted to precisely identify biomarkers in various clinical settings. [Table: see text]

Serial analysis of ESR1 and PIK3CA mutations in cell-free DNA from metastatic breast cancer during 1st line endocrine therapy.

e13060 Background: Activating mutations ofESR1 and PIK3CA genes are known mechanisms of endocrine resistance. Recent analysis supported benefit of early intervention at presence of ESR1 mutation in blood (bESR1) before clinical progression. We aimed to investigate ESR1 and PIK3CA mutations detected in cell-free DNA (cfDNA) from patients with hormone receptor-positive, HER2-negative metastatic breast cancer (MBC) and its impact on progression-free survival (PFS). Methods: Thirty patients undergoing 1st line endocrine therapy (ETx) were identified from a prospective cohort. Seven ESR1 hotspot mutations in the ligand binding domain were tested in cfDNA with droplet digital polymerase chain reaction (ddPCR) assay (Genopeaks Co., Ltd). Eleven PIK3CA hotspot mutations were tested using amplicon-targeted sequencing method (Genopeaks Co., Ltd) in both tumor and cfDNA. PFS analyses were performed using Kaplan-Meier methods and compared with the log-rank test. Results: We examined serial cfDNA samples from 26 patients collected every 3-6 months. ESR1 mutations were found in 61.5% (16/26) patients at any timepoint during 1st line endocrine therapy, with D538G being the most common followed by Y537C and 68.8% (11/16) being polyclonal. Among the 50.0% (8/16) bESR1 positive patients with clinical progression, 4 had bESR1 detected before clinical progression while 4 were diagnosed clinical progression concurrently, suggesting the necessity of closer intervals of cfDNA analyses. While presence of bESR1 did not affect PFS overall, 18.8% of patients with bESR1 detected within 6 months after 1st line ETx, displayed worse outcome (median PFS 5 vs 52 months). Also, patients with clearance of bESR1 mutation in subsequent cfDNA (81.3%, 13/16) were shown to have a better outcome (median PFS 52 vs 41 months). Among 14 patients with clinical progression, 8 had bESR1 mutation. Median PFS between patients with bESR1 positive vs negative was 14 vs 9 months. PIK3CA mutations were detected in 69.5% (16/23) of cfDNA at diagnosis of distant metastasis, among them 53.3% (8/15) had co-occurrence with bESR1 mutations. While presence of PIK3CA mutation in primary tumor tissue did not affect disease-free survival nor PFS, patients with blood PIK3CA mutation at diagnosis of metastasis displayed significantly worse outcome (p = 0.041). Conclusions: Substantial number of hormone receptor-positive, HER2-negative MBC patients were detected with ESR1 and PIK3CA mutation in plasma. Though it was a small-sized analysis to accurately assess statistical significance, we observed worse outcome in patients with 1) early detection (<6 months) of bESR1 and with 2) sustained bESR1 during palliative ETx. Patients with clinical progression without bESR1 mutation displayed shorter PFS than patients with bESR1. PIK3CA mutations in cfDNA were prognostic supporting the benefit of combined targeted therapy.

Evaluating the significance of next-generation sequencing (NGS) testing in Indian patients with advanced malignancies: An observational study on clinical outcomes and treatment costs.

e15091 Background: The widespread use of Next-Generation Sequencing (NGS) panels to guide targeted therapy in advanced malignancies has raised questions regarding their real-world impact on clinical outcomes and affordability. Methods: This observational study included 126 patients, conducted between January 2022 and December 2023 aimed to assess the utility of NGS testing in both the initial and subsequent lines of treatment for Indian patients facing advanced malignancies. Results: In the first-line setting, NGS was extensively employed for patients with metastatic Colorectal Cancer (mCRC) and Non-Small Cell Lung Cancer (NSCLC). Analysis of RAS and BRAF mutations in mCRC patients (n=21) informed the choice between EGFR and VEGFR-directed therapy, resulting in a median Progression-Free Survival (PFS) of 7.5 months. In NSCLC patients (n=37), the NGS identified targetable mutations in 30 cases, leading to significantly improved mPFS (12 months vs 6 mo with chemotherapy alone). NGS was also employed for HRR, HRD and BRCA testing in advanced breast (n=45) and ovarian carcinoma (n=11), which helps in determining prognosis, role of targeted therapy and genetics. No adverse events noted till date. For patients undergoing subsequent lines of treatment (n=12), NGS detected targetable mutations in 9 cases. NGS revealed actionable mutations (ERBB2, CD74/NRG1fusion and KRAS12C mutation) in metastatic NSCLC patients (n=3), leading to appropriate treatment and PFS of 4-6 months. In metastatic NSCLC patients, T790M mutation was detected in 1 patient confering PFS of 6 months with change to Osimertinib and EGFR exon 20 insertion mutation detected in another, survival didn't improve with addition of amivantamab due to poor general condition. Alpelisib was started for PIK3CA mutation in metastatic Ca Breast (HR+,HEr2-), which was discontinued due to poor tolerance after 3 months. In Metastatic Neuroendocrine Carcinoma (NEC) Lung, detection of EGFR l858R mutation did not confer a PFS benefit despite the addition of Geftinib to chemotherapy. Due to financial constraints, targeted therapy couldn't be started for 2 patients with pathogenic target detected. Conclusions: The study's findings suggest that NGS testing offers tangible clinical benefits in both initial and subsequent lines of treatment for advanced malignancies in the Indian population. Despite the varying costs of NGS in India (ranging from 240 to 1800 USD), the data implies an overall affordability, making it a viable option for patients with advanced malignancies. However, NGS didn't improve outcomes in patients with rapidly progressing disease, poor general condition and financial constraints.

Advancing drug development by leveraging microdissection for proteogenomic analysis of histological sections.

e14660 Background: Combining laser capture microdissection (LCM) with proteogenomics in cancer research offers a targeted way to way to explore the intricate interactions between the tumor microenvironment and tumor cells proper. For immuno-oncology (IO) research, this is especially important, since improvements in the predictability of patient response to IO-based drugs is greatly needed. An improved understanding of the spatial relationships involving tumor cells, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: From fresh frozen tissue, LCM is used to isolate and obtain distinct histological cell types. Nucleic acids and proteins are extracted from captured cells for in-depth multi-modality molecular profiling assays. This includes multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing the analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, DNA sequencing can be performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. RNA-seq may be performed for gene expression. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. Our novel proteogenomics approach is focused around a customized enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: A proteogenomic approach is reported that is utilizing amount limited tissue quantities from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Comparative analysis of targetable mutations and genomic-directed therapy by tumor genomic profile in soft tissue sarcoma: A single-center retrospective study.

e23534 Background: The use of tumor genomic profile (TGP) has led to improved genomic-directed therapies in many solid cancers. Due to its heterogeneity, soft tissues sarcomas (STS) have not benefited much from these genomic advances. Emerging research indicates that about 30% STS have actionable alterations which can lead to potential treatment options (Gounder et al. Nature 2022). Our study aimed to compare the differences in genomic-directed therapy between Non-Hispanic White (NHW) and Hispanic (H) patients with STS at Dignity Health Cancer Institute. Methods: We performed a retrospective analysis of medical records using the electronic medical record. Our study specifically examined patients diagnosed with STS, with a sample size of 14 NHW and 19 H individuals.The eligibility criteria consisted of known diagnosis of STS with completed TGP. We conducted a descriptive analysis using GraphPad Prism Software, where mutations were evaluated as continuous variables. The statistical significance of differences between groups was assessed using an unpaired t-test, with a significance level of < 0.05. Results: We found 73 tumor alterations, with 11 potentially targetable (15.2%). Most patients had negative to low tumor mutational burden (0-2 mut/Mb) with only 2 patients with high TMB ( > 10 mut/Mb). The most common mutations were TP53 and CDK4 with 9 patients (12%) each. The NHW group had an average of 2.57 mutations (standard deviation = 2.87), whereas the H group had an average of 1.42 mutations (standard deviation = 1.35). The median values were 2 and 1, respectively, with no statistically significant difference (p = 0.21). The average for mutations of unknown significance was 1.54 in NHW individuals, while it was 1.00 in Hispanics. In the NHW group showed 11 males and 3 females, while the H group included 14 males and 5 females. There was no significant difference in gender representation between the two groups (p = 1). 3 patients (9%) received non-FDA approved treatments based on TGP (1 CDKN2A-abemaciclib, 1 CDK4-ribociclib and 1 IDH1-ivosidenib) and 2 patients (6.6%) were enrolled in clinical trials. Conclusions: Our investigation reveals that there is no statistically significant disparity in the frequency of mutations between NHW and H groups in our sample. Nevertheless, there was an evident inclination towards an increased frequency of mutations in NHW. Similarly, the occurrence of mutations with uncertain significance was slightly higher in NHWs, although the difference was not statistically significant. Only a minority of patients had targetable mutations or were enrolled in clinical trials. However, the routine use of genomic profiles may lead to more personalized treatments. Our research emphasizes the need to expand the use of comprehensive genomic profiles to increase directed treatments for patients with STS.

