Detection Of PIK3CA Mutations Research Articles

Detection of hotspot PIK3CA mutations in primary tumors and plasma-cfDNA of breast cancer patients using a novel highly sensitive CE-IVD KIT

Detection of hotspot PIK3CA mutations in primary tumors and plasma-cfDNA of breast cancer patients using a novel highly sensitive CE-IVD KIT

Abstract 2407: Detection of hotspot PIK3CA mutations in primary tumors and plasma-cfDNA of breast cancer patients using a novel highly sensitive CE-IVD kit

Abstract Introduction: Detection of PIK3CA mutations in primary tumors (FFPEs) and liquid biopsy samples is of increasing importance for treatment decisions in breast cancer. Oncolipsy PIK3CA kit (Pharmassist Ltd, Greece) is based on the combination of allele-specific priming, asymmetric PCR, and melting curve analysis. The CE-IVD kit can detect the presence of four PIK3CA hotspot mutations (E542K, E545K, E545Q, H1047R). The aim of the present study was to: a) evaluate the performance of this novel CE-IVD kit both in FFPEs and plasma-cfDNA samples from patients with metastatic breast cancer (MBC) and b) directly compare the derived results using the Oncolipsy PIK3CA kit with the cobas® PIK3CA mutation test (CE-IVD, Roche Diagnostics, Germany) in identical gDNAs isolated from FFPEs samples. Patients and methods: We analyzed 42 primary tumors (FFPEs) and 29 plasma-cfDNA samples from patients with estrogen receptor (ER) positive metastatic breast cancer. Genomic DNA was isolated from FFPEs using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), while cfDNA was extracted from 2.00 mL of plasma using the QIAamp DSP cNA Kit (Qiagen, Hilden, Germany). PIK3CA hotspot mutations were detected in the LightCycler® 2.0 (IVD instrument, Roche Diagnostics, Germany) using the highly sensitive Oncolipsy PIK3CA kit (analytical sensitivity 0.05%). A direct comparison study was performed using the cobas® PIK3CA Mutation Test on the cobas z 480 analyzer (IVD instrument, Roche Diagnostics, Germany). Results: In primary tumors the E545K mutation was detected in 22/42 (52.4%), the E542K and H1047R mutations were detected in 11/42 (26.2%) and 4/42 (9.5%) of primary tumor samples, respectively, while the E545Q mutation was not detected in any FFPE sample tested. At least one PIK3CA mutation was detected in 14/29 (48.3%) plasma-cfDNA samples. More specifically the E545K mutation was detected in 11/29 (38%), the H1047R mutation in 3/29 (10.4%) and the E542K mutation in 2/29 (6.9%) while the E545Q mutation was not detected in any plasma-cfDNA sample tested. In FFPEs, as expected, PIK3CA mutation frequency was higher compared to plasma-cfDNA. Direct comparison of the results derived using these two commercially available kits, the Oncolipsy CE-IVD kit and the cobas PIK3CA mutation test, using the same gDNA isolated from 42 FFPEs revealed a concordance of 34/42 (81%). Conclusions: The commercially available Oncolipsy PIK3CA CE-IVD kit is highly sensitive and specific for the detection of four PIK3CA hotspot mutations in primary tumors and plasma-cfDNA. Acknowledgement: This study has been financially supported by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T1RCI-02935). Citation Format: Stavroula Smilkou, Dimitra Stergiopoulou, Eleni Tzanikou, Loukas Kaklamanis, Ioanna Balgkouranidou, Helena Linardou, Flora Zagouri, Evangelia Razi, Stylianos Kakolyris, Amanda Psyrri, Christos Papadimitriou, Ioanna Koukli, Athina Markou, Evi Lianidou. Detection of hotspot PIK3CA mutations in primary tumors and plasma-cfDNA of breast cancer patients using a novel highly sensitive CE-IVD kit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2407.

Abstract 5039: Preanalytical workflow enabling cfDNA analysis from urine samples

Abstract Introduction: Urine has become an important source of information in the liquid biopsy field. In contrast to blood, specifications for the collection, storage, transport and processing of urine intended for molecular examination are not widely established. Preanalytical specifications were published for urine cell-free DNA (cfDNA) only recently (CEN/TS 17811:2022). In this study, we investigated post-collection changes to cfDNA profiles in urine samples and present the performance of an optimized preanalytical workflow for cfDNA analysis. Methods: Urine from apparently healthy, consented female individuals was spiked with processed cell-free male urine or with a cfDNA standard containing PIK3CA mutations (SensID). The urine was stabilized with a urine collection/stabilization solution under development at PreAnalytiX or was left unstabilized. Urine samples were stored for varying durations at different temperatures in a time course experiment to simulate different urine storage conditions. After storage, urine samples were centrifuged to remove cells and cfDNA was isolated from the supernatant via the QIAsymphony® platform (QIAGEN). Autosomal and male-specific targets were quantified using the Rotor-Gene® Q instrument and the therascreen® PIK3CA qPCR Kit (both QIAGEN) or the QIAcuity® Digital PCR System and a dPCR LNA PIK3CA Mutation Assay (both QIAGEN). Fragment size distribution was determined by TapeStation® Cell-free DNA ScreenTape® (Agilent Technologies). Results: Analysis of cfDNA profiles in unstabilized urine stored for varying durations and temperatures highlighted changes which create artificial cfDNA profiles. The yield of cfDNA drastically decreased over time. Furthermore, the size distribution of cfDNA isolated from unstabilized urine was impacted by release of gDNA as well as DNA degradation. Urine stabilization minimized cfDNA degradation and gDNA release and allowed isolation of cfDNA that was analyzed with qPCR and dPCR even after urine storage. Analysis of isolated cfDNA revealed positive detection of PIK3CA mutations in spiked urine samples. Conclusions: Changes in post-collection cfDNA profile can hinder downstream analysis, resulting in artificial or failed outcomes. Urine stabilization with a collection/stabilization solution under development at PreAnalytiX minimized DNA degradation and gDNA release. It enabled urine storage, allowed analysis of urine cfDNA and target detection by qPCR and dPCR. Citation Format: Daniela Mancarella, Julia Rutsch, Lisa Erkelenz, Moritz Meyer, Laura Sofie Witthaus, Moritz Rath, Thorsten Voss. Preanalytical workflow enabling cfDNA analysis from urine samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5039.

Genomic Alterations of Tumors in HER2-Low Breast Cancers.

The aim of this study was to elucidate molecular profiling in HER2-low tumors based on a promising dataset. A total of 615 consecutive HER2-negative breast cancer samples were assayed. The genomic mutations in the two groups with different HER2 expression levels (HER2-0 vs. HER2-low) were compared. The mutation types obtained via next-generation targeted sequencing were correlated with the clinicopathological features of the patients with HER2-0 and HER2-low breast cancer. The results showed that there was a significantly higher percentage of receptor-positive (ER/PR) tumors and more low-level Ki-67 tumors, but a lower incidence of stage I/II tumors in the HER2-low group compared to the HER2-0 group. There was a significantly higher frequency of 17.62% (65/369) for PIK3CA_SNA in the HER2-low group than in the HER2-0 group, which had a frequency of only 9.35% (23/246) (p = 0.006). When the called gene alterations in the triple-negative breast cancer (TNBC) group were compared with those in the luminal-like breast cancer group, there was a significantly high frequency of 28.17% (140/497) for ERBB2_SNA in a luminal-like group than in the TNBC group(16.95% (20/118)).We conclude that the early detection of PIK3CA mutations is likely to be important and might help therapeutic decision making in patients with HER2-low tumors.

2261P A head-to-head comparison for detecting PIK3CA mutations in circulating tumor DNA of advanced HR+/HER2- breast cancer patients

2261P A head-to-head comparison for detecting PIK3CA mutations in circulating tumor DNA of advanced HR+/HER2- breast cancer patients

Development and Validation of a Novel Dual-Drop-off ddPCR Assay for the Simultaneous Detection of Ten Hotspots PIK3CA Mutations.

