Next-generation Sequencing Profiling Research Articles

Exploratory miRNA profiling from serum and bone tissue of mice with T1D-induced bone loss.

Type 1 diabetes (T1D) represents a significant health burden worldwide, with associated complications including bone fragility. Current clinical methods and biomarkers for assessing bone health and predicting fracture risk in T1D are limited and lack accuracy. MicroRNAs (miRNAs) have emerged as potential biomarkers for predicting T1D-induced bone loss, although comprehensive profiling studies are lacking. Previous investigations have indicated a link between dysregulated miRNA expression levels and impaired bone health in T1D. Therefore, in this study, we explored differential miRNA expression levels in serum and bone tissue of mice with T1D-induced bone loss using Next Generation Sequencing (NGS). T1D was induced using streptozotocin in male wild-type mice. Serum and bone tissues were analyzed at 14 weeks of age, following the prior characterization of bone loss in this mouse model. MiRNA profiling was conducted using two-independent NGS analyses and validated through quantitative RT-PCR. NGS profiling identified differential expression of miRNAs in serum and bone tissue of T1D mice compared to controls. The first NGS analysis revealed 24 differentially expressed miRNAs in serum and 13 in bone tissue. Especially, miR-136-3p was consistently downregulated in both serum and bone tissue. However, the second NGS analysis presented a distinct set of dysregulated miRNAs, with miR-206-3p overlapping in both tissues but exhibiting differential expression patterns. Surprisingly, miR-144-5p, miR-19a-3p, and miR-21a-5p displayed contrasting regulatory patterns between NGS and qPCR analyses. Finally, gene network analysis identified associations between dysregulated miRNAs and pathways involved in bone physiology, including TGF-beta, PI3-Akt signaling, and osteoclast differentiation in humans. In conclusion, our study offers initial insights into dysregulated miRNAs associated with T1D-induced bone loss, but also highlights the lack of consistency in the results obtained from miRNA sequencing in different cohorts. Thus, further investigation is needed to better understand the complexities of miRNA analyses before they can be established as reproducible biomarkers for predicting bone health in T1D.

Prenatal Ultrasound Findings and Chromosomal Outcomes of Pregnancies with Mosaic Embryo Transfer.

To investigate prenatal ultrasound findings and the chromosomal outcomes of mosaic embryo transfer. This retrospective study was conducted on pregnant women who underwent mosaic embryo transfer following blastocyst-stage preimplantation genetic testing for aneuploidy (PGT-A) at CHA Gangnam Medical Center from January 2021 to July 2024. Trophectoderm biopsy specimens were collected using standard protocols, and next-generation sequencing profiles were defined as mosaics when displaying copy number counts in the 20-80% range. The results of the PGT-A, the amniocentesis results, the findings of prenatal ultrasounds, and the pregnancy outcomes were analyzed. A total of 88 mosaic embryos were transferred, of which 77 embryos were successfully implanted. Sixty-seven embryo-maintained pregnancies went beyond 11 weeks (87.0%), all among 58 patients with singleton pregnancies. The chaotic subtype showed the lowest ongoing pregnancy rate, and high-level mosaicism was less frequent in the ongoing group, compared to the total study group and the successful implantation group. Amniocentesis was performed on 33 mothers (56.9%), revealing two cases with abnormal findings that did not correlate with the PGT-A results. Two cases showed abnormalities in the second trimester detailed ultrasound, and both subsequently demonstrated normal findings in the third trimester and after birth. The average gestational age at birth was 38.4 weeks, and the average birth weight was 3313 g. No congenital anomalies were detected in 16 postnatal cases. Our study indicated that mosaic embryos can develop into euploid healthy infants with various levels or types of mosaicism, although the postnatal follow-up data are limited. This study is invaluable for counseling clinical results after mosaic embryo transfer, reassuring that, if patients do not have euploid embryos available, mosaic embryos can also be a viable option for transfer.

Systematic review and feasibility study on pre-analytical factors and genomic analyses on archival formalin-fixed paraffin-embedded breast cancer tissue

Formalin-fixed paraffin-embedded (FFPE) tissue represents a valuable source for translational cancer research. However, the widespread application of various downstream methods remains challenging. Here, we aimed to assess the feasibility of a genomic and gene expression analysis workflow using FFPE breast cancer (BC) tissue. We conducted a systematic literature review for the assessment of concordance between FFPE and fresh-frozen matched tissue samples derived from patients with BC for DNA and RNA downstream applications. The analytical performance of three different nucleic acid extraction kits on FFPE BC clinical samples was compared. We also applied a newly developed targeted DNA Next-Generation Sequencing (NGS) 370-gene panel and the nCounter BC360® platform on simultaneously extracted DNA and RNA, respectively, using FFPE tissue from a phase II clinical trial. Of the 3701 initial search results, 40 articles were included in the systematic review. High degree of concordance was observed in various downstream application platforms. Moreover, the performance of simultaneous DNA/RNA extraction kit was demonstrated with targeted DNA NGS and gene expression profiling. Exclusion of variants below 5% variant allele frequency was essential to overcome FFPE-induced artefacts. Targeted genomic analyses were feasible in simultaneously extracted DNA/RNA from FFPE material, providing insights for their implementation in clinical trials/cohorts.

Tissue- and liquid-biopsy based NGS profiling in advanced non-small-cell lung cancer in a real-world setting: the IMMINENT study.

To date, for all non-small cell lung cancer (NSCLC) cases, it is recommended to test for driver alterations to identify actionable therapeutic targets. In this light, comprehensive genomic profiling (CGP) with next generation sequencing (NGS) has progressively gained increasing importance in clinical practice. Here, with the aim of assessing the distribution and the real-world frequency of gene alterations and their correlation with patient characteristics, we present the outcomes obtained using FoundationOne (F1CDx) and FoundationLiquid CDx (F1L/F1LCDx) NGS-based profiling in a nationwide initiative for advanced NSCLC patients. F1CDx (324 genes) was used for tissue samples, and F1L (70 genes) or F1LCDx (324 genes) for liquid biopsy, aiming to explore the real-world occurrence of molecular alterations in aNSCLC and their relationship with patients' characteristics. Overall, 232 advanced NSCLC patients from 11 Institutions were gathered [median age 63 years; never/former or current smokers 29.3/65.9%; adenocarcinoma/squamous 79.3/12.5%; F1CDx/F1L+F1LCDx 59.5/40.5%]. Alterations were found in 170 different genes. Median number of mutated genes per sample was 4 (IQR 3-6) and 2 (IQR 1-3) in the F1CDx and F1L/F1LCDx cohorts, respectively. TP53 (58%), KRAS (22%), CDKN2A/B (19%), and STK11 (17%) alterations were the most frequently detected. Actionability rates (tier I and II) were comparable: 36.2% F1CDx vs. 34% ctDNA NGS assays (29.5% and 40.9% F1L and F1LCDx, respectively). Alterations in KEAP1 were significantly associated with STK11 and KRAS, so as TP53 with RB1. Median tumor mutational burden was 6 (IQR 3-10) and was significantly higher in smokers. Median OS from metastatic diagnosis was 23 months (IQR 18.5-19.5) and significantly lower in patients harboring ≥3 gene mutations. Conditional three-year survival probabilities increased over time for patients profiled at initial diagnosis and exceeded those of individuals tested later in their clinical history after 12 months. This study confirms that NGS-based molecular profiling of aNSCLC on tissue or blood samples offers valuable predictive and prognostic insights.

Next-generation sequencing profiling of miRNAs in individuals with 22q11.2 deletion syndrome revealed altered expression of miR-185-5p

BackgroundThe 22q11.2 deletion syndrome (22q11.2DS) is a microdeletion syndrome with highly variable phenotypic manifestations, even though most patients present the typical 3 Mb microdeletion, usually affecting the same ~ 106 genes. One of the genes affected by this deletion is DGCR8, which plays a crucial role in miRNA biogenesis. Therefore, the haploinsufficiency of DGCR8 due to this microdeletion can alter the modulation of the expression of several miRNAs involved in a range of biological processes.ResultsIn this study, we used next-generation sequencing to evaluate the miRNAs profiles in the peripheral blood of 12 individuals with typical 22q11DS compared to 12 healthy matched controls. We used the DESeq2 package for differential gene expression analysis and the DIANA-miTED dataset to verify the expression of differentially expressed miRNAs in other tissues. We used miRWalk to predict the target genes of differentially expressed miRNAs. Here, we described two differentially expressed miRNAs in patients compared to controls: hsa-miR-1304-3p, located outside the 22q11.2 region, upregulated in patients, and hsa-miR-185-5p, located in the 22q11.2 region, which showed downregulation. Expression of miR-185-5p is observed in tissues frequently affected in patients with 22q11DS, and previous studies have reported its downregulation in individuals with 22q11DS. hsa-miR-1304-3p has low expression in blood and, thus, needs more validation, though using a sensitive technology allowed us to identify differences in expression between patients and controls.ConclusionsThus, lower expression of miR-185-5p can be related to the 22q11.2 deletion and DGCR8 haploinsufficiency, leading to phenotypic consequences in 22q11.2DS patients, while higher expression of hsa-miR-1304-3p might be related to individual genomic variances due to the heterogeneous background of the Brazilian population.

Assessment of CDKN2A homozygous and hemizygous deletion status in gliomas.

e14021 Background: Homozygous deletion of CDKN2A (Homdel) has been included in the WHO CNS5 classification as a grade 4 definition in IDH mutated gliomas without the 1p-/19q-codeletion ( IDH mu-noncodel). Hemizygous CDKN2A deletion (Hemdel) has also been reported to be associated with poor prognosis. The identification methods for CDKN2A deletion include next generation sequencing (NGS), methylation array, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). However, at present, there is no gold standard threshold for FISH to judge the homozygous and hemizygous deletion. Methods: We collected formalin fixed paraffin-embedded (FFPE) tissue samples from 99 patients with adult-type diffuse glioma and all samples were tested using DNA-based NGS profiling (Simceredx). In addition, CDKN2A status was also detected by FISH and IHC (P16 and MTAP). Based on the CDKN2A status determined by NGS, the FISH were evaluated using ROC curves, including Area Under Curve (AUC), sensitivity, specificity, and cut-off values of Homdel and Non-Homdel, and deletion (Homdel and Hemdel) and non-deletion. The consistency of CDKN2A status identified by NGS, FISH IHC-P16 and IHC-MTAP was compared by Kappa test and Cramer V coefficient. Results: In 99 enrolled patients, NGS results showed 49 patients with CDKN2A Homdel, 18 patients with CDKN2A Hemdel, and 32 patients with CDKN2A Normal. There were no significant differences in age, gender, location of occurrence, IDH mutation status, or receiving chemoradiotherapy among the three groups. There were statistically significant differences in cancer subtype (p value=0.015) and grade (p value=0.010) among the three groups: oligodendroglioma and 2-3 grade was more likely to be normal in CDKN2A and grade 4 significantly predisposed to be Homdel. The ROC curve determines that cutoff of Homdel and Non- Homdel by FISH is 23%, and greater than 23% is Homdel. The ROC curve showed that the AUC of FISH method using 23% cutoff is 0.958, and the sensitivity and specificity were 85.11% and 100%, respectively. The consistency between FISH and NGS was 0.854. For the inconsistent samples, all NGS results were Homdel, and all FISH were deletion except one. Compared the results with FISH, we found that the results of NGS and IHC-P16 were more consistent. The ROC curve determines that the cutoff of deletion and non- deletion by FISH is 39%, and greater than 39% is deletion. The ROC curve showed that the AUC of FISH method with 39% cutoff is 0.962 and the sensitivity and specificity were 95.31% and 90.62%, respectively. Consistency comparison of the four methods showed that the consistency between FISH and NGS in determining Homdel and Non-Homdel, and deletion and non-deletion were the highest, 0.841 and 0.86, respectively. Conclusions: In our study, four methods were used to determine the status of CDKN2A, and results showed that the consistency between FISH and NGS was the highest.

