PIK3CA Exon Research Articles

Clinical activity of FOLFIRI plus cetuximab according to extended gene mutation status by next-generation sequencing: findings from the CAPRI-GOIM trial

Treatment with antiepidermal growth factor receptor (anti-EGFR) monoclonal antibodies has been restricted to metastatic colorectal cancer (mCRC) patients with RAS wild-type tumors. Next-generation sequencing (NGS) allows the assessment in a single analysis of a large number of gene alterations and might provide important predictive and prognostic information. In the CAPRI-GOIM trial, 340 KRAS exon 2 wild-type mCRC patients received first-line FOLFIRI plus cetuximab. Tumor samples (182/340, 53.5%) were assessed by NGS to search for mutations in 22 genes involved in colon cancer. Objective responses in the NGS cohort were observed in 104/182 patients [overall response rate (ORR) 57.1%; 95% confidence interval (95% CI) 52% to 66.4%] with a median progression-free survival (mPFS) of 9.8 (95% CI 8.7-11.5) months. NGS analysis was successfully completed in all 182 samples. One or more gene mutations (up to five) were detected in 124/182 (68.1%) tumors within 14/22 genes for a total of 206 mutations. KRAS exon 2 mutations were identified in 29/182 (15.9%) samples, defined as wild type by local laboratory assessment. Frequently mutated genes were: TP53 (39.6%), KRAS exons 3/4 (8.8%), NRAS exons 2/3 (7.1%), PIK3CA exons 9/20 (13.2%), BRAF (8.2%). FOLFIRI plus cetuximab treatment determined ORR of 62.0% (95% CI 55.5% to 74.6%) with mPFS of 11.1 (95% CI 9.2-12.8) months in patients with KRAS and NRAS wild-type tumors. Conversely, ORR was 46.6% (95% CI 39.9-57.5%) with mPFS of 8.9 (95% CI 7.4-9.6) months in patients with KRAS or NRAS mutations. Similarly, the subgroup of patients carrying KRAS, NRAS, BRAF, or PIK3CA mutations showed a worse outcome, although this might be due to a prognostic effect. This study demonstrates that NGS analysis in mCRC is feasible, reveals high level of intra and intertumor heterogeneity, and identifies patients that might benefit of FOLFIRI plus cetuximab treatment.

407P - Pik3Ca Mutations and Loss of Pten Expression in Circulating Tumor Cells (Ctcs) in Patients with Metastatic Breast Cancer (Mbc)

ABSTRACT Aim: Currently, several methods are available to detect CTCs in peripheral blood; the enumeration of CTCs has emerged as a promising biomarker. CTCs shed from metastatic cancers may allow the non-invasive analysis of the evolution of tumor genomes during treatment and disease progression through “liquid biopsies” 1. PIK3CA mutations and loss of PTEN expression are being analyzed in many studies in breast cancer. Frequent deregulation and aberration of this pathway have been implicated not only in breast cancer development and progression, but also in breast cancer therapy resistance, and as a potential predictor of response to new treatments 2,3. The objective of this study is to analyze genomic alterations of the PTEN gene and PIK3CA (exon 20) in CTCs in patients with MBC. 1. Cristofanilli M, et al. N Engl J Med 2004;351:781-91. 2. Sarah-Jane Dawson, et al. N Engl J Med 2013;368:1199-1209. 3. Cancer Genome Atlas Network. Nature 2012;490:61-70. Methods: We carried out a descriptive study in patients with MBC currently undergoing treatment at Hospital Universitario de Salamanca (Spain). CTCs were quantified by means an automated fluorescence method (MetaFer-MetaCyte of MetaSystem), allowing selection of cells by size and genetic alterations. We analyzed genomic alterations in PTEN gene in CTCs by FISH and High Resolution Melting (HRM) was performed to measure PIK3CA mutations in circulating tumor DNA. Finally, we studied the association between PTEN deletion and PIK3CA mutations. Results: In this preliminary analysis, a total of 30 samples have been obtained. CTCs (size >7 µm) were detected in 27 of 30 women. Of 27 samples, there were 12 cases (44,4%) of PTEN loss detected by FISH, and there were 10 cases (37,1%) in which PIK3CA mutations were detected by HRM. In 5 samples, there were association between PTEN deletion and PIK3CA mutations. These 5 cases were Luminal (A and B) tumors. Conclusions: Monitoring and isolation of CTCs represents an opportunity to study specific genomic alterations. In this study, we carried out the analysis of two genes involved in the same signaling pathway: PTEN and PIK3CA; which may have additive or synergistic effects through non-canonical functional pathways. Disclosure: All authors have declared no conflicts of interest.

Occurrence of low frequency PIK3CA and AKT2 mutations in gastric cancer

The PI3K/AKT signal transduction pathway has distinct functional roles in tumor progression. PIK3CA was reported to harbor the hot-spot in many types of tumor. Akt, the downstream of PI3K, its family members especially AKT2 activation in human cancer has been extensively studied, but its activation by mutation was less reported. The occurrence of PIK3CