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.

Detection of genetic mutations in 855 cases of papillary thyroid carcinoma by next generation sequencing and its clinicopathological features.

To investigate the genetic mutations in patients with papillary thyroid carcinoma (PTC) and their clinicopathological features by next generation sequencing (NGS). NGS technology was used to detect genetic mutations in PTC patients, and clinicopathological features were collected. ①Among 855 PTC patients, 810 patients had genetic mutations, and 45 patients had no genetic mutation. ②BRAF mutation was associated with tumor diameter (P < 0.001) and histological subtypes (P = 0.002). The abundance of V600E mutation was associated with gender (P = 0.004), tumor diameter (P < 0.001), bilateral presentation (P = 0.001), extrathyroidal extension (P < 0.001), lymphatic metastasis (P < 0.001), histological subtypes (P = 0.002) and TNM staging (P = 0.000); The different mutation abundance of V600E was associated with tumor diameter (P < 0.001), multifocal presentation (P = 0.047), bilateral presentation (P = 0.001), extrathyroidal extension (P = 0.001), lymphatic metastasis (P < 0.001), histological subtypes (P = 0.022) and TNM staging (P = 0.000). ③RET fusion was associated with tumor diameter (P < 0.001) and lymphatic metastasis (P = 0.005). ④TERT mutation was associated with gender (P = 0.043), tumor diameter (P < 0.001), extrathyroidal extension (P = 0.028) and TNM staging (P = 0.017). ⑤RAS mutation was associated with histological subtypes (P < 0.001). ⑥NTRK and PIK3CA mutations were not associated with clinicopathological features. NGS technology can comprehensively analyze the genetic mutations in PTC patients, which provides important prompts for the occurrence, development, diagnosis and treatment of PTC. In addition, BRAF V600E mutation, RET fusion and TERT mutation are associated with a number of high-risk clinicopathological features. Detection of genetic mutations in PTC patients by NGS is of great significance.

Molecular Analysis of PIK3CA in Metastatic Hormone Receptor-Positive Breast Cancer in Chile: Clinical and Pathological Insights

Breast cancer is the most common cancer among women and a leading cause of cancer-related deaths. PIK3CA gene mutations, which are often present in advanced HR+ breast cancer, can be targeted by alpelisib. However, data on PIK3CA mutations in Chile are limited. Here, we aim to assess the mutational status of PIK3CA in metastatic breast cancer tissues from Chilean patients and describe their clinicopathological characteristics and survival outcomes. We analyzed 102 formalin-fixed, paraffin-embedded metastatic breast cancer samples from 96 patients diagnosed at three Chilean hospitals between 2007 and 2023. PIK3CA mutations were identified using targeted sequencing, and clinicopathological data were collected. We evaluated associations between mutational status, clinicopathological features, and survival. The median age at diagnosis was 56 years. The most common metastatic sites were liver (29.4%), bone (17.6%), and lung/pleura (16.7%). Most patients were HR+ HER2− (83.3%), with 57.3% showing HER2-low status. PIK3CA mutations were present in 40.6% of patients, mainly in exons 7, 9, and 20. No significant associations were found between PIK3CA mutations and clinicopathological characteristics or survival. Our study reveals a high frequency of PIK3CA mutations in HR+ metastatic breast cancer, consistent with global data. The majority of mutations are targetable with alpelisib. The proportion of HER2-low status patients suggests potential benefits from novel HER2-targeted therapies. These findings highlight the need for routine molecular diagnostics in Chile to improve personalized treatment and address economic and access challenges.

Olfactory Neuroblastoma With Divergent Differentiation: Contemporary Management of Unusual Pathology and Literature Review.

Olfactory neuroblastoma (ON; Esthesioneuroblastoma) is a malignant tumor that arises from the olfactory neuroepithelium. Very rarely, ON can histologically display a biphenotypic pattern, with only 7 cases reported in the literature to date. We describe a case of this poorly understood entity and review the patient's histology, pathology, and treatment. An 85-year-old man presented with endoscopy and imaging findings of a sinonasal mass arising from the olfactory cleft. Biopsy and further pathological review established Hyams grade 3 and Kadish stage C tumor. The patient underwent combined endoscopic and open-approach resection due to the involvement of the nasal bone and subsequent adjuvant radiation therapy. Specimen contained Flexner-Wintersteiner rosettes and stained positive for the neuroendocrine marker synaptophysin consistent with ON. A second, epithelial component pattern of goblet cells and mucin was observed suggesting divergent differentiation. We are the first group to report next-generation sequencing of this tumor, which revealed a pathogenic mutation in PIK3CA and a likely pathogenic variant in RUNX1 (AML1). ON with divergent differentiation is very rare, and more robust studies characterizing molecular drivers and pathology may aid in clinical management.