Breast cancer is the leading cause of cancer-related deaths in women worldwide. Approximately 40% of patients with hormone receptor-positive, human epidermal growth factor receptor-2-negative breast cancer have activating mutations in the PIK3CA gene. We developed a highly sensitive, specific, cost-effective, and reproducible dual-drop-off droplet digital polymerase chain reaction (PCR) assay for the simultaneous detection of ten hotspots of PIK3CA mutations in plasma cell-free (cf) DNA. We first evaluated the analytical specificity, sensitivity, limit of blank, repeatability, and reproducibility of the assay, which simultaneously detects seven mutations in exon9 and three in exon20. We further applied this assay in 11 gDNA and 18 plasma cfDNA samples from healthy donors and 35 plasma cfDNA samples from metastatic breast cancer patients. The assay is highly sensitive, specific, and applicable for clinical samples containing at least 1-5% mutant DNA. We detected PIK3CA mutations in 9/35(26%) plasma cfDNA samples in exon 9 and in 9/35(26%) in exon 20. Direct comparison of the developed assay with amplification refractory mutation system-based PCR (using plasma samples) and with the Food and Drug Administration-approved cobas PIK3CA mutation assay (using formalin fixed paraffin embedded samples) showed high concordance of our developed assay with the cobas PIK3CA assay. The developed assay is cost-effective and can reliably and simultaneously detect ten hotspot PIK3CA mutations in plasma cfDNA. The clinical performance of the assay will be further evaluated in liquid biopsy samples.

Targeted next-generation sequencing for detection of PIK3CA mutations in archival tissues from patients with Klippel–Trenaunay syndrome in an Asian population

BackgroundKlippel–Trenaunay syndrome (KTS) is a rare slow-flow combined vascular malformation with limb hypertrophy. KTS is thought to lie on the PIK3CA-related overgrowth spectrum, but reports are limited. PIK3CA encodes p110α, a catalytic subunit of phosphatidylinositol 3-kinase (PI3K) that plays an essential role in the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway. We aimed to demonstrate the clinical utility of targeted next-generation sequencing (NGS) in identifying PIK3CA mosaicism in archival formalin-fixed paraffin-embedded (FFPE) tissues from patients with KTS.ResultsParticipants were 9 female and 5 male patients with KTS diagnosed as capillaro-venous malformation (CVM) or capillaro-lymphatico-venous malformation (CLVM). Median age at resection was 14 years (range, 5–57 years). Median archival period before DNA extraction from FFPE tissues was 5.4 years (range, 3–7 years). NGS-based sequencing of PIK3CA achieved an amplicon mean coverage of 119,000x. PIK3CA missense mutations were found in 12 of 14 patients (85.7%; 6/8 CVM and 6/6 CLVM), with 8 patients showing the hotspot variants E542K, E545K, H1047R, and H1047L. The non-hotspot PIK3CA variants C420R, Q546K, and Q546R were identified in 4 patients. Overall, the mean variant allele frequency for identified PIK3CA variants was 6.9% (range, 1.6–17.4%). All patients with geographic capillary malformation, histopathological lymphatic malformation or macrodactyly of the foot had PIK3CA variants. No genotype–phenotype association between hotspot and non-hotspot PIK3CA variants was found. Histologically, the vessels and adipose tissues of the lesions showed phosphorylation of the proteins in the PI3K/AKT/mTOR signaling pathway, including p-AKT, p-mTOR, and p-4EBP1.ConclusionsThe PI3K/AKT/mTOR pathway in mesenchymal tissues was activated in patients with KTS. Amplicon-based targeted NGS could identify low-level mosaicism from low-input DNA extracted from FFPE tissues, potentially providing a diagnostic option for personalized medicine with inhibitors of the PI3K/AKT/mTOR signaling pathway.

Prevalence of PIK3CA mutations in Taiwanese patients with breast cancer: a retrospective next-generation sequencing database analysis.

Breast cancer is the most common cancer type that affects women. In hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) is the most frequently mutated gene associated with poor prognosis. This study evaluated the frequency of PIK3CA mutations in the Taiwanese breast cancer population. This is a retrospective study; patient data were collected for 2 years from a next-generation sequencing database linked to electronic health records (EHRs). The primary endpoint was the regional prevalence of PIK3CA mutation. The secondary endpoints were to decipher the mutation types across breast cancer subtype, menopausal status, and time to treatment failure after everolimus (an mTOR inhibitor) or cyclin-dependent kinase 4/6 (CDK4/6) inhibitor treatment. PIK3CA mutations were identified in 278 of 728 patients (38%). PIK3CA mutations were reported in 43% of patients with HR-/HER2+ subtype and 42% of patients with HR+/HER2- postmenopausal status. A lower prevalence of PIK3CA mutations was observed in triple-negative (27%) and HR+/HER2- premenopausal patients (29%). The most common mutation was at exon 20 (H1047R mutation, 41.6%), followed by exon 9 (E545K mutation, 18.9% and E542K mutation, 10.3%). Among patients treated with CDK4/6 inhibitors, the median time to treatment failure was 12 months (95% CI: 7-21 months) in the PIK3CA mutation cohort and 16 months (95% CI: 11-23 months) in the PIK3CA wild-type cohort, whereas patients receiving an mTOR inhibitor reported a median time to treatment failure of 20.5 months (95% CI: 8-33 months) in the PIK3CA mutation cohort and 6 months (95% CI: 2-9 months) in the PIK3CA wild-type cohort. A high frequency of PIK3CA mutations was detected in Taiwanese patients with breast cancer, which was consistent with previous studies. Early detection of PIK3CA mutations might influence therapeutic decisions, leading to better treatment outcomes.