Co-occurring EGFR p.E709X Mutation Mediates Primary Resistance to the Third-Generation EGFR-TKIs in EGFR p.G719X-Mutant Patients with Advanced NSCLC.

The current National Comprehensive Cancer Network (NCCN) guidelines recommend afatinib or osimertinib as the preferred first-line treatment strategy for patients with advanced NSCLC harboring EGFR p.G719X mutation. However, in the absence of head-to-head trials comparing afatinib with osimertinib in EGFR p.G719X-mutant patients, it is unclear which regimen is the preferred treatment option. A large cohort of 4,228 treatment-naïve patients with lung cancer who underwent targeted next-generation sequencing (NGS) testing was screened for EGFR p.G719X mutation. A multicenter cohort involving 68 EGFR p.G719X-mutant patients with advanced NSCLC and NGS profiling was retrospectively enrolled to evaluate clinical responses to afatinib (n = 37) and the third-generation EGFR-TKIs (n = 31). Ba/F3 cells stably expressing the EGFR p.G719A mutation were created to investigate the response to EGFR-TKIs in vitro. Concurrent EGFR p.E709X mutations, being the most frequent co-occurring EGFR mutation in EGFR p.G719X-mutant NSCLC (∼30%), exerted a detrimental effect on outcomes in patients treated with third-generation EGFR-TKI [G719X/E709X vs. G719X; objective response rate (ORR): 0.00% vs. 47.62%, P < 0.001; mPFS: 7.18 vs. 14.2 months, P = 0.04, respectively]. Conversely, no significant difference was found in the treatment efficacy of afatinib between EGFR p.G719X/E709X and EGFR p.G719X patients (G719X/E709X vs. G719X; ORR: 71.43% vs. 56.67%, P = 0.99; mPFS: 14.7 vs. 15.8 months, P = 0.69, respectively). In vitro experiments elucidated a resistant drug sensitivity and poor inhibition of EGFR phosphorylation in Ba/F3 cells expressing EGFR p.G719A/E709K mutation upon the third-generation EGFR-TKI treatment. Co-occurring EGFR p.E709X mutation mediated primary resistance to the third-generation EGFR-TKIs in EGFR p.G719X-mutant patients but remained sensitive to afatinib. A personalized treatment strategy should be undertaken based on the coexisting EGFR p.E709X mutation status.

Digital counting of tissue cells for molecular analysis: the QuANTUM pipeline.

The estimation of tumor cellular fraction (TCF) is a crucial step in predictive molecular pathology, representing an entry adequacy criterion also in the next-generation sequencing (NGS) era. However, heterogeneity of quantification practices and inter-pathologist variability hamper the robustness of its evaluation, stressing the need for more reliable results. Here, 121 routine histological samples from non-small cell lung cancer (NSCLC) cases with complete NGS profiling were used to evaluate TCF interobserver variability among three different pathologists (pTCF), developing a computational tool (cTCF) and assessing its reliability vs ground truth (GT) tumor cellularity and potential impact on the final molecular results. Inter-pathologist reproducibility was fair to good, with overall Wk ranging between 0.46 and 0.83 (avg. 0.59). The obtained cTCF was comparable to the GT (p = 0.129, 0.502, and 0.130 for surgical, biopsies, and cell block, respectively) and demonstrated good reliability if elaborated by different pathologists (Wk = 0.9). Overall cTCF was lower as compared to pTCF (30 ± 10 vs 52 ± 19, p < 0.001), with more cases < 20% (17, 14%, p = 0.690), but none containing < 100 cells for the algorithm. Similarities were noted between tumor area estimation and pTCF (36 ± 29, p < 0.001), partly explaining variability in the human assessment of tumor cellularity. Finally, the cTCF allowed a reduction of the copy number variations (CNVs) called (27 vs 29, - 6.9%) with an increase of effective CNVs detection (13 vs 7, + 85.7%), some with potential clinical impact previously undetected with pTCF. An automated computational pipeline (Qupath Analysis of Nuclei from Tumor to Uniform Molecular tests, QuANTUM) has been created and is freely available as a QuPath extension. The computational method used in this study has the potential to improve efficacy and reliability of TCF estimation in NSCLC, with demonstrated impact on the final molecular results.

A pilot study to investigate the clinically predictive values of copy number variations detected by next-generation sequencing of cell-free deoxyribonucleic acid in spent culture media

To investigate the positive predictive value and false positive risk of copy number variations (CNV's) detected in cell free deoxyribonucleic acid (DNA) from spent culture media for nonviable or aneuploid embryos. Diagnostic/prognostic accuracy study. Patients aged 35 and younger with an indication for IVF-ICSI and elective single frozen embryo transfer at a single, private IVF center. Embryo selection was performed according to the conventional grading, blinded to noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) results. After clinical outcomes were established, spent culture media samples were analyzed. Prognostic accuracy of CNVs according to niPGT-A results to predict nonviability or clinical aneuploidy. One hundred twenty patients completed the study. Interpretations of next-generation sequencing (NGS) profiles were as follows: 7.5% (n = 9) failed quality control; 62.5% (n = 75) no CNVs detected; and 30% (n = 36) abnormal copy number detected. Stratification of abnormal NGS profiles was as follows: 15% (n = 18) whole chromosome and 15% (n = 18) uncertain reproductive potential. An intermediate CNV was evident in 27.8% (n = 5) of the whole chromosome abnormalities. The negative predictive value for samples with no detected abnormality was 57.3% (43/75). Whole chromosome abnormality was associated with a positive predictive value of 94.4% (17/18), lower sustained implantation rate (5.6%, 1/18), and higher relative risk (RR) for nonviability compared with no detected abnormalities (RR 2.21, 95% CI: 1.66-2.94). No other CNVs were associated with significant differences in the sustained implantation or RRs for nonviability. Unequal sex chromosome proportions suggested that maternal contamination was not uncommon. A secondary descriptive analysis of 705 supernumerary embryos revealed proportions of NGS profile interpretations similar to the transferred cohort. Significant median absolute pairwise differences between certain subcategories of CNV abnormalities were apparent. Whole chromosome abnormalities were associated with a high positive predictive value and significant RR for nonviability. Embryos associated with other CNVs had sustained implantation rates similar to those with no abnormalities detected. Further studies are required to validate the clinical applicability of niPGT-A. clinicaltrials.gov (NCT04732013).