Single-cell and bulk sequencing analysis to evaluate the oncogenic role of PDE3A in pan-cancer and validation in breast cancer.

e15128 Background: PDE3A is involved in critical physiological processes through the hydrolysis of cycle AMP (cAMP) and GMP (cGMP). Its role in the occurrence and progression of cancer has recently attracted attention, but carcinogenic mechanism in pan-cancer has not been fully elucidated. Methods: We explored the expression, prognostic and diagnostic value, mutation, methylation, immune infiltration, and signaling pathway enrichment analysis of PDE3A in pan-cancer by combining multiple bioinformatics databases and single-cell sequencing analysis. Furthermore, we verified the protein and mRNA expression levels of PDE3A and investigated its oncogenic role in the progression of breast cancer (BRCA). Results: PDE3A was expressed at lower levels than normal tissues in most cancers and showed a wonderful prognostic value. PDE3A was strongly correlated with immune cells, especially cancer-associated fibroblast (CAF), and was involved in various vital signaling pathways. Furthermore, PDE3A could facilitate BRCA progression by promoting cells invasion and migration, and it may be associated with the efficacy of endocrine therapy in ER+ BRCA. Conclusions: We provided new insights into the carcinogenic effect of PDE3A in pan-cancer. PDE3A may be a potential biomarker for cancer diagnosis and prognosis, and it is also a promising target for cancer immunotherapy.

Comprehensive clinical characteristics and ctDNA mutational profile analysis of endocrine resistance/sensitivity to adjuvant ET therapy in luminal breast cancer from the GEICAM/2014-03_RegistEM registry.

1011 Background: Patients (pts) with hormone receptor-positive (HR) early breast cancer (BC) may develop resistance to adjuvant endocrine therapies (ET), with relapse occurring months (m) to decades after surgery. Our study explores clinical characteristics, prognosis, and oncogenic drivers in circulating tumor DNA (ctDNA) at relapse in HR+ BC pts, based on their sensitivity to adjuvant ET. Methods: In the REGISTEM study, we identified 805 primary HR-positive BC pts who developed metastatic relapse. These cases were classified based on their sensitivity to adjuvant ET, according to the 5th ESO-ESMO ABC Guidelines: 1ET-resistance (1ET-R, relapse within the first 2 years of adj ET; n=138), 2ET-resistance (2ET-R, relapse after the first 2 years or <12 m after completing adj ET; n=284), and ET-sensitive (ET-S, relapse after >12 m of completing adj ET; n=383). Out of this cohort, 139 individuals had plasma samples obtained prior to the initiation of the first-line treatment for advanced disease, and these samples were analyzed using the AVENIO ctDNA assay. Results: Pts with 1ET-R were significantly more likely to have grade 3, stage III, and highly proliferative tumors (Ki67>20%). The median time to distant relapse was 24 m for 1ET-R, 52.8 m for 2ET-R, and 124 m for ET-S cases. For first-line treatment, the median PFS was 8.1 m for 1ET-R, 13 m for 2ET-R, and 21.2 m for ET-S relapse (p<0.0001). With a median follow-up in the advanced setting of 34 m, median OS was 28 m for 1ET-R, 46 m for 2ET-R, and 55 m for ET-S relapses (p<0.0001). Pts with ET resistance had more mutations (mut) and higher mut allele frequency than ET-sensitive relapse. Pts with ET resistance showed higher incidence of mut in TP53, ESR1, PTEN, andhigh copy number in EGFRand ERBB2 than ET-sensitive, but similar incidence of mut in PIK3CA.Mut in ESR1 and high copy number in EGFR were correlated with poorer PFS, while ESR1 mut were additionally associated with worse OS, although these associations were not independent of the ET relapse groups (Table). Furthermore, mut in PIK3CA were associated to worse PFS only in ET-R pts. Conclusions: In HR-positive BC, both primary and secondary resistance to adjuvant ET represents a highly aggressive disease with enriched actionable genetic alterations detected at the time of diagnosis of metastatic relapse. Addressing these alterations has the potential to mitigate the unfavorable prognosis associated with these cases. Clinical trial information: NCT02819882 .[Table: see text]