Rezultati multicentričnog testiranja somatskih mutacija gena PIK3CA kod uznapredovalog luminalnog raka dojke s negativnim HER2 receptorom

Aim: Activating somatic mutations in p110α catalytic subunit of PIK3-kinase (PIK3CA) are present in about 40% of breast cancers (BC), and are involved in oncogenesis and cancer growth. PIK3CA inhibitor therapy is approved for patients with advanced HR+/HER2- endocrine-resistant BC with somatic PIK3CA mutations. Such targeted therapy improved patient disease-free survival. Method: Analyses of PIK3CA gene mutations were performed in five University Hospital Centers in Croatia. Qualitative detection of PIK3CA gene mutations was performed on DNA extracted from 507 breast cancer samples, by the real-time PCR method using Cobas® PIK3CA mutation test. Results: PIK3CA gene mutations were detected in 209 (42.2%) out of 495 successfully analyzed cases. In 12 cases (2.4%), the tumor tissue was not of sufficient quality for analysis. Most mutations were detected in the helical (41.2%) and kinase domains (46.9%) of the PIK3CA protein. The most frequently detected mutations were H1047L/R/Y (46.4%), E545A/D/G/K (26.3%) and E542K (13.9%), followed by N345K (8.1%). PIK3CA mutations were detected in 44.8% of primary BC cases and in 39.6% of metastatic lesions (χ2=0.647; P=0.421). The age of the tissue did not significantly affect the percentage of detected mutations (χ2=0.543; P =0.461). Institutions differed in the number of analyzed cases, but not in the percentage of detected PIK3CA mutations (χ2=6.23; df=4; P=0.183) nor in the frequency of the most common mutations (χ2=2.65; df=4; P=0.618). Conclusion: The frequencies of PIK3CA mutations correspond to those reported in the literature. We have shown that the tissue of primary BC as well as metastatic lesions are suitable for analysis, and that the age of the tissue is not a significant obstacle in the analysis.

Highly Sensitive Detection of PIK3CA Mutations by Looping-Out Probes-Based Melting Curve Analysis.

PIK3CA mutations have important therapeutic and prognostic implications in various cancer types. However, highly sensitive detection of PIK3CA hotspot mutations in heterogeneous tumor samples remains a challenge in clinical settings. To establish a rapid PCR assay for highly sensitive detection of multiple PIK3CA hotspot mutations. We described a novel melting curve analysis-based assay using looping-out probes that can enrich target mutations in the background of excess wild-type and concurrently reveal the presence of mutations. The analytical and clinical performance of the assay were evaluated. The developed assay could detect 10 PIK3CA hotspot mutations at a mutant allele fraction of 0.05-0.5% within 2h in a single step. Analysis of 82 breast cancer tissue samples revealed 43 samples with PIK3CA mutations, 28 of which were confirmed by Sanger sequencing. Further testing of 175 colorectal cancer tissue samples showed that 24 samples contained PIK3CA mutations and 19 samples were confirmed by Sanger sequencing. Droplet digital PCR supported that all mutation-containing samples undetected by sequencing contained mutations with a low allele fraction. The rapidity, ease of use, high sensitivity and accuracy make the new assay a potential screening tool for PIK3CA mutations in clinical laboratories.