Impact of somatic genomic alterations in patients with metastatic colorectal cancer in a minority-rich academic medical center.

215 Background: Genomic alterations in metastatic colorectal cancer (mCRC) play a pivotal role in the clinical manifestations of the disease. Mutations have been associated with the age of onset of mCRC, tumor location, and metastatic sites. We conducted a retrospective study to explore the relationship between the genomic profile and clinical phenotype in a distinct patient population predominantly comprised of Hispanic and Black patients with mCRC. Methods: Patients with mCRC with available next-generation sequencing (NGS) treated at Montefiore Einstein Comprehensive Cancer Center (MECCC) who were diagnosed from January 2000 - January 2023 were included. Molecular testing was collected from: Perthera, Guardant, Caris, and Foundation One. The following parameters were collected: race, ethnicity, age of CRC diagnosis, age of metastatic diagnosis, metastatic sites, NGS profile, tumor location, and lines of treatment. Mutations were categorized into one of eight distinct pathways: cell cycle, MAP Kinase, WNT, homologous recombination, DNA damage repair, epigenetic modifications, transcription, &amp; cell death. The primary endpoint was to examine the relationship between individual genetic alterations or any of these eight pathways and the clinical parameters. Results: 169 patients with microsatellite stable (MSS) mCRC were included (median age at diagnosis 59 years, 42% Hispanic, 38% Black). 148 distinct genetic alterations were discovered across our population, of which roughly half fit into one of eight clinical pathways. In concordance with previously published data, we found a significant association between PTEN and right-sided tumors (38% mutated in right-sided vs. 16% mutated in left-sided, p&lt;.001). Cell cycle pathway mutations and SMAD4 mutations were associated with an earlier age of both primary CRC (p=.001 &amp; p=.042, respectively) and metastatic diagnosis (p=&lt;.001 &amp; p=.013, respectively). CDK8 mutations were associated with atypical sites of metastases (p=.045). Conclusions: This analysis sought to deepen the understanding of how genomic alterations impacted the clinical phenotype in our population of mCRC patients. We identified previously undescribed associations between mutations in the cell cycle pathway and SMAD4 versus age of CRC diagnosis. As molecular sequencing in CRC continues to advance, these relationships may be further elucidated, which can potentially help guide treatment and identify higher-risk patients.

#249 : Spotlight on the Artefacts in Next Generation Sequencing in PGT-A: Possible Reason for High Mosaicism Reporting

Background and Aims: Advancement in Next Generation Sequencing (NGS) has led to the extensive utilization of Preimplantation Genetic testing for Aneuploidy. But one challenge, in reporting of aligned short read sequencing data, is the ability to discriminate between embryonic NGS profiles with genuine pathological copy number variations (CNVs) from artefacts arising from erroneous DNA amplification/library preparation. Do technical errors in VeriSeq-NGS lead to artefacts in CNV charts resulting in falsely increased reporting of chromosomal mosaicism? Method: After ethical clearance and informed consent, patients [Formula: see text] 35 years, [Formula: see text]1 implantation failures and severe male factor infertility underwent PGT-A. Those with aneuploid embryos on TE biopsy, donated their embryos for research in year 2022. The DNA extraction and amplification was carried out using Sureplex-DNA amplification system. The libraries were prepared using VeriSeqTM-PGS Library Preparation kit. A total of 24 libraries were pooled, denatured and subjected to NGS using Illumina MiSeq system. CNV visualization and analysis for each sample was carried out using BlueFuse Multi Software (Illumina, USA). The aneuploid whole blastocyst were subjected to the same protocol. Results: NGS profiles generated from the aneuploid whole blastocysts, were manually analyzed for CNVs and for any common genomic artifacts. The quality control was performed using Illumina-Quality Control requirements and only samples satisfying these requirements were used for analysis in the study. Common artifacts were identified affecting chromosome 7,11,16 and 19. Out of 22 blastocysts, NGS profiles of 21 blastocysts had artifact at chromosome 19, and 15 had artifacts at chromosome 7,11,16. These artifacts may be introduced during whole genome amplification and/or suboptimal NGS library preparation or is inherent weakness of the VeriSeq-NGS kit. Conclusion: The four commonly occurring artefacts involved chromosome 7,11,16 and 19, which could be introduced during steps of whole genome amplification or library preparation. The awareness of these artefacts is important to reduce the false positive/inconclusive rate.

New Pattern of Emerging Somatic Mutations in Optimal Responders Following Tyrosine Kinase Inhibitor Therapy in Chronic Myeloid Leukemia Patients Evidenced from Mutational Kinetic Analysis Based on Pairwise Comparison

Introduction Recent advances in genomics in chronic myeloid leukemia (CML) patients (pts) provide new insights in understanding somatic mutation (SM) and their prognostic implications following tyrosine kinase inhibitor (TKI) therapy. DNMT3A, TET2, or ASXL1 (DTA) mutations are most frequently detected following TKI therapy and have adverse prognosis. Five longitudinal patterns of changes in SM after TKI therapy were suggested with its clinical significance (Kim, Blood 2017): Pattern 1, persistent mutation burden despite optimal TKI response; Pattern 2, emerging mutation which correlates with TKI resistance/CML progression; Pattern 3, mutational clearance with mixed clinical outcomes; and Pattern 4 and 5 having some mutation in germline control as well as CML cells. Clonal evolution (CE) in Philadelphia negative (Ph -) clone is found in up to 10% of CML pts who respond to TKI therapy optimally but develop +8 or -7 changes in Ph - clone. This is diagnosed only by metaphase cytogenetic test on bone marrow samples. Alternatively, fluorescence in situ hybridization (FISH) test can be used. With recent advances in next-generation sequencing (NGS), we hypothesize that SM profiling can capture a clone carrying a SM in the Ph - population. We sequenced and analyzed mutation profiles in paired samples taken prior to TKI therapy and at longitudinal follow-up. Patients and methods The present study adopted single molecule-tagging and molecular inversion probe (smMIP)-based sequencing methods to analyze the DNA extracted from the mononuclear cell fraction of peripheral blood samples collected from CML pts at initial diagnosis and during follow-up. While conventional NGS has a relatively high intrinsic sequencing error rate and is limited in detecting mutations with a variant allele frequency (VAF) of less than 2%, smMIP-based sequencing overcomes this issue by tracking all sequence reads from a single strand and can detect mutations with a limit of detection up to 0.1%. The in-house CML-specific smMIP panel used in this study included 40 genes with 332 amplicon probes covering genes involved in epigenetic modifiers, activation signaling, myeloid transcription factor, spliceosome, tumor suppressor, cohesion, and miscellaneous functions. To evaluate kinetics of mutations, we calculated doubling time of mutations. Using the mutational VAFs from 2 time points, t1 and t2, the doubling time was calculated according to the following equation: = (-2 -1) log(2) /log(-2) - log(-1), where VAF1 and VAF2 is the VAF of the mutant at time points t1 and t2, respectively. Results We performed targeted deep sequencing on a total of 119 serial samples from 51 CML pts (median of 405.5 days following TKI therapy). 41 pts had an optimal response, 7 were non-responders, and 3 were non-evaluable for response. Pattern 2 was observed in 1 of 7 non-responders. In 12 pts pattern 3 (mutation clearance with mixed clinical outcomes) was observed. 22 pts carried 36 mutations. At baseline 9 pts had somatic mutations. Emergence of somatic mutations in optimal responders was observed in 13 out of total 51 (25%) pts, with DNMT3A (n=5), TET2 (n=4), ASXL1 (n=4), and EZH2 (n=2). While JAK2, SF3B1, U2AF1, PHF6, TP53, BRAF, and CBL were observed in one patient each (Figure 1). Particularly, those with emerging DTA mutation were all optimal responders to TKI therapy. This pattern of mutational change is novel and has never been described before. The median doubling time was 77 days (range 21-1909 days) in the group with emergence of somatic mutation in follow-up samples. The doubling time was 56.1, 93.7, 74.8 days in DNMT3A, ASXL1, and TET2 mutations, respectively. Based on kinetics of mutations (doubling time), to capture 10-fold increase (i.e. 1 log increase) of certain mutation, NGS profile can be monitored every 6-7 months. This pattern of emergence of mutations (mostly DTA mutation) in pts responding optimally to TKI therapy is new and does not fit into the five patterns mentioned in our previous paper (Kim, Blood 2017). Thus, we propose this sixth novel pattern called “Emerging mutational clones in CML pts responding optimally to TKI therapy”. Conclusion We suggest a new pattern of emerging mutations in optimal responders to TKI therapy, which could derive from expansion of Ph- clone carrying some clones with emerging mutations. Longitudinal NGS monitoring in CML patients is feasible with frequency every 6-7 months.

Circulating Tumor DNA Dynamics as Prognostic Markers in Locally Advanced and Metastatic Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is a deadly disease with frequent recurrence. There are unmet needs for prognostic biomarkers for dynamically monitoring disease progression and detecting minimal residual disease. To examine whether circulating tumor DNA is clinically useful as a prognostic biomarker for ESCC recurrence and patient survival. This single-center, population-based cohort study consecutively enrolled 147 patients receiving curative (n = 74) or palliative (n = 73) treatment at the surgery and clinical oncology departments of Queen Mary Hospital in Hong Kong from August 1, 2016, to September 31, 2021. Patients were followed up for 2 years. Plasma samples were collected at different longitudinal time points for a prospective circulating tumor DNA (ctDNA) next-generation sequencing profiling study of 77 actionable genes. Patients were treated with up-front surgery, neoadjuvant chemoradiotherapy plus surgery with or without adjuvant therapy, or palliative chemotherapy (CT). Detection of circulating tumor DNA (ctDNA), progression-free survival (PFS), and overall survival (OS). A total of 478 serial plasma samples from 147 patients with locoregional or metastatic ESCC were prospectively analyzed. Among the 74 patients in the curative group (median [range] age, 66 [46-85] years; 56 [76.0%] male), 44 (59.5%) relapsed and 36 (48.6%) died. For patients receiving curative surgical treatment, a high ctDNA level (hazard ratio [HR], 7.84; 95% CI, 1.87-32.97; P = .005) and ctDNA alterations (HR, 5.71; 95% CI, 1.81-17.97; P = .003) at 6 months postoperation were independently associated with poor OS. Among patients receiving neoadjuvant chemoradiotherapy, postneoadjuvant ctDNA alterations were associated with poor PFS (HR, 3.16; 95% CI, 1.17-8.52; P = .02). In the 73 patients in the palliative group (median [range] age, 63 [45-82] years; 63 [86.0%] male), 71 (97.3%) had disease relapse and 68 (93.2%) died. Detectable pre-CT NFE2L2 alterations were independently associated with PFS (HR, 2.99; 95% CI, 1.35-6.61; P = .007) and OS (HR, 28.39; 95% CI, 7.26-111.03; P = 1.52 × 10-6), whereas high ctDNA levels (HR, 2.41; 95% CI, 1.18-4.95; P = .02) and alterations in pre-cycle III ctDNA (HR, 1.99; 95% CI, 1.03-3.85; P = .04) showed weaker associations with PFS. Alterations in pre-CT ctDNA were independently associated with OS (HR, 4.46; 95% CI, 1.86-10.69; P = 7.97 × 10-4). The findings of this cohort study indicate that prognostic models incorporating ctDNA features are useful in ESCC. Both ctDNA level and NFE2L2 alterations pre-CT and before cycle III were found to be important prognostic factors in palliative groups, and ctDNA alterations after treatment and at 6 months after surgery may define high-risk groups for recurrence in the curative group. High-risk patients can benefit by a timely switch to the next therapeutic options.

