PIK3CA Mutations Research Articles

An immortalized adipose-derived stem cells line from the PIK3CA-related overgrowth spectrum: Unveiling novel therapeutic targets

PIK3CA-related overgrowth spectrum (PROS) encompasses several rare conditions that lead to overgrowth of various body parts resulting from activating variants in PIK3CA. The absence of ideal cell models significantly impedes progress in PROS research. In this study, we focused on facial infiltrating lipomatosis (FIL) (A disorder within PROS) and aimed to establish and characterize an immortalized PROS cell line. Primary adipose-derived stem cells of FIL were immortal-ized through the transfection of simian virus 40 large T antigen (SV40LT). No significant mor-phological differences were observed in immortalized FIL-ADSCs (Im FIL-ADSCs). Im FIL-ADSCs expressed original mesenchymal surface markers, confirmed by flow cytometry. It harbored PIK3CA mutation and an increased level of PI3K/AKT activation, revealed by sanger sequencing and Western blot respectively. Karyotype analysis revealed a stable chromosome in Im FIL-ADSCs. Higher adipogenic potential and lower osteogenic differentiation properties were de-tected in Im FIL-ADSCs. The proliferative potential of Im FIL-ADSCs increased, whereas malig-nant transformation was not observed in the tumorigenesis assay. Moreover, RNA sequencing further elucidated the role of the transcription factor E2F1 in Im FIL-ADSCs. Drug screening unveiled that STAT3, HSP, EGFR, and NF-kB might be potential therapeutic targets for FIL. This study provided a valuable cellular resource for exploring the underlying pathogenic mechanisms and developing new targeted therapeutic options for PROS.

PATH-13. MOLECULAR AND BIOLOGY CORRELATIVE RESULTS FROM THE PHASE 1B TRIAL OF UNESBULIN (PTC596) WITH RADIOTHERAPY IN CHILDREN NEWLY-DIAGNOSED WITH DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AND HIGH-GRADE GLIOMA (HGG): A REPORT FROM THE COLLABORATIVE NETWORK FOR NEURO-ONCOLOGY CLINICAL TRIALS (CONNECT)

Abstract BACKGROUND Within the CONNECT1702 trial of unesbulin (tubulin-binding agent causing BMI-1 modulation) with radiotherapy in patients with DIPG/HGG, tumor tissue was analyzed at diagnosis and/or autopsy for biology correlatives, enabling comprehensive, longitudinal molecular characterization. METHODS Whole genome sequencing, RNA-sequencing, and Western blot analyses were performed on tumor, normal brain, and peripheral blood mononuclear cells (PBMCs). RESULTS Tumor tissue from diagnosis (n=11) and/or autopsy (n=7) was analyzed for 16 patients (DIPG=9; HGG=7). Western blot analyses demonstrated 1) BMI-1 overexpression in baseline tumor compared to normal brain and 2) primary on-target effect of BMI1-modulation causing M-phase mitotic arrest when comparing pre- and post-treated PBMCs (increased H3S10-phosphorylation). Among 9 DIPG patients, alterations in the following genes were identified: H3-3A (H3.3) (n=5), H3C2 (H3.1) (n=2, both with co-occurring ACVR1 and PIK3CA mutations), TP53 (n=5), ATRX (n=2), and PPM1D (n=2). Tumors from two longer DIPG survivors were H3.1-, ACVR1-mutant (overall survival [OS]=26 months) and MET-amplified (OS=18 months); one patient with H3.3K27M-DIPG had germline MSH6 mutation (Lynch syndrome) (OS=12.6 months). Among 7 HGG patients, alterations in the following genes were identified: H3-3A (n=5), TP53 (n=3), NF1 (n=3), PDGFRA (n=1), EGFR (n=1), and PIK3CA (n=1). A BRAFV600E mutation was observed in the thalamic HGG patient (H3.3K27M) with greatest progression-free survival (>23 months). Early leptomeningeal metastases occurred in one DIPG patient with BCOR alteration and one HGG patient, with MYCN amplification identified in the spinal metastasis, but not intracranial disease. From gene set enrichment analyses, pathways enriched in patients with longer survival included telomerase regulation and ATP synthesis, whereas pathways enriched in patients with shorter survival included interneuron development, neuron semaphorin signaling, and gamma aminobutyric signaling. CONCLUSIONS Comprehensive molecular profiling of diagnostic and post-mortem tumor tissue provides valuable early biological insight, elucidating potential genomic biomarkers predictive of outcome and response to M-phase alteration and BMI-1 modulation in DIPG/HGG.

PATH-45. TREATMENT OF FGFR1 – ALTERED WHO GRADE 1 GLIONEURONAL TUMORS WITH FGFR INHIBITOR ERDAFITINIB

Abstract INTRODUCTION Low-grade mitogen-activated protein kinase (MAPK) pathway-altered glioneuronal tumors carry distinct molecular alterations, which may represent therapeutic opportunities to avoid or delay toxic treatments like radiation. Erdafitinib is a pan-FGFR tyrosine kinase inhibitor, regulatorily approved in recurrent urothelial carcinoma, and is under clinical investigation for recurrent FGFR-altered gliomas (NCT05859334). METHODS We report two patients with FGFR1-altered rosette-forming glioneuronal tumors (RGNT) treated with erdafitinib. RESULTS Patient 1 is a 32-year-old woman who presented with obstructive hydrocephalus and a non-enhancing pineal tumor. She underwent a subtotal resection, with histopathology initially consistent with a low grade dysembryoplastic neuroepithelial tumor. She was under observant management for 7 years, and developed worsening symptoms with slow non-enhancing growth, although stable per RANO low grade criteria. The initial tissue was sent for next-generation sequencing (NGS) and DNA methylation profiling, revealing an FGFR1 pathogenic variant with DNA methylation profiling matching with RGNT. Patient began Erdafitinib 8 mg daily, and had stable disease per RANO at 6 months. She developed CTCAE grade 1 toxicities of subretinal fluid, fatigue, dry skin, and paronychia. Patient 2 is a 34-year-old man diagnosed 1 year prior with a tectal tumor, who underwent biopsy due to non-measurable/ non-enhancing progression. Histopathology was consistent with RGNT. NGS revealed FGFR1 mutation, PIK3CA mutation, and NF1 pathogenic variant, described in the literature as characteristic of RGNT. There was insufficient tissue for DNA methylation profiling. Patient started erdafitinib 8 mg daily and remains clinically stable for 1 month. He developed asymptomatic hyperphosphatemia (CTCAE grade 1). Both patients have tolerated treatment without interruptions or dose reductions. CONCLUSION Erdafitinib is a well-tolerated and potentially efficacious treatment of FGFR1- altered RGNT, and may avoid the need for radiation. Diagnosis of MAPK- altered low-grade glioneuronal tumors, specifically RGNT, is increasingly reliant on molecular data, and can significantly impact treatment options.

NCOG-35. IDENTIFYING PROGNOSTIC FACTORS IN PEDIATRIC DIFFUSE MIDLINE GLIOMAS, H3 K27-ALTERED

Abstract INTRODUCTION Diffuse midline gliomas, H3 K27-altered (DMG) are rare, grade 4 tumors localized to the diencephalon, brainstem, and cervical spine. DMGs, characterized by a K27M missense mutation in histone H3, often cannot be safely resected and predominantly present with poor outcomes in children. Few prognostic factors for DMGs have been identified and/or need to be further elucidated, including adolescent age of onset, re-irradiation status, and tumor molecular profile (i.e., PIK3CA mutation). Herein, we describe the clinical outcomes of DMG patients at our institution. METHODS Pediatric patients (<18 y/o) between 2015 and 2023 with biopsy-proven DMG were reviewed. Demographic, treatment, and tumor characteristics (histologic and molecular) were recorded. Adolescent-onset DMGs were defined as patients ≥10 y/o at diagnosis and early-onset DMGs were defined as patients <10 y/o. Progression free survival (PFS) and overall survival (OS) were calculated according to the Kaplan Meier-method. Multivariate regression analyses were performed. RESULTS Twenty-one patients were included (median age=9 y/o). Early-onset DMGs (n=13) progressed significantly earlier and experienced significantly greater mortality than adolescent-onset DMGs (n=8); PFS and OS at 12 months was 7.7% and 23% for early-onset DMGs and 50% and 87.5% for adolescent-onset DMGs (PFS: p<0.01, OS: p<0.02). Patients that received re-irradiation for progressive disease (n=9, mean=84Gy) had an OS of 75% at 12 months, which was significantly greater than an OS of 23% at 12 months (p<0.05) for patients that received no re-irradiation (n=12, mean=53Gy). PIK3CA mutation status was identified as an independent predictor of survival, with PIK3CA-mutant DMGs (n=7) having a median survival of 4.1 months versus 9.7 months for PIK3CA-WT DMGs. CONCLUSION We describe a pediatric cohort that presents several possible prognostic factors of survival in DMGs. Our findings on adolescent age of onset, if further corroborated, has the potential to inform the design and enrollment of DMG clinical trials.

CTNI-43. MULTI-CENTER PHASE I STUDY OF CONCURRENT PAXALISIB AND RADIATION THERAPY (RT) IN PATIENTS WITH SOLID TUMOR BRAIN METASTASES (BM) OR LEPTOMENINGEAL METASTASES (LM) HARBORING PI3K PATHWAY ALTERATIONS