247P Efficacy of therapy directed against PI3K and cyclin inhibitors based on the location of PI3K mutations

Activation of the phosphatidylinositol-3-kinase (PI3K) pathway is responsible of activating the phosphorylation cascade that regulates cell survival and metabolism. Mutations in this pathway may occur in 28-46% of HR+/HER2- advanced breast cancers. It is known that the presence of this mutation confers a worse prognosis. We set out to analyze if PI3K mutations in different exons confer resistance to treatments directed against this target and to cyclin inhibitors (inh-CDK4/6). We have analyzed 96 patients with advanced breast cancer HR+/HER2- subsidiaries of treatment with PI3K-targeted therapy in the hospital of Jaén from February/20 to January/23. To detect PIK3CA mutations we used the cobas ® diagnostic kit that detects: exons 1 (R88Q), 4, 7 (C420R), 9 (E542K, E545K/A/D/G, Q546E/R) and 20 (H1047R/Y/L). We obtained 26% mutations in PI3K (5% exon 1, 40% exon 9, 55% exon 20). All in women with a mean age at diagnosis of 48 years and 56 at diagnosis of metastases, located at bone 65%, lung 35%, liver 23% and CNS 4% All patients have been treated with inh-CDK4/6. The median progression-free survival (mSLP) is 17 months (1-60). In patients with exon 1 mutation, mSLP is 25 months, in exon 9, 13 (2-43) and in exon 20, 19 (1-60) 60% of the patients have progressed to inh-CDK4/6 and as next treatment we used alpelisib. In 58% corresponds to 2 line in metastatic disease. The mSLP is 5 months (1-22) with a median follow-up of 14 months (1-34) and 69% of events. In patients with mutation in exon 1 the mSLP is 2 months, in exon 9, 5 (1-13) and in exon 20, 9 (2-22). Treatment was associated with an antiestrogen (77%) with a mSLP 7 months (1-22) or an aromatase inhibitor with a mSLP 3 months (2-10). 53% suffered grade 3 adverse effects (G3 AE): hyperglycemia (80% exon 9, 16% exon 20), diarrhea (20% exon 9) and skin rash (16% exon 20). 14% discontinued treatment due to toxicity. The PFS with alpelisib is lower than reported in the SOLAR1 study and is similar to Bylieve study, whose population is most similar to our sample. Exon 20 carriers are those who obtain a higher PFS, as well as those who receive it with an antiestrogen. Exon 9 carriers have a higher rate of G3 AE With respect to inh-CDK4/6, the PFS corresponds to the literature, with exon 20 carriers obtaining a higher PFS.

Novel Method for Detection of PIK3CA Mutations in Circulating Tumor DNA of Patients with Colorectal Cancer.

The PIK3CA mutation is considered a potential target for treatment of colorectal cancer. We evaluated a PIK3CA mutation assay on plasma cell-free DNA (cfDNA) using a newly developed PCR with restriction digestion integrated and followed by Sanger's sequencing. We analyzed PIK3CA mutation in plasma with our newly developed assays and in matching tumor tissues by routine methods. We detected the PIK3CA gene mutation status by both methods in samples from 40 colorectal cancer patients. Three H1047R mutations of PIK3CA gene were detected in the cfDNA of the 40 patients by restriction digestion PCR. Neither E545K nor H1047R mutations were detected in the cfDNA by routine PCR/sequencing. The PIK3CA H1047R and E545K mutations in cfDNA can be sensitively detected with our newly developed assays. The colorectal cancer has been used as a clinical example in testing our new assays, which indicates that the new assays may have wider applications in detecting mutations in precision oncology. Trial registration: Current Controlled Trials ChiCTR-DDT-12002848, 8 October 2012.

Abstract CT249: A study to evaluate the safety and tolerability of the covalent phosphoinositide-3-kinase (PI3K)-alpha Inhibitor, TOS-358, in adult subjects with select solid tumors

Abstract Background: TOS-358 is an orally available, highly selective covalent inhibitor of PI3Kα capable of achieving a deep and durable inhibition of PI3K-AKT signaling with virtually no off-target effects. In patient-derived xenograft models, TOS-358 has shown anti-cancer activity in several different tumor types including breast, colorectal, gastric, head and neck, bladder, and esophageal cancer, and its’ activity was superior to non-covalent compounds targeting PI3Kα. Administered at the equivalent dose, TOS-358 has shown a good safety profile in 28-day repeat dose toxicity studies in rats and dogs. Methods: The TOS-358-001 study is a multicenter dose escalation (phase 1) and dose expansion (phase 1b) clinical study evaluating the safety, tolerability, and preliminary efficacy of TOS-358. Eligible subjects are adults with one of protocol-defined tumor types, known PIK3CA mutation or amplification, adequate organ function, ECOG performance status 0 or 1, available archived or fresh tumor tissue for PIK3CA mutation detection and no prior treatment with PI3K, AKT or mTOR inhibitors (except subjects with breast cancer). The phase 1 portion of the study follows a 3+3 dose escalation design and is intended to determine the minimum effective dose (MED) and the maximum tolerated dose (MTD) of TOS-358 when administered orally on a once daily (QD) or a twice daily (BID) schedule, and the recommended phase 2 dose (RP2D). Cohort expansions at the MTD and MED to support the RP2D selection are planned. The phase 1b portion of the study will enroll subjects with tumor PIK3CA mutations or amplifications in several tumor-specific cohorts (colorectal, gastric, non-small cell lung, breast, squamous cell carcinoma of the head and neck, urothelial and select gynecological cancers) to further assess the safety and tolerability of TOS-358 and to evaluate the initial efficacy of the investigational agent. Enrolment to the study commences in US with European sites planned to open later in 2023. Clinical trial information: NCT05683418. Citation Format: Marwan Fakih, Davendra P. Sohal, Navid Hafez, Jordan D. Berlin, Kimberly Hari, Ovid C. Trifan. A study to evaluate the safety and tolerability of the covalent phosphoinositide-3-kinase (PI3K)-alpha Inhibitor, TOS-358, in adult subjects with select solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT249.