A retrospective, descriptive analysis of a single-center experience with patients with metaplastic breast cancer.

e13173 Background: Metaplastic breast cancer (MpBC) is a subtype of breast cancer (BC) that is commonly triple negative (TN) and chemotherapy (CT) resistant. Despite CT resistance, standard of care therapy is administered according to receptor status, regardless of metaplastic histology. Randomized data regarding optimal treatment of this rare histologic subtype are lacking. Methods: This was a retrospective analysis of MpBC patients (pts) treated at Miami Cancer Institute (MCI) between 2017 and 2021. Pts were identified using COTA real-world Analytics, an analytics platform enabling rapid investigation of longitudinal real-world data. Pt demographics, pathologic characteristics, staging, treatment, and survival outcomes were collected and analyzed. Results: 46 pts with MpBC were identified. Median age at diagnosis was 60.5 years, and 83.7% were post-menopausal. 84.8% were White, 10.9% were Black, and 54.3% were Hispanic (all White). 80.4% (n=37) had TNBC (ER < 1% and HER2 negative by ASCO CAP guidelines). 13.0% of pts (n=6) had spindle cell morphology and these pts had numerically lower 5-yr overall survival (OS) compared to those with non-spindle cell morphology; (83.3%; 95% CI 53.5%-100% vs 91.7%; 95% CI 82.7%-100%). Of 21 pts (45.7%) who underwent germline genetic testing, a pathogenic variant was detected in 2 (9.5%); (BRCA2, PALBB2). At diagnosis, 93.5% (n=43) had early-stage disease, 2.2% (n=1) had de novo metastatic breast cancer (MBC), 2 were unknown. Of the early-stage pts, 12 (27.9%) had stage 1, 19 (44.1%) stage 2, and 6 (14.0%) stage 3 BC. Of 43 pts with treatment records available, 34 (79.1%) received CT and 20 (46.5%) received anthracycline-based CT in the early-stage setting. Of those who received CT, 50% (n=17) were treated in the neoadjuvant setting, and 2 pts (11.8%) achieved pathologic complete response (pCR). Median distant recurrence free survival (DRFS) and OS were not reached in the overall cohort. Two-year OS was 90.5% (95% CI 81.6%-99.4%) and two-year DRFS was 78.9% (95% CI 66.6%-91.2%). Of the 43 pts with early-stage MpBC at diagnosis, 11 (25.6%) developed MBC with lung as the most common site of metastasis. Only 15.2% (n=7) of tumors underwent somatic mutation analysis and 71.4% (5/7) were PIK3CA-mutated. Of the pts who developed MBC, median progression-free survival with cytotoxic chemotherapy was 9.0 months (95% CI 5.0-13.0). Conclusions: MpBC represents an aggressive phenotype of BC and optimal guidance regarding treatment is lacking. Consistent with published literature, a high proportion of MpBC in this study were TN (>80%). Our dataset represents the largest cohort to date of Hispanic pts with MpBC. Despite the poor outcomes historically reported with MpBC, median DRFS and OS were not reached in this cohort, with short follow up. Further prospective randomized data on the optimal treatment of MpBC is warranted and a larger multi-institution review is planned.