Abstract INTRODUCTION Partial or whole brain RT is a standard treatment for solid tumor BM and LM. However, tumors harboring PI3K pathway mutations have poorer CNS control after RT alone. Combining RT with paxalisib, a CNS-penetrant PI3K/mTOR inhibitor, may overcome radioresistance. METHODS This multi-center, phase I trial (NCT04192981) studied concurrent paxalisib with cranial RT (30Gy in 10 fractions) for PI3K pathway-altered BM/LM. Part A was a 3 + 3 dose escalation cohort of paxalisib at 45, 60 or 75mg to establish maximum tolerated dose (MTD). Part B was an expansion at MTD to further validate safety and early efficacy. CNS response was assessed using Response Assessment in Neuro-Oncology Brain Metastases (RANO-BM) criteria. RESULTS From 3/2020-2/2024, 21 patients (median age 58y, n=18 with BM, n=10 with breast cancer) were enrolled. PIK3CA mutations were the most common pathway alteration (n=15, 71%) followed by PTEN deletion (n=3, 14%). Part A enrolled 12 patients of which 9 were evaluable (3 did not complete protocol therapy). There were no dose-limiting toxicities (DLTs) at 45mg, and 2 DLTs at 60mg: grade 3 nausea and grade 4 neutropenic enterocolitis. Subsequently, 9 additional patients were enrolled during Part B at MTD of 45mg, of which 1 withdrew consent prior to completion of study interventions. No additional DLTs were observed during Part B. In total, there were 14 evaluable patients who received 45mg/day paxalisib concurrently with RT, two of whom died of intercurrent extracranial progression prior to CNS restaging. Of the remaining 12, there was robust response signal. At 1-month post-treatment, 6/12 (50%) had partial response (PR) and the remaining had stable disease (SD). To date, best CNS response is 67% PR (8/12) and 33% SD (4/12). CONCLUSIONS A MTD of 45 mg/day has been confirmed for paxalisib with concurrent brain RT for PI3K pathway-altered BM/LM. Given significant unmet need, this novel therapeutic combination warrants further investigation given promising activity signal.

BIOM-24. GLIOBLASTOMA DNA ALTERATIONS AND RAPID EARLY PROGRESSION AFTER SURGERY

Abstract Glioblastoma is the most common primary brain cancer with overall poor prognosis. In the weeks following initial resection, approximately 50% of glioblastoma patients will demonstrate rapid early progression (REP) on MRI prior to the start of radiation therapy (RT). REP is associated with worse overall survival (OS), however associations between REP and biomarkers in glioblastoma have yet to be evaluated. A single institution sample of 100 patients (age 61.2 years median, 57 males) with a primary diagnosis of glioblastoma were included in this study. Patients underwent gross or subtotal resection followed by RT within 60 days of their surgery. Patients who received biopsy only were excluded. For this analysis, REP was defined as increased nodular enhancement at the border of the resection cavity between the post-operative MRI and RT planning MRI. DNA mutations were collected from commercial next generation sequencing (NGS) results. Biomarkers with a mutational incidence of >10% were used in the analysis, which included TERT promoter (N=80), EGFR (N=38), PTEN (N=40), TP53 (N=36), CDKN2A/2B (N=41), NF1 (N=16), MTAP (N=24), PIK3CA (N=14), and MGMT methylation status (N=32). Statistical analysis was performed using SPSS Statistics 28.0. Of the patients sampled, 45 met the classification of REP. The presence of EGFR alterations (p=0.001) and MGMT methylation (p=0.016) strongly correlated with REP. After multivariate linear regression, only EGFR alterations significantly correlated with REP (p=0.006). There was no significant correlation between REP or overall survival and mutations in TERT promoter, PTEN, TP53, CDKN2A/2B, NF1, MTAP, and PIK3CA. This is the first study to analyze molecular contributors to REP. We identified that EGFR alterations are most strongly correlated with REP. This suggests that patients with EGFR alterations identified on pathology should move rapidly to adjuvant therapies given their propensity to quickly re-grow after resection.

TMOD-13. IDENTIFYING MOLECULAR SIGNATURES OF PPM1D-MUTANT DIPGS

Abstract Diffuse Intrinsic Pontine Glioma (DIPGs) are universally fatal pediatric tumors with a median survival of only 9-12 months. PPM1D-truncating mutations (PPM1Dtr), which promote its phosphatase activity and suppress TP53 function, are found in approximately 15% of DIPG tumors. PPM1Dtr are mutually exclusive to TP53 mutations, and present unique therapeutic opportunities to reactivate wild-type TP53. Another distinct feature of PPM1Dtr DIPGs is the co-occurrence of PI3K/AKT/mTOR pathway alterations, such as PIK3CA. There is an unmet need to elucidate the underlying molecular mechanisms of DIPG and identify novel therapeutic vulnerabilities that will improve outcomes of this devastating tumor. We hypothesize that PPM1D-mutant DIPGs can be therapeutically targeted by reactivating the p53 signaling pathway and other complementary pathways which cooperate with PPM1Dtr. To investigate this, we have generated Trp53- and Ppm1d-mutant isogenic DIPG murine models to determine how specific oncogenic alterations function in DIPG formation, growth and treatment sensitivity. Current studies aim to examine differences in sensitivity to standard of care treatment modalities and leverage next generation sequencing techniques to uncover additional vulnerabilities. We found notable differences upon addition of PIK3CA mutations in the context of PPM1Dtr DIPG tumor formation, growth, survival, and lineage identity. Ongoing studies using whole genome transcriptomics to compare tumor models will provide insight into the molecular signals and cell state changes driven by individual genetic alterations. Furthermore, by comparing isogenic TP53 and PPM1Dtr mutant DIPG models, we aim to further delineate the molecular mechanisms that drive differential sensitivity of TP53 wild-type tumors. The immune-competent nature of our murine models also provides us with a unique opportunity to delineate tumor immune microenvironment changes. Leveraging this information and our novel murine models, our long-term goal is to develop combinatorial therapeutic strategies to improve outcomes for this deadly disease.