P17.06.B TARGETED THERAPY MATCHED TO GENOMIC ALTERATIONS (BRAFV600E, NTRK, FGFR, ROS1, PIK3CA, PTEN) IN PATIENTS WITH RECURRENT IDH WILDTYPE GLIOBLASTOMA: A REAL-LIFE COHORT ANALYSIS FROM VENETO INSTITUTE OF ONCOLOGY, PADUA (ITALY)

Abstract BACKGROUND The use of next-generation sequencing (NGS) allows the identification of subgroups of patients that may respond to personalized targeted therapies (TT). Despite the acquired deep knowledge about biology, there are only a few data available regarding TT efficacy and feasibility for patients with glioblastoma (GBM). MATERIAL AND METHODS We retrospectively analyzed a cohort of patients with recurrent GBM treated at Veneto Institute of Oncology, Padua (Italy) with TT based on their individual NGS profile. NGS was obtained with FoundationOne®CDx on formalin-fixed paraffin-embedded (FFPE) tissue samples. We identified 6 druggable genomic alterations, classified according to ESCAT (ESMO Scale for Clinical Actionability of molecular Targets): BRAFV600E mutation (IB), NTRK1-2-3 fusions (IC), FGFR1-2-3 alterations (IIB), ROS1 fusions (IIIA), PIK3CA mutations (IIIA) and PTEN loss/mutations (IIIA). TT was given as agnostic approval, part of compassionate use programs or in clinical trials. Response assessment was based on RANO (Response Assessment in Neuro-Oncology) criteria. RESULTS Between March 2020 and December 2022, 34 patients with recurrent glioblastoma received TT (19 males, 15 females, median age of 54 years at time of TT initiation). ECOG PS was 0-1 in 29 pts. All patients received radiotherapy and temozolomide as first-line therapy. The median line of therapy with TT was 3 (range 2-7). TT was dabrafenib/trametinib (9 pts), larotrectinib (2 pts), erdafitinib (4 pts), entrectinib (1 pt), alpelisib (6 pts), ipatasertib+/-atezolizumab (12 pts). At data cut-off (March 2023), 19 patients had died. In the entire cohort, median overall survival and progression-free survival (PFS) after starting TT was 8.72 and 2.14 months, respectively. The dabrafenib/trametinib subgroup had the longest median PFS (5.23 months), a disease control rate (DCR) of 77%, an objective response rate of 22%, and a median duration of response of 22.5 months. 7/9 pt had died and 2 pts are continuing dabrafenib/trametinib. The pt with ROS1-GOCP fusion maintained a complete response for 12 months with entrectinib. Among the others, no complete/partial responses were detected. DCR due to stable disease was 50% in larotrectinib and erdafitinib, 8.3% in ipatasertib+/-atezolizumab, 0% in alpelisib subgroup. No toxicities were reported in dabrafenib/trametinib subgroup. Among all patients, no grade 4 drug-related adverse events were observed and in any case TT was interrupted for toxicity. CONCLUSION Our results confirm the activity of dabrafenib/trametinib in BRAFV600E mutant glioblastoma pts and might suggest deeper explorations in targeting ROS1 and FGFR. Further correlation among patients' outcome, molecular characteristics and TT timing are still under investigation.

P16.01.A THE CASE OF AGGRESSIVE DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR IN ADULT TREATED WITH CRANIOSPINAL IRRADIATION AND TEMOZOLOMIDE

Abstract BACKGROUND Diffuse leptomeningeal glioneuronal tumor (DLGNT) is a rare tumor, which was introduced as a separate entity in the 2016 World Health Organization classification of central nervous system tumors. It is most common in pediatric age, but very rare in adults. Literature demonstrates limited reports regarding treatment strategy in adult. We present a case of young female affected by DLGNT with aggressive behavior. MATERIAL AND METHODS A 28-year-old woman presented with severe headache and vomiting. Brain MRI showed diffuse leptomeningeal enhancement with hydrocephalus. At first, she was under suspicion of tuberculosis meningitis with mild increased protein level in CSF study. Her symptom rapidly improved after ventriculoperitoneal shunt and she started anti-tuberculosis medication. Despite 8-month of medication, her neurological status gradually deteriorated with paraparesis and follow-up MR images showed progression of disease. She underwent open biopsy of superficial brain lesions. RESULTS Intraoperatively, multiple nodular masses were attached inner side of dura mater and diffuse adhesion on subarachnoid space of brain surface. Histological examination revealed small round cells with oligodendroglial-like aspects. Immunohistochemistry showed positivity for Olig2, synaptophysin and S100. Chromosome 1p deletion was confirmed by FISH. Next-generation sequencing profile disclosed mutation of H3K28M, FGFR1 and ATRX. Diagnosis was consistent with DLGNT. She received craniospinal irradiation of 36 Gy in 20 fractions, then started chemotherapy with temozolomide (TMZ) at a dose 200mg/m2/day for 5 days in 4 weeks. After 4 cycles of TMZ, MR images demonstrated stable disease. CONCLUSION DLGNT is a very rare tumor in adult. Diagnosis needed high level of suspicion and histological confirmation. We present a very rare adult DLGNT patient with aggressive clinical features who showed stable state treated with craniospinal irradiation and TMZ chemotherapy.

From seed to harvest: 18-month experience of molecular tumor board (MTB)-based precision oncology practice at the Verona University Hospital.

e15065 Background: Next generation Sequencing (NGS) allows the identification of potentially actionable molecular alterations that may constitute a therapeutic opportunity beyond standard systemic treatments for advanced cancer patients (pts). The interpretation of NGS profiling and its introduction in clinical practice can be guided by systematic implementation of Molecular Tumor Board (MTB). Methods: Between May 2020 and January 2023, 663 cancer pts accessed NGS profiling, in the context of investigator-driven and/or industry-sponsored research programs, using the CORE panel (in-house developed in collaboration with the International Cancer Genome Consortium - ICGC, covering 174 clinically relevant genes, structural variations and genomic signatures; 411 pts) and commercial FoundationOne CDx or FoundationOne Liquid assays (131 and 27 respectively). Pts with potentially clinically relevant molecular alterations were prescreened by Pathology and Oncology dedicated professionals and within multidisciplinary disease-oriented teams. Here we describe the results of MTB discussions at the Verona University Hospital. Results: Sixty-seven pts (10% of those profiled) were discussed at MTB bi-weekly meetings, between July 2021 and January 2023. Further diagnostic tests were requested in 19 pts (28%; IHC: 7, molecular analyses: 10, re-biopsies: 3). In 1 case histological diagnosis was changed upon re-biopsy (acinar pancreatic cancer to breast cancer NOS). Oncogenetic counselling was proposed in 10 pts (15%). Forty pts (60% of those discussed) were deemed eligible for NGS-informed treatment. Among these, twenty-two pts (55%) did not start MTB-indicated therapy, due to currently non-progressive disease (11), clinical deterioration (7), other Center referral (3) or refusal (1). Eighteen pts (45%) started MTB-recommended therapy in first (17%), second (28%) or &gt; = third line (55%). Treated pts were affected by rare tumors (8), pancreatic cancer (5), NSCLC (2), biliary tract, colon, and breast cancer (1 patient each). Types of treatment were: 12 targeted-, 2 immuno-, 2 chemo-, 2 combined-therapies. Among twelve evaluable pts, one complete response (8%), seven partial responses (58%), two stable (16%) and two progressive diseases (16%) were observed (RECIST v1.1); median PFS in treated pts was 12.7 [0.1-17.7] months. Five pts (41%) are currently awaiting response evaluation. Conclusions: Systematic identification and discussion of potentially actionable gene alterations brought to NGS-informed, MTB-endorsed therapeutic indications in 6% of profiled pts, 65% of whom experienced relevant clinical benefit from treatment. Increased and timely (preferably at diagnosis) access to NGS profiling, revised criteria for and more extensive MTB discussions should be implemented to increase the impact of Precision Oncology practices on pts' outcome.

Potential therapeutic implications of next generation sequencing (NGS)-based molecular profiling in HR+/HER2- metastatic breast cancer (mBC).

e13079 Background: BC is the most frequent malignancy in women and is potentially sensitive to hormonal treatment (HR+, HER2+ or -) in approximately 70% of cases. PIK3CA is among the most frequently mutated genes in BC (approximately 40% of cases) and its mutations portend sensitivity to PI3K inhibitors, such as alpelisib, in combination with hormonal treatment in HR+/HER2- cases. However, extended NGS-based testing is not currently recommended by national and international guidelines. Methods: Women aged 18 years or older, with cytological or histological diagnosis of HR+/HER2- mBC, for whom suitable tissue samples were available, were subjected to extended NGS profiling using the FoundationOne, analyzing 324 genes and gene signatures, such as tumor mutational burden (TMB) and microsatellite instability (MSI); and Illumina TruSight Oncology 500, analyzing 500 genes. DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) samples, from either the primary or metastatic site. Results: Patients (pts) were analyzed (ductal histology: 52%; lobular: 15%; mixed ductal-lobular: 12%; NOS: 21%), among which 7 (12%) had inadequate pathological material and were considered screening failures. A full molecular report was obtained in 53 patients. The most frequent pathogenic and likely pathogenic alterations (class 5 and 4), listed in descending order of prevalence, are: PIK3CA in 51% of pts (E545K&gt;H1047R&gt;N345K&gt;E542K); CDH1 in 21% (prevalence in lobular carcinoma: 75%; in ductal carcinoma: 3,5%). CCND1 in 28% (all amplifications). FGFR1-2 in 21%, TP53 in 15%, WHSC1L1 and ZNF703 in 13%, RAD21 and TBX3 in 9%. Homologous recombination-related genes, such as BRCA1/2 and PALB2 were altered in &lt;5% while ATM altered in 9% of cases. Many other pathogenic mutations (affecting 69 individual genes) were identified, all with a prevalence &lt;10%. TMB was high (&gt;17 mutations/Mb) in 13% of cases, in one of which MSI was also detected. Conclusions: In our analysis PIK3CA was confirmed to be the most frequently mutated gene (51% of cases); however, due to the specific European regulatory constraints, it was deemed to be actionable in a minority of cases, suggesting that NGS profiling should be performed early on after the diagnosis of metastatic disease, in order to inform the overall therapeutic strategy. Other potentially actionable alterations (FGFR1/2, AKT, HER2 ex 20 mutations) emerged from extended NGS profiling, suggesting its potential utility in selected HR+/HER2- mBC pts.

The impact of DNA damage repair gene mutations on therapeutic response among Chinese patients diagnosed with advanced biliary tract cancer.

e16190 Background: Biliary tract cancer (BTC) is a fatal disease characterized by molecular heterogeneity, which results in variations in biological behavior. Despite this, the knowledge of pathogenic alterations in the Chinese BTC population using next-generation sequencing (NGS) profiles remains limited. Therefore, we performed a retrospective investigation to assess the predictive significance of DNA damage repair (DDR) mutations in facilitating precision medicine for this disease. Methods: NGS profiling was carried out on resected tissues obtained from Chinese patients diagnosed with advanced BTC. In addition, baseline clinical and genetic characteristics were collected, along with data on their survival outcomes. Results: A total of 68 Chinese patients with BTC who received first-line platinum-containing chemotherapies were enrolled in this study [intrahepatic cholangiocarcinoma (ICC), 33; extrahepatic cholangiocarcinoma (ECC), 13; gallbladder carcinoma (GBC), 22]. Genetic alterations were observed in 67 patients (98.5%), with TP53 (50.0%), KRAS (26.8%), ARID1A (16.2%), and CDKN2A (16.2%) being the most commonly mutated genes. Patients with TP53 mutations demonstrated a trend towards reduced median OS (16.6 vs. 20.1 months, P=0.145). Mutations in DDR genes were identified in 29 (42.6%) of overall patients, primarily involving ATM (11.8%) and BAP1 genes (8.8%), and were significantly associated with longer mPFS (6.4 vs. 4.8 months, P=0.022) and mOS (21.2 vs. 14.3 months, P=0.011) in patients who received first-line platinum-containing chemotherapies. DDR-mutated patients treated with PD-1 blockades showed a tendency towards improved overall survival compared to the DDR-naive group (20.8 vs. 14.3 months, P=0.297). Furthermore, tumor mutational burden (TMB) could be estimated in 63 cases and was significantly higher in those with DDR mutations (P=0.029). Conclusions: Genetic information can aid in identifying a subgroup of BTC patients who may benefit from targeted therapy, this retrospective study demonstrated the predictive significance of DDR mutations for platinum-based chemotherapy efficacy in Chinese patients. Nonetheless, additional research is necessary to explore the potential of DDR mutations in predicting sensitivity to PD-1 blockade.