Prognostic significance of ctDNA mutation analysis in patients with HER2-positive breast cancer undergoing neoadjuvant chemotherapy.

e12654 Background: Neoadjuvant chemotherapy (NACT) combined with dual anti-HER2 agents followed by surgical resection is the standard treatment for stage II-III HER2- positive breast cancer (BC). Despite the availability of effective therapies such as ado-trastuzumab emtansine (T-DM1) and neratinib, identifying patients at high risk of disease recurrence remains challenging. To evaluate whether circulating tumor DNA (ctDNA) can be used as a biomarker to assess treatment response in patients with HER2-positive EBC treated with neoadjuvant anti-HER2 therapy. ctDNA burden can serve as a useful determinant for escalating or de-escalating (neo)adjuvant strategy in EBC patients. This study aimed to assess the role of ctDNA mutation analysis using low-pass whole-genome sequencing (lpWGS) and whole exome sequencing (WES) in evaluating blood copy-number burden (bCNB), genomic alterations, mutational signatures, blood tumor mutational burden (bTMB), and minimal residual disease (MRD) monitoring. Methods: A total of 46 HER2-positive BC patients who underwent NACT combined with dual anti-HER2 agents and surgical resection were enrolled, including 28 clinical stage II and 17 III patients. Plasma samples were collected at four time points: before NACT, after 3 cycles, at the completion of NACT, and after surgery. lpWGS was utilized to assess bCNB, while WES was performed to evaluate genomic alterations, mutational signatures, and bTMB. Mutations selected from the baseline sample were monitored for MRD. In cases where lpWGS yielded negative results, targeted NGS sequencing was conducted. Somatic mutations are identified by whole exome sequencing across 20,000 genes (with boosted sequencing coverage for 600 cancer-related genes). Between 4-50 personalized somatic mutations or fusions are selected for each patient. This personalized panel together with a fixed MRD core panel is utilized for MRD detection and monitoring. Results: Among the enrolled patients, nodal metastasis was observed in 29 patients and 29 were hormone receptor-positive, and 36 achieved pCR (78.2%). Positive ctDNA was detected 58.8% of patients. Higher nodal stage was associated with ctDNA positivity at baseline. MRD positivity after chemotherapy was observed in 4 patients and 2 patients achieved pCR TP53 was the most frequently altered gene, followed by PIK3CA, MAP3K1, REV3L, MYC, MAP2K4, ERBB3, ATRX, ATM and ALK. This pattern is comparable to that observed in previous studies on BC. Non-PCR patients commonly exhibit MAP3K1 and MAP2K4 alteration is their ctDNA. Conclusions: The identification of MRD, regardless of pCR status, can be valuable for risk stratification and personalized treatment decision-making. Further studies are warranted to validate these findings and explore the clinical implications of ctDNA mutation analysis in HER2-positive BC management.

Characterization of the dual regulation by a c-di-GMP riboswitch Bc1 with a long expression platform from Bacillus thuringiensis.

A riboswitch generally regulates the expression of its downstream genes through conformational change in its expression platform (EP) upon ligand binding. The cyclic diguanosine monophosphate (c-di-GMP) class I riboswitch Bc1 is widespread and conserved among Bacillus cereus group species. In this study, we revealed that Bc1 has a long EP with two typical ρ-independent terminator sequences 28 bp apart. The upstream terminator T1 is dominant in vitro, while downstream terminator T2 is more efficient in vivo. Through mutation analysis, we elucidated that Bc1 exerts a rare and incoherent "transcription-translation" dual regulation with T2 playing a crucial role. However, we found that Bc1 did not respond to c-di-GMP under in vitro transcription conditions, and the expressions of downstream genes did not change with fluctuation in intracellular c-di-GMP concentration. To explore this puzzle, we conducted SHAPE-MaP and confirmed the interaction of Bc1 with c-di-GMP. This shows that as c-di-GMP concentration increases, T1 unfolds but T2 remains almost intact and functional. The presence of T2 masks the effect of T1 unwinding, resulting in no response of Bc1 to c-di-GMP. The high Shannon entropy values of EP region imply the potential alternative structures of Bc1. We also found that zinc uptake regulator can specifically bind to the dual terminator coding sequence and slightly trigger the response of Bc1 to c-di-GMP. This work will shed light on the dual-regulation riboswitch and enrich our understanding of the RNA world.IMPORTANCEIn nature, riboswitches are involved in a variety of metabolic regulation, most of which preferentially regulate transcription termination or translation initiation of downstream genes in specific ways. Alternatively, the same or different riboswitches can exist in tandem to enhance regulatory effects or respond to multiple ligands. However, many putative conserved riboswitches have not yet been experimentally validated. Here, we found that the c-di-GMP riboswitch Bc1 with a long EP could form a dual terminator and exhibit non-canonical and incoherent "transcription-translation" dual regulation. Besides, zinc uptake regulator specifically bound to the coding sequence of the Bc1 EP and slightly mediated the action of Bc1. The application of SHAPE-MaP to the dual regulation mechanism of Bc1 may establish the foundation for future studies of such complex untranslated regions in other bacterial genomes.