Abstract 2314: Development of a 6-color multiplex Crystal Digital PCR assay for the detection of ESR1 and PIK3CA mutations in the plasma of metastatic breast cancer patients

Abstract Blood-based liquid biopsies have become a real asset for patient treatment management in the field of precision oncology over the past few years, as they allow quick and easy access to tumor genetic alterations of interest. The analysis of circulating tumor DNA (ctDNA) from these liquid biopsies is a particularly well-suited strategy for longitudinal monitoring of patients. In patients with HR+/HER2- metastatic breast cancer (MBC), early detection of ESR1 and PIK3CA mutations is a key element to better personalize disease management. Among the technologies currently used for liquid biopsies, digital PCR (dPCR) has the advantage of being among the fastest and least expensive, providing robust results along with unmatched sensitivity as compared to other methods. Until recently, however, dPCR has been previously limited by multiplex capability. The recent availability of the 6-color version of the naica® platform (Stilla Technologies) has allowed us to develop a single-well multiplexed assay that combines an 11 pathogenic mutation ESR1 assay with a 21 mutation PIK3CA assay, for a combined total of 32 ESR1 and PIK3CA mutations. Here we present data on this new high-plex dPCR panel. As ESR1 and PIK3CA analyses are often requested together, this new screening assay represents a significant saving of time and resources for the analysis of our MBC patients with a relative ease of implementation and analysis. Citation Format: Veronique Quillien, Julien Corne, Thibault De la Motte Rouge, Fanny Le Du, Florence Godey. Development of a 6-color multiplex Crystal Digital PCR assay for the detection of ESR1 and PIK3CA mutations in the plasma of metastatic breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2314.

Development of Multiplex Drop-Off Digital PCR Assays for Hotspot Mutation Detection of KRAS, NRAS, BRAF, and PIK3CA in the Plasma of Colorectal Cancer Patients

The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. We assessed the analytical performance of the assays using synthetic oligonucleotides, which were further validated on plasma cfDNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cfDNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring.

Abstract P5-02-46: Targeted capture sequencing allows sensitive genomic profiling of circulating tumor DNA in advanced HR+/HER- breast cancer patients