Updates in Treatment of HER2-positive Metastatic Breast Cancer.

The therapeutic landscape for HER2-positive metastatic breast cancer has exploded in the last two decades following the initial advent of trastuzumab, a monoclonal antibody. While the first line treatment has remained a combination of dual HER2 blockade with taxane chemotherapy, we now have several exciting options in the second line and beyond. The introduction of antibody-drug conjugates, in specific trastuzumab deruxtecan, has resulted in the best progression-free survival among patients with this subtype of breast cancer. Given the excellent outcomes of these drugs, clinical trials are now evaluating the role of ADCs in the front-line setting in previously untreated patients. In addition, there are also clinical trials evaluating the role of other targets in patients with HER2-positive cancers, including PI3KCA mutations, PD-L1 and CDK4/6. Given the predilection for brain metastases in this population, there is enthusiasm to identify the optimal combination of effective treatments. Tucatinib, capecitabine, and trastuzumab combination represent one such promising strategy. With the increasing longevity of these patients, important clinical questions include optimal treatment sequencing, the role of de-escalation of treatment in excellent responders, and the associated financial toxicity. Despite the aggressive nature of this subtype of breast cancer, the outcomes continue to improve for these patients with the evolving treatments.

The genomic landscape of papillary thyroid carcinoma on next-generation sequencing in patients undergoing total thyroidectomy.

Thyroidectomy is increasingly performed for suspected malignancy. This cohort study aimed to identify genetic markers associated with malignancy and determine the molecular landscape of papillary thyroid carcinoma (PTC) through next-generation sequencing (NGS) in patients undergoing total thyroidectomy. Among 116 surgical candidates, 103 patients (age=42.9±13.7years; Male: Female=14:89) with benign or malignant thyroid nodules were eligible. Live thyroid tissue samples harvested intraoperatively with adequate DNA and RNA yield were subjected to NGS on the Illumina NovaSeq 6000 platform for genomic and transcriptomic analysis, respectively. Histopathology comprised 20 malignant (19.4%) and 83 benign (80.6%) cases, including 16 PTC (15.5%) cases. On NGS, single nucleotidepolymorphisms (SNPs) in NTRK1 at NC_000001.11:156879016 on chromosome 1 and ALK at NC_000002.12:29717663 on chromosome 2 were frequent in malignant lesions (p<0.05). A SNP in ALK at NC_000002.12:29193706 was consistently a homozygous alternate alleleacross the cohort. DNA-sequencing of PTC lesions identified recurrentsomatic mutations in BRAF (100%), ALK (56.3%), RET (18.8%), PIK3CA (12.5%), NTRK1 (12.5%), NTRK2 (87.5%), NTRK3(12.5%), NRAS(6.3%), and PTCH1 (31.3%) genes. RNA sequencing revealednovelfusiongenes, including MKRN1-BRAF, RN7SL1-CDH1, IRF2BPL-MED12, MED12-IRF2BPL, CPM-MDM2, and AC005895.3-PDGFRB. Inreceiveroperative characteristics analysis, the AUCs ofALKmutationpredictingrecurrence and metastases were 0.818 and 0.783. This Indian study identified novel somatic mutations and fusion genes in PTC, revealing a distinct genomic landscape with implications in precision diagnostics and personalized therapies. NGS with intraoperative live sampling shows promise in prognostication and therapeutic optimization of advanced/metastatic PTC cases. CTRI/2020/09/027607dt 04/09/2020; REF/2020/08/036119.

Targeted Sequencing of HER2-Positive Breast Cancer Mutations Revealed a Potential Association between PIK3CA and Trastuzumab Resistance.

Different molecular subtypes, including HER2-positive, have been identified in breast cancer. The overexpression of HER2 triggers downstream signaling pathways such as the PI3K/AKT/mTOR pathway. Until recently, trastuzumab has been used as a single HER2-targeted therapy in Egypt. However, resistance to trastuzumab has been reported. Previous studies have demonstrated the genetic variants that affect the trastuzumab response. However in Egypt, few studies investigated molecular biomarkers such as p53 that might affect the trastuzumab response. Therefore, we aimed to extend the genetics workup of Her2 + BC to include important oncogenes and other vital cancer pathways. Formalin-fixed paraffin-embedded samples were collected from 24 HER2+ BC Egyptian patients, twelve patients in complete remission for 2 years or more from the start of trastuzumab and twelve resistant patients who relapsed or developed metastasis within 2 years from the start of trastuzumab. Somatic mutations in hotspot regions of 17 genes were further investigated using next-generation sequencing. Among the total number of identified variants (106 variants), PIK3CA showed the most frequent variants, with more variants occurring in the resistant group than in the responsive group (P= 0.004). The frequency of PIK3CA mutations was greater in resistant patients than in responsive patients (P= 0.036). Additionally, there was a significant correlation between PIK3CA mutations and pathological complete response (pCR) (P=0.036). Most of PIK3CA variants in resistant patients were detected in exon 9 and 20. The PIK3CA variants His1047Tyr, Glu545Lys, His701Pro, Lys111Glu, Val344Gly and Tyr1021Cys were found only in the resistant patients, suggesting that they are associated with trastuzumab resistance. PIK3CA variants were more frequent in resistant HER2+ BC patients than in responsive patients, with a significant correlation between PIK3CA mutation and a lower pCR rate. PIK3CA variants within exon 9 and 20 (such as Glu545Lys and His1047Tyr respectively) were associated with trastuzumab resistance.