Molecular profiling reveals novel therapeutic targets and clonal evolution in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) has a disproportionately high incidence among women in East Asia. Patients diagnosed with OCCC tend to experience worse clinical outcomes than those with high-grade serous carcinoma (HGSC) at advanced stages. The unfavorable prognosis of OCCC can be partly attributed to its frequent resistance to conventional chemotherapy. Within a precision medicine framework, we sought to provide a comprehensive molecular characterization of OCCC using whole-exome sequencing to uncover potential molecular targets that may inform novel therapeutic strategies. We performed whole-exome sequencing analysis on tumor-normal paired samples from 102 OCCC patients. This comprehensive genomic characterization of a substantial cohort of OCCC specimens was coupled with an analysis of clonal progression. On analyzing 102 OCCC samples, ARID1A (67%) and PIK3CA (49%) emerged as the most frequently mutated driver genes. We identified tier 1 or 2 clinically actionable molecular targets in 40% of cases. This included DNA mismatch repair deficiency (n = 1), as well as BRCA2 (n = 1), PIK3CA (n = 36), KRASG12C (n = 1), and ATM (n = 4) mutations. Furthermore, 45% of OCCC samples displayed ARID1A biallelic loss. Interestingly, we identified previously unreported mutations in the 5' untranslated region of the TERT gene that harbored an adverse prognostic significance. Clock-like mutational processes and activated APOBECs were major drivers of somatic point mutations. Mutations arising from DNA mismatch repair deficiency were uncommon. Reconstruction of clonal evolution revealed that early genetic events likely driving tumorigenesis included mutations in the ARID1A, PIK3CA, TERT, KRAS, and TP53 genes. Our study provides a comprehensive characterization of the genomic landscape and clonal evolution in OCCC within a substantial cohort. These findings unveil potentially actionable molecular alterations that could be leveraged to develop targeted therapies.

Molecular alterations influencing the prognostic outcome in small pancreatic cancer (≤ 2 cm)

Abstract Purpose Pancreatic cancer (PC) is the most lethal cancer. The prognosis of small PC (tumor smaller than 20 mm) is better than that of larger PC tumors, indicating the importance of detecting early-stage PC for patient outcome. The aim of this study was to elucidate the molecular features in small PC (≤ 20 mm). Methods This study included 79 PC tumors (≤ 20 mm in pathological examination) resected between 2004 and 2022. c-Myc, Caveolin-1, Smad4, and Thrombospondin-1 were examined by immunostaining. These molecular alterations were compared in PC patients with tumor size ≤10 mm (n = 11) (14%) and 10 mm &lt; tumor size ≤20 mm (n = 68) (86%). Mutation analyses of KRAS, PIK3CA, and BRAF were performed by pyrosequencing in 22 PCs. Results PC with 10 mm &lt; tumor size ≤20 mm showed significantly worse overall survival and disease-free survival than PC with tumor size &lt;10 mm (P = 0.024 and P = 0.028). Tumor c-Myc and stromal Caveolin-1 expressions were significantly increased in tumors larger than 10 mm (P = 0.02 and P = 0.04). c-Myc and Caveolin-1 expressions were associated with poor disease-free survival and overall survival. KRAS, PIK3CA, and BRAF mutation status did not differ between the two groups. Conclusions Tumor c-Myc and stromal Caveolin-1 overexpressions were detected in tumors larger than 10 mm. Their overexpressions were associated with worse prognosis even in small PC. These molecular alterations in small PC may be a clue for the detection of early-stage PC.

Use of 3′ Rapid Amplification of cDNA Ends (3′ RACE)-Based Targeted RNA Sequencing for Profiling of Druggable Genetic Alterations in Urothelial Carcinomas

Targeted treatment of advanced or metastatic urothelial carcinomas (UCs) requires the identification of druggable mutations. This study describes the development of a 3′ Rapid Amplification of cDNA Ends (3′ RACE)-based targeted RNA sequencing panel which accounts for the status of all genes relevant to UC treatment, namely, FGFR1-4, KRAS, NRAS, BRAF, ERBB2 (HER2), CD274 (PD-L1) and PIK3CA. FGFR2/3-activating point mutations or fusions were found in 54/233 (23.2%) tumors. FGFR3 rearrangements were identified in 11 patients, with eight of them being undetectable by commonly used PCR kits. In addition, one tumor contained a high-copy FGFR2 gene amplification accompanied by strong overexpression of the gene. Mutations in RAS/RAF genes were present in 30/233 (12.9%) UCs and were mutually exclusive with alterations affecting FGFR2/3 genes. On the contrary, activating events in the HER2 oncogene (point mutations and overexpression), as well as PIK3CA mutations, which were relatively common, occurred with similar frequencies in RAS/RAF- or FGFR2/3-positive vs. negative samples. High PD-L1 mRNA expression was associated with advanced disease stage and was not observed in tumors with increased HER2 mRNA expression or in UCs with evidence for FGFR2/3 activation. Three of the studied carcinomas had high-level microsatellite instability (MSI). Overall, more than half of the UCs had potentially druggable genetic alterations. The proposed NGS panel permits comprehensive and cost-efficient analysis of UC-specific molecular targets and may be considered in clinical routine.

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 (&amp;lt;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 &amp;lt;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&amp;lt;0.01, OS: p&amp;lt;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&amp;lt;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 &amp;gt;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.

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.