Abstract Introduction: CDK4/6 inhibitors provide substantial benefits as 1st or 2nd line treatments and are now the standard of care for patients with advanced HR-positive, HER2-negative breast cancer. Recently, we demonstrated that a high-resolution SiMSen-seq assay (SSS) provides a sensitive and robust method for detecting 11 PIK3CA hotspot mutations in cell-free circulating DNA, allowing the identification of patients eligible for alpelisib treatment. Unfortunately, all patients progress at some time point due to intrinsic or acquired resistance. Therefore, detecting additional genomic biomarkers for treatment resistance beyond PIK3CA mutations is crucial. Targeted panel sequencing offers a promising strategy to profile circulating tumor DNA (ctDNA) for genetic alterations in multiple genes associated with treatment response and disease progression. This ongoing study aims to show that a commercial NGS assay (AVENIO ctDNA Expanded Kit, Roche Diagnostics) can (1) detect PIK3CA mutations with similar sensitivity as our high-resolution SSS assay and can (2) simultaneously identify additional genetic alterations in multiple genes, possibly associated with treatment resistance or disease progression. Material and Methods: To this end, we collected plasma samples from 46 metastatic HR+/HER2- breast cancer patients before starting 1st (32 patients) or 2nd (14 patients) line treatment. Samples were analyzed using SSS and the AVENIO ctDNA Expanded Kit, enriching for 77 clinically relevant cancer genes. PIK3CA mutation detection and variant allele frequencies (VAF) were compared between the two methods. Additionally, mFAST-SeqS was used to estimate the tumor fractions in plasma samples. Results: The median z-score from mFAST-SeqS analyses was 2.38 [25–75th percentile: 1.23–4.5], and 17/46 (37%) patients had z-scores 3, indicating elevated tumor fractions (>5%). Patients starting 2nd line treatment had significantly higher z-scores than those starting 1st line treatment (median 2.2 vs. 3.8, rank-sum p-value 0.042). One sample repeatedly failed with the SSS assay, leaving 45 samples for a head-to-head comparison. Considering only PIK3CA hotspot mutations covered by both assays, 16 alterations were detected in 14 patients (31%) by the SSS assay and 19 alterations in 17 patients (38%) by the AVENIO ctDNA Expanded Kit. Both assays detected the identical co-occurrence of two PIK3CA mutations in two samples. Two of three mutations only detected with the AVENIO ctDNA Expanded Kit were also observed with the SSS assay but below the pre-defined detection limit. One mutation was only detected by the AVENIO ctDNA Expanded Kit. Overall, we found an excellent concordance rate of 94% between the two assays, confirming the high sensitivity of the panel sequencing assay. Moreover, the VAF of SSS and AVENIO kit were highly correlated (Spearman’s rho = 0.97, p < 0.001). Using the AVENIO kit, a large number of additional mutations in 40 genes could be identified in 42/46 (91%) patients with a median of 2.0 variants (range 1-13) per sample. The most frequently mutated genes included PIK3CA (43%), followed by ESR1 (20%), TP53 (20%), MET (17%), SMAD4 (17%), ERBB2 (11%), and BRCA2 (11%). The median VAF was 0.64% (range 0.1-29.8). Further analyses are still ongoing. Conclusion: The AVENIO ctDNA Expanded Kit revealed a high sensitivity and concordance rate for detecting PIK3CA hotspot mutations in plasma samples compared with the high-resolution SSS assay. A major advantage of panel sequencing over a single gene approach is that the interrogation of multiple genes can indicate a true negative PIK3CA result if other variants are present with a high VAF. Moreover, other actionable targets or mechanisms of resistance can be captured simultaneously, thus improving the effective precision treatment of metastatic breast cancer patients. Citation Format: Nadia Dandachi, Ricarda Graf, Nina Potocnik, Lara Pancheri, Eva Klocker, Christoph Suppan, Philipp Jost, Hanno Gerritsmann, Ellen Heitzer, Marija Balic. Targeted capture sequencing allows sensitive genomic profiling of circulating tumor DNA in advanced HR+/HER- breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-46.

Abstract ED9-2A: Can ctDNA substitute tissue testing

Abstract Circulating tumor DNA (ctDNA) is at the forefront of liquid biopsy technology. A key advantage of ctDNA is being able to achieve genomic profiling from a blood test rather than from a more invasive tissue biopsy, reducing risk and discomfort for patients as well as costs and logistical complexity. Increasing evidence supports ctDNA as a useful clinical tool in specific indications, for example in the detection of PIK3CA mutations to identify patients suitable for alpelisib plus fulvestrant.The expectation is that indications for routine ctDNA testing will expand as more data become available and ctDNA is embedded into clinical trials and paired with novel therapies. Biological and technical factors both contribute to the feasibility of ctDNA expansion into the clinic. Representation of heterogeneity might be a key strength of ctDNA analysis over tumour biopsy analysis, as sampling the circulation compartment can in principle allow representation of a wider array of disease sites than a tumor sample acquired with a single needle. However, the factors influencing how different metastases might contribute to the circulating compartment are poorly understood, presenting a challenge to clinical interpretation. Moreover, the limit of detection of ctDNA assays, the timing of treatment and stochastic effects can contribute to uncertainty, particularly in the interpretation of negative results. Recent work analyzing tumor mutational burden using ctDNA has highlighted these challenges as applied to biomarker development. Circulating tumour DNA is likely to complement tissue biopsy in the future, and may substitute tumor biopsy in specific indications supported by the data. Ultimately, clinical utility for different ctDNA indications, as opposed to tissue biopsy, will need ongoing confirmation within clinical trials. Citation Format: Ben O’Leary. Can ctDNA substitute tissue testing [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr ED9-2A.