KRASG12D-Mutated Metastatic Colorectal Cancer: Clinical, Molecular, Immunologic, and Prognostic Features of a New Emerging Targeted Alteration.

KRASG12D mutation (mut) occurs in about 10%-12% of metastatic colorectal cancer (mCRC). Recently, novel KRASG12D inhibitors have been developed and are currently under investigation in phase I/II clinical trials in solid tumors including mCRC. We aimed at performing a comprehensive characterization of clinical, molecular, immunologic, and prognostic features of KRASG12D-mutated mCRC to inform the design and the interpretation of future trials. We performed a pooled analysis of phase III TRIBE and TRIBE2 studies comparing 5-fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI)/bevacizumab (bev) to doublets (5-fluorouracil, leucovorin, and oxaliplatin or 5-fluorouracil, leucovorin, and irinotecan)/bev. One hundred and thirty-six (16%) of 854 patients with available KRASG12D mutational status were KRASG12D mutated. KRASG12D-mutated patients had more frequently right-sided primary tumor and were less likely to present liver-only metastases with respect to other RAS mutated and all-wild-type (wt) patients. Compared with the BRAFV600E-mutated group, KRASG12D-mutated patients had more frequently left-sided primary tumor, resected primary tumor at the time of diagnosis, and Eastern Cooperative Oncology Group performance status 0. KRASG12D-mutated patients had better prognosis than BRAFV600E-mutated and worse prognosis than all wt patients. No prognostic difference was evident between KRASG12D mut and other RAS mut patients overall or according to other specific KRAS or NRAS hotspot mutations. No interaction effect was observed between KRASG12D mut and the benefit provided by FOLFOXIRI/bev compared with doublets/bev. PIK3CA mut were reported more frequently among KRASG12D-mutated tumors compared with both other RAS mut and all wt. A detail estimation of KRASG12D mut mCRC patients' characteristics and expected outcomes may be useful when planning future studies in this subgroup. The high prevalence of PI3K/PTEN/Akt pathway activating alterations may affect the efficacy of targeted strategies.

Sialadenopapillary Ductal Tumors: Unifying the Spectrum of Sialadenoma Papilliferum-like Tumors With Low Malignant Potential.

Sialadenoma papilliferum is a tumor characterized by surface papillary projections and glandular/microcystic proliferation at the lesion base. Cases in which surface involvement is absent have been termed "sialadenoma papilliferum-like intraductal papillary tumor." Similar tumors that are present in the mandible have been termed "tubulopapillary hidradenoma-like tumor of the mandible." While previously considered benign, these tumors demonstrate variable clinical behavior and likely exist on a spectrum, rather than as discrete entities. In this study, we present a detailed clinicopathologic and molecular analysis of these lesions and propose a unifying diagnostic term: sialadenopapillary ductal tumor (SDT). Twenty-two cases with similar histologic features were reviewed, with special attention being paid to the clinicopathologic features. Immunohistochemistry for BRAF V600E and molecular testing were performed where material was available. The cases had varying diagnoses, ranging from benign to malignant. Six cases involved bone, 1 of which metastasized to a local lymph node. Of the 20 cases tested for BRAF V600E by immunohistochemistry, 18 were positive. Molecular testing was performed in 5 cases, where BRAF, PTPN11, and PIK3CA mutations were identified, predominantly members of the RAS-RAF-MEK-ERK pathway. In addition, 1 case was reclassified as an intraductal carcinoma after the identification of an NCOA4::RET gene fusion. Tumors on the SDT spectrum all share morphologic and molecular commonalities with unreliable distinguishing features. These tumors demonstrate the potential for aggressive local growth and regional metastasis. We propose a unifying diagnostic term for these lesions to reflect their common morphologic and molecular features and, most importantly, low malignant potential.

Detection of PIK3CA hotspot mutations in canine mammary tumors using droplet digital PCR: tissue validation and liquid biopsy feasibility

Domestic dogs (Canis lupus familiaris) serve as valuable translational models for human cancer research due to their biological similarities. Canine mammary tumors (CMTs), frequently diagnosed in female dogs, share various characteristics with human breast cancers. This study investigates the PIK3CA (H1047R) mutation in CMTs using droplet digital PCR (ddPCR) and explores the potential of liquid biopsy for non-invasive detection. We analyzed 80 formalin-fixed, paraffin-embedded (FFPE) CMT tissue samples and compared ddPCR results with next-generation sequencing (NGS) data, achieving high concordance. Plasma and serum samples were also assessed for mutation concordance with tissue results. Our findings indicate a higher frequency of the PIK3CA (H1047R) mutations in benign and grade I malignant CMTs compared to more aggressive malignancies. The ddPCR assay demonstrated high sensitivity and specificity, with plasma testing showing 78.6% sensitivity and 87.5% specificity, and serum testing showing 66.7% sensitivity and 90.0% specificity. These results highlight the viability of liquid biopsy as a minimally invasive method for monitoring PIK3CA mutations in canine patients. The study suggests that liquid biopsy techniques hold significant promise for improving the early detection and monitoring of canine cancers, warranting further research to refine these methods and explore their applications in canine cancer diagnostics and treatment.

Higher risk of recurrence in early-stage breast cancer patients with increased levels of ribosomal protein S6

PI3K/AKT/mTOR pathway is implicated in breast cancer progression and recurrence. The identification of PIK3CA and AKT1 mutations and loss of PTEN serve as selection criterion for targeted therapies involving selective inhibitors. However, they do not consistently align with pathway activation, and high-cost determinations limit their routine application. PI3K-downstream epigenetic regulatory mechanisms broaden the alterations that amplify pathway activity and, consequently, sensitivity to selective inhibitors. In this retrospective observational study, conducted within a cohort of early-stage breast cancer patients, we determined phosphorylated ribosomal protein S6 (pS6) at Ser240/244 by immunohistochemistry as an indicator of PI3K pathway activation. Log-Rank test and Cox proportional hazards regression were used to analyze the clinical relevance of pS6, alone and together with clinicopathological variables, regarding recurrence-free survival. ROC curves and the area under the curves were used to evaluate the calibration and discrimination properties of uni- and multivariate models. Our results show that a high percentage of pS6 positive tumor cells was associated with an unfavorable prognosis in a cohort of 129 hormone receptor positive/HER2 negative breast cancer patients (Hazard Ratio = 5.92; Log-Rank p = 9.5e-08; median follow-up = 53 months). When assessed in combination with lymph node status, the predictive capacity was higher compared to both univariate models individually. In conclusion, pS6 could represent a novel independent marker for predicting recurrence risk in luminal breast cancer.

The potential role of next-generation sequencing in identifying MET amplification and disclosing resistance mechanisms in NSCLC patients with osimertinib resistance.

Osimertinib, one of the third-generation EGFR-tyrosine kinase inhibitors (TKIs) designed to target EGFR T790M mutation, significantly improves the prognosis of lung cancer. However, drug resistance still happens and MET amplification is responsible for one of the main causes. Fluorescence in situ hybridization (FISH) is the gold standard for MET amplification detection, but fundamentally limited by observer subjectivity. Herein, we assessed the value of next-generation sequencing (NGS) method in MET amplification detection in non-small cell lung cancer (NSCLC), as well as revealed the mutation profiling of NSCLC patients with osimertinib resistance to provide some valuable clues to the mechanisms of resistance. A total of 317 cancer tissue samples from 317 NSCLC patients at time of progression following osimertinib were submitted to NGS and only 96 tissues were tested by FISH simultaneously. With FISH results as gold standard, enumeration algorithm was applied to establish the optimal model for identifying MET amplification using gene copy number (GCN) data. The optimal model for identifying MET amplification was constructed based on the GCN of MET, BRAF, CDK6 and CYP3A4, which achieved a 74.0% overall agreement with FISH and performed well in identifying MET amplification except polysomy with a sensitivity of 85.7% and a specificity of 93.9%. The inconsistency between NGS and FISH occurred mainly in polysomy subtype, while MET GCN ≥ 5 could be reliably recognized by NGS. Moreover, the most frequently mutated genes in NSCLC patients with osimertinib resistance were EGFR (59.94%), followed by TP53 (43.85%), NRG1 (9.46%), PIK3CA (6.31%), and ATM (5.36%). The known resistance mechanisms, including MET amplification, EGFR (C797S, L718Q/R), TP53, CDK4, CDK6, CDKN2A, BRAF, KRAS, NRAS and PIK3CA mutations were also disclosed in our cohort. NGS assay can achieve a high concordance with FISH in MET amplification detection and has advantages in portraying various genetic alterations, which is of worthy in clinical promotion.

Comprehensive liquid biopsy analysis for monitoring NSCLC patients under second-line osimertinib treatment.

The heterogeneous and complex genetic landscape of NSCLC impacts the clinical outcomes of patients who will eventually develop resistance to osimertinib. Liquid biopsy (LB) analysis as a minimally invasive approach is a key step to efficiently identify resistance mechanisms and adjust to proper subsequent treatments. In the present study, we combined plasma-cfDNA and CTC analysis from 30 NSCLC patients in samples collected before treatment and at the progression of disease (PD). We detected molecular alterations at the DNA mutation (EGFR, PIK3CA, KRAS G12C, BRAF V600E), DNA methylation (RASSF1A, BRMS1, FOXA1, SLFN1, SHISA3, RARβ,, WIF-1, RASSF10 and APC), gene expression (CK-19, CK-18, CK-8, AXL, TWIST-1, PD-L1, PIM-1, Vimentin, ALDH-1, and B2M) and chromosomal level (HER2 and MET amplification) as possible resistance mechanisms and druggable targets. We also studied the expression of PD-L1 in single CTCs using immunofluorescence. In some cases, T790M resistance EGFR mutation was detected at baseline in CTCs but not in the corresponding plasma cfDNA. PIK3CA mutations were detected only in plasma-cfDNA but not in corresponding CTCs. KRAS G12C and BRAF V600E mutations were not detected in the samples analyzed. MET amplification was detected in the CTCs of two patients before treatment whereas HER2 amplification was detected in the CTCs of three patients at baseline and in one patient at PD. DNA methylation analysis revealed low concordance between CTCs and cfDNA, indicating the complementary information obtained through parallel LB analysis. Results from gene expression analysis indicated high rates of vimentin-positive CTCs detected at all time points during osimertinib. Moreover, there was an increased number of NSCLC patients at PD harboring CTCs positive in PD-L1. AXL and PIM-1 expression detected in CTCs during treatment suggesting new possible therapeutic strategies. Our results reveal that comprehensive liquid biopsy analysis can efficiently represent the heterogeneous molecular landscape and provide prominent information on subsequent treatments for NSCLC patients at PD since druggable molecular alterations were detected in CTCs.

Clear Cell Carcinoma Arising in Vaginal Endometriosis in a Patient 33 Years after Total Hysterectomy and Bilateral Salpingo-oophorectomy: A Case Report

Abstract Introduction/Objective Endometriosis, a common gynecological disorder characterized by ectopic endometrial tissue, is associated with an increased risk of extraovarian malignancies. Vaginal endometriosis is a rare condition, and the occurrence of vaginal malignancies arising from endometriosis is anticipated to be exceedingly rare. Here, we present a rare case of clear cell carcinoma arising in vaginal endometriosis, occurring three decades post-total hysterectomy and bilateral salpingo-oophorectomy. Methods/Case Report A 71-year-old female presented with abnormal Cologuard results, leading to a colonoscopy revealing a mass in the recto-sigmoid colon. She had undergone total hysterectomy and bilateral salpingo- oophorectomy for leiomyoma and endometriosis 33 years prior. Histopathological examination revealed clear cell carcinoma arising in vaginal endometriosis, with tumor extension through the colon wall to the colonic mucosal surface (Figure 1). Notably, the vaginal mucosal surface was negative for tumor. Immunohistochemistry staining demonstrated positivity for Pax8, CK7, AE1/3, racemase, and p16, while negtive for ER, PR, CDX2, CK20, CAIX, SatB2, RCC, WTI, Napsin, and calretinin (Figure 2). The staining for p53 was patchy (wild type, not mutated). Cancer-type relevant biomarkers reveal BRAF mutation (p.D594G), ARID1A mutation (p.M1564fs), PD-L1 mutation (Positive, CPS:80), and PIK3CA mutation (p.H1047L). The histologic and immunohistochemistry findings are consistent with clear cell carcinoma arising in a long-standing area of endometriosis (previous surgery in 1990). Results (if a Case Study enter NA) NA Conclusion Clear cell adenocarcinoma arising from endometriosis is exceptionally rare. Pathologists must consider the possibility of tumors arising from endometriosis when evaluating bowel or mesenteric neoplasms, even decades post-total hysterectomy and bilateral salpingo-oophorectomy. Immunohistochemistry plays a crucial role in the differential diagnosis between carcinoma arising from endometriosis and primary colonic carcinoma. Additionally, Sampson’s criteria are valuable for diagnosing malignant transformations in endometriosis. The identification of specific cancer-type relevant biomarkers, such as BRAF, ARID1A, PD-L1, and PIK3CA mutations, provides additional insights into the molecular characteristics of the tumor, which may have implications for prognosis and treatment selection.

Parathyroid carcinoma: molecular pathology and correlation with clinicopathologic features, a single center experience

Abstract Introduction/Objective Parathyroid Carcinomas are rare malignant neoplasms, accounting for less than 1% of primary hyperparathyroidism cases. Parathyroid carcinomas are characterized by markedly elevated levels of PTH, severe hypercalcemia and established target organ damage. The authors report the experience of a single center regarding the clinic-pathologic and genetic analysis of parathyroid carcinoma cases. Methods/Case Report The study is a retrospective review of all patients with parathyroid carcinoma that were evaluated at a tertiary oncologic center from 2001 until 2023. Results (if a Case Study enter NA) A total of nine cases (6 male and 3 female) were identified, with a mean age at diagnosis of 52 ± 16 years and a median follow-up of 16.5 years. Most patients presented with hypercalcemia (n = 7), with a mean serum calcium concentration of 13.5 mg/dl (9.6-16.5) and a mean PTH of 1123 pg/ml (&amp;lt;a href=“tel:276- 2500”&amp;gt;276-2500&amp;lt;/a&amp;gt;). En bloc surgical resection was performed in 6 patients and 3 patients underwent adjuvant radiotherapy. Recurrence was observed in 4 cases (44.4%) after a median of 24 months following surgery. Negative immunohistochemical staining of parafibromin was noted in 3/4 cases of recurrence. Parathyroid carcinoma was diagnosed on the basis of finding lesions with vascular or perineural invasion, capsular penetration, and/or documented metastases. Immunohistochemical staining for parafibromin, Ki-67, and E-cadherin was performed on formalin-fixed, paraffin-embedded tissue samples. Next generation sequencing (NGS) was performed in 6/9 cases and CDC73 mutations were present in 38.5% of analyzed patients. Loss of TP53 and RB1 alleles and CCND1 amplification were other reported genetic alterations in the study cohort. Conclusion Parathyroid carcinoma is associated with a significant rate of recurrence and limited effective treatment beyond initial complete surgical resection. Inactivating CDC73 alterations which include truncating or frameshift mutations, as well as missense mutations lead to the loss of parafibromin immunoreactivity. Other molecular alterations like PTEN and PIK3CA mutations may represent potential targets for molecular therapy. An IHC panel of parafibromin, Ki-67 and E-cadherin may help to distinguish parathyroid carcinoma from other parathyroid neoplasms.