PIK3CA Hotspot Mutations in Saliva as a Diagnostic Marker in Oral Squamous Cell Carcinoma Patients.

This study aimed at the analogous detection of PIK3CA mutations, common in oral squamous cell carcinoma (OSCC), in matched tumor and saliva samples. Tissue and saliva samples were obtained from 29 patients diagnosed with primary OSCC. Saliva samples were obtained preoperatively; tissue specimens were acquired during tumor resection. Tumor DNA was extracted from both tissue and saliva samples. All samples were controlled for DNA quantity and quality and genetic matching of sample pairs was confirmed using the iPlex Pro Exome QC Panel. Variant detection was performed using the MassARRAY® System, a mass-spectrometry based detection system. Mutational analysis in tissue tumor DNA was made using the multiplexed ClearSEEK™ PIK3CA v1.0 Panel covering 20 hotspot mutations in PIK3CA. In saliva samples, variants were analyzed using both the ClearSEEK™ and the UltraSEEK® Lung v1.1 Panel, with a higher limit of detection but covering less PIK3CA variants. Overall, a PIK3CA variant was found in seven of the 29 tumor tissue samples (24%) by ClearSEEK™; UltraSEEK® additionally confirmed the variant in four of these seven positive samples. Of the three variants not detected by UltraSEEK®, two were not included in the panel and one was included but not detected. Of the seven variants found in tissue, five could also be detected in the matching saliva samples (71%), either by utilizing ClearSEEK™ or UltraSEEK® Conclusion: The detection of PIK3CA hotspot mutations in OSCC and their simultaneous occurrence in saliva underline the potential benefit of liquid biopsies for non-invasive cancer detection and follow-up care of OSCC patients.

A computational approach for structural and functional analyses of disease-associated mutations in the human CYLD gene

Tumor suppressor cylindromatosis protein (CYLD) regulates NF-κB and JNK signaling pathways by cleaving K63-linked poly-ubiquitin chain from its substrate molecules and thus preventing the progression of tumorigenesis and metastasis of the cancer cells. Mutations in CYLD can cause aberrant structure and abnormal functionality leading to tumor formation. In this study, we utilized several computational tools such as PANTHER, PROVEAN, PredictSNP, PolyPhen-2, PhD-SNP, PON-P2, and SIFT to find out deleterious nsSNPs. We also highlighted the damaging impact of those deleterious nsSNPs on the structure and function of the CYLD utilizing ConSurf, I-Mutant, SDM, Phyre2, HOPE, Swiss-PdbViewer, and Mutation 3D. We shortlisted 18 high-risk nsSNPs from a total of 446 nsSNPs recorded in the NCBI database. Based on the conservation profile, stability status, and structural impact analysis, we finalized 13 nsSNPs. Molecular docking analysis and molecular dynamic simulation concluded the study with the findings of two significant nsSNPs (R830K, H827R) which have a remarkable impact on binding affinity, RMSD, RMSF, radius of gyration, and hydrogen bond formation during CYLD-ubiquitin interaction. The principal component analysis compared native and two mutants R830K and H827R of CYLD that signify structural and energy profile fluctuations during molecular dynamic (MD) simulation. Finally, the protein–protein interaction network showed CYLD interacts with 20 proteins involved in several biological pathways that mutations can impair. Considering all these in silico analyses, our study recommended conducting large-scale association studies of nsSNPs of CYLD with cancer as well as designing precise medications against diseases associated with these polymorphisms.