Genomic profiling in advanced NSCLC in Italy: First results of NERoNE (NSCLC Emilia Romagna Next Generation Sequencing Evaluation) project.

e21077 Background: The NERoNE Project evaluates the use of Next Generation Sequencing (NGS) in molecular profiling of advanced NSCLC (aNSCLC) patients (pts) in three Oncology Units (OU) of Emilia Romagna Region aiming to clarify the impact of such extensive tool in characterizing different NSCLC subpopulations. Methods: Since January 2020 to December 2022, 970 sequential aNSCLC pts from three OU (AUSL-IRCCS of Reggio Emilia, Modena University Hospital and IRST Meldola) were profiled by NGS. Samples from Modena and Reggio Emilia were analyzed at Modena University Hospital Molecular Pathology Lab using Myriapod IL- 56G, Cancer Panel DNA, Cancer Panel RNA, Oncomine DX Target Test Assay, Oncomine Focus Assay. Samples from IRST Meldola were processed at its Molecular Diagnostic Unit by Myriapod NGS cancer panel DNA e RNA, Oncomine Focus Assay and Oncomine Comprehensive Assay v3.We considered molecular alterations with available target therapies in Italy: EGFR mutations (mut) (exon 18,19,20,21), KRAS mut, BRAF V600E mut, ALK, ROS1, RET and NTRK rearrangements, MET exon 14 skipping mut, ErbB2 mut, FGFR 1-3 mut. PDL1 expression was assessed by standard immunohistochemistry on 486 pts. Results: Among 970 pts, 501 with at least one mut (52%) were considered for analysis: 266 were female (53%), mean age was 69 ± 11 years (min-max: 33-94) and 473 pts (94%) had adenocarcinoma. Molecular features are listed in table 1. Distribution of mut among ages, categorized as &lt; 60, 60-70 and &gt; 70 y, was similar, EGFR activating mut were most prevalent in females; KRAS mut (G12C and non G12C) were most prevalent in males; 41 pts had co-mutations (8.2%): the most frequent co-mut were EGFR mut (27%). PDL1 expression was &lt; 1% 129 (27%), 1-49% 211 (43,4%) and ≥50% 146 pts (30%). The majority of pts with EGFR activating mut had PDL1 &lt; 50%. Conclusions: NERoNE evaluates data from NGS profiling of three OU mapping key actionable molecular alterations in a large population of aNSCLC also accounting for PDL1 status. This preliminary analysis will be the starting point for further evaluations in order to improve the personalized approach to aNSCLC. [Table: see text]

Prevalence of next generation sequencing testing and targetable mutations in cholangiocarcinoma.

e16194 Background: Cholangiocarcinoma (CCA) is an aggressive cancer with a poor prognosis. However, next-generation sequencing (NGS) of tumors and new mutation-targeted therapies have the potential to greatly improve treatment outcomes while reducing the systemic toxicities of traditional chemotherapy. In this study, we examined clinical characteristics, treatment outcomes and the use of NGS profiling in a diverse South Florida population of CCA patients to identify mutations as potential therapeutic targets. Methods: We included patients treated at Jackson and University of Miami Health Systems who were diagnosed with CCA between 2018 and 2021. Clinical and demographic characteristics were obtained through retrospective chart review and entered into a REDCap database. We analyzed the association between clinical characteristics, treatment outcomes, use of NGS and race/ethnicity using Pearson’s Chi-square and Kruskal Wallis tests. Results: The majority of our sample, n = 140, had intrahepatic CCA (iCCA), n = 97, followed by extrahepatic CCA (eCCA), n = 27, and perihilar CCA (pCCA), n = 16. Stage, based on AJCC 8th edition, was most advanced in iCCA and pCCA; 56% had stage III or higher, compared to only 26.2% of eCCA patients. In addition, the median size of the largest iCCA tumor, 7 cm, was significantly larger than that of pCCA, 2.4 cm, and eCCA, 2.2 cm, p = 0.001. NGS was performed in significantly more iCCA cases, 72.2%, compared to pCCA, 37.5%, and eCCA, 59.3%, p = 0.04. TP53 mutations were most common across all CCA subtypes. Other common mutations included CDKN2A, CDKN2B, ERBB2, KRAS, FGFR2, IDH1, and ARID1A, of which only IDH1 and FGFR2 are targetable mutations. There were no significant differences across CCA subtypes in receipt of targeted therapy; overall, 17.1% of patients received targeted therapy. There were no racial differences in stage at presentation or survival in any CCA subtype. While there were numerical differences in the proportion of patients receiving targeted therapy (13.6% of Black, 12.8% of White, and 21.3% of Hispanic), these differences were not statistically significant. Conclusions: There are significant differences in the presentation of CCA subtypes. Examination of NGS results in a larger sample of CCA patients will help expand knowledge on CCA’s mutational profile and permit examination of potential racial disparities in survival or rates of NGS. This work also highlights the need for further targeted therapies against other common mutations in CCA. [Table: see text]