Multigene Panel Next-Generation Sequencing Techniques in the Management of Patients with Metastatic Colorectal Carcinoma: The Way Forward for Personalized Treatment? A Single-Center Experience

The efficacy and cost-effectiveness of Multigene Panel Next-Generation Sequencing (NGS) in directing patients towards genomically matched therapies remain uncertain. This study investigated metastatic colorectal cancer (mCRC) patients who underwent NGS analysis on formalin-fixed paraffin-embedded tumor samples. Data from 179 patients were analyzed, revealing no mutations in 39 patients (21.8%), one mutation in 83 patients (46.4%), and two or more mutations in 57 patients (31.8%). KRAS mutations were found in 87 patients (48.6%), including KRAS G12C mutations in 5 patients (2.8%), PIK3CA mutations in 40 patients (22.4%), and BRAF mutations in 26 patients (14.5%). Less common mutations were identified: ERBB2 in five patients (2.8%) and SMO in four patients (2.2%). Additionally, MAP2K1, CTNNB1, and MYC were mutated in three patients (2.4%). Two mutations (1.1%) were observed in ERBB3, RAF1, MTOR, JAK1, and FGFR2. No significant survival differences were observed based on number of mutations. In total, 40% of patients had druggable molecular alterations, but only 1.1% received genomically guided treatment, suggesting limited application in standard practice. Despite this, expanded gene panel testing can identify actionable mutations, aiding personalized treatment strategies in metastatic CRC, although current eligibility for biomarker-guided trials remains limited.

Malignant Peripheral Nerve Sheath Tumor of the Pancreas with Molecular Confirmation: A Case Report with Comprehensive Histopathologic and Molecular Characterization

Abstract Introduction/Objective Malignant peripheral nerve sheath tumors (MPNSTs) are rare soft tissue sarcomas arising from a peripheral nerve or exhibiting nerve sheath differentiation. They are often associated with neurofibromatosis type 1, and most often arise in the trunk, extremities, or head and neck region. In patients without neurofibromatosis type 1, sporadic de novo or radiotherapy-associated MPNSTs can pose a diagnostic challenge. MPNSTs arising from visceral organs are exceedingly rare. Here we report a case of MPNST arising in the pancreas. Methods/Case Report The patient is a 75-year-old female, MUTYH mutation carrier, with prior history of breast and bladder cancers, each treated with respective local excision and radiation therapy. Magnetic resonance imaging (MRI) performed for pancreatic cancer screening showed a 4.7 cm heterogeneous mass in the pancreatic tail suggestive of pancreatic neoplasm. Fine needle aspiration cytology was non-diagnostic. However, due to the radiologic concern for malignancy, the patient proceeded with surgical resection by distal pancreatectomy with splenectomy. Macroscopically, a 6.5 cm, tan-white, partially necrotic mass was identified in the pancreatic tail. Microscopic examination showed a spindle cell neoplasm arranged in a vague fascicular fashion with moderate nuclear pleomorphism, geographic necrosis, and frequent mitotic figures (15 per 10 high power fields). Fifteen lymph nodes were negative for metastasis. The tumor was confined to the pancreas grossly and microscopically. Immunohistochemical stains showed the neoplastic spindle cells to be positive for TLE1 (diffuse) and S100 (patchy), while negative for SOX10, CD117, DOG1, desmin, actin, MyoD1, ERG, STAT6, and ALK1. In addition, H3K27me3 showed loss of nuclear expression. Next generation sequencing (NGS) revealed gene mutations in NF1, TP53, PIK3CA, and PTEN, and copy number losses involving CDKN2A and CDKN2B. Genome-wide DNA methylation analysis was performed which revealed copy number losses of CDKN2A/B and gains of PTCH1 and MDM2. However, the Sarcoma DNA Methylation Classifier did not provide any diagnostic matches. Overall, the morphologic features, loss of H3K27me3 expression, presence of NF1 and TP53 mutations, and copy number losses in CDKN2A/B supported a diagnosis of high-grade MPNST. The patient has been followed clinically without adjuvant therapy. Results (if a Case Study enter NA) N/A Conclusion We report an extremely rare case of MPNST of the pancreas with molecular confirmation.