Proficiency testing of PIK3CA mutations in HR+/HER2-breast cancer on liquid biopsy and tissue

Precision oncology based on specific molecular alterations requires precise and reliable detection of therapeutic targets in order to initiate the optimal treatment. In many European countries—including Germany—assays employed for this purpose are highly diverse and not prescribed by authorities, making inter-laboratory comparison difficult. To ensure reproducible molecular diagnostic results across many laboratories and different assays, ring trials are essential and a well-established tool. Here, we describe the design and results of the ring trial for the detection of therapeutically relevant PIK3CA hotspot mutations in HR+/HER2-breast cancer tissue and liquid biopsy (LB). For PIK3CA mutation detection in tissue samples, 43 of the 54 participants (80%) provided results compliant with the reference values. Participants using NGS-based assays showed higher success rate (82%) than those employing Sanger sequencing (57%). LB testing was performed with two reference materials differing in the length of the mutated DNA fragments. Most participants used NGS-based or commercial real-time PCR assays (70%). The 167 bp fragments led to a successful PIK3CA mutation detection by only 31% of participants whereas longer fragments of 490 bp were detectable even by non-optimal assays (83%). In conclusion, the first ring trial for PIK3CA mutation detection in Germany showed that PIK3CA mutation analysis is broadly established for tissue samples and that NGS-based tests seem to be more suitable than Sanger sequencing. PIK3CA mutation detection in LB should be carried out with assays specifically designed for this purpose in order to avoid false-negative results.

Chip-based digital Polymerase Chain Reaction as quantitative technique for the detection of PIK3CA mutations in breast cancer patients

BackgroundPIK3CA is a gene frequently mutated in breast cancer. With the FDA approval of alpelisib, the evaluation of PIK3CA for activating mutations is becoming routinely. Novel platforms for gene analysis as digital PCR (dPCR) are emerging as a potential replacement for the traditional Sanger sequencing. However, there are still few studies on chip-based dPCR to detect mutations in tumor samples. Thus, this cross-sectional study aimed to assess the sensibility of a chip-based dPCR to detect and quantify PIK3CA mutations and compare its performance with Sanger sequencing. Materials and MethodsTumor samples from 57 breast cancer patients (22 pre-treatment samples, 32 tumors after neoadjuvant chemotherapy, and three lymph nodes) were collected and analyzed by Sanger sequencing and dPCR for the three PIK3CA most relevant mutations (p.E545K, p. H1047R, and p. H1047L). Digital PCR sensitivity, specificity, and overall performance were estimated by contingency tables, receptor operator characteristic (ROC), and area under the curve (AUC). Association of PIK3CA mutations with clinicopathological variables was conducted. ResultsSanger sequencing identified PIK3CA mutations in six patients (10.5%), two with p. H1047R, and four with p. E545K. Digital PCR confirmed those mutations and identified 19 additional patients with at least one mutation. Comparison between dPCR and Sanger sequencing showed a sensitivity of 100% (95% CI 53–100%), and a specificity of 84.2% (95% CI 83–84.2%). Besides, p. H1047R mutation detected by dPCR showed a significant association with breast cancer phenotype (p = 0.019) and lymphatic nodes infiltration (p = 0.046). ConclusionsDigital PCR showed a high sensitivity to detect mutations in tumor samples and it might be capable to detect low-rate mutations and tumor subpopulations not detected by Sanger sequencing.

144P Development of a 6-color multiplex crystal digital PCR assay for the simultaneous detection of ESR1 and PIK3CA mutations in the plasma of metastatic breast cancer patients

Blood-based liquid biopsies have become a real asset for patient treatment management in the field of precision oncology over the past few years, has it allows quick and easy access to tumor genetic alterations of interest. Indeed, circulating tumor DNA (ctDNA) analysis is particularly well suited for longitudinal monitoring with the advantage of covering the potential genetic heterogeneity of metastatic lesions. For Hormone Receptor-positive (HR+) Metastatic Breast Cancer (MBC) patients, mutations on the ESR1 (encoding the estrogen receptor (ER) alpha), and PIK3CA (encoding the phosphatidylinositol-3-kinase (PI3K) catalytic subunit p110-alpha) genes are emerging clinical biomarkers that can both guide clinicians in their choice of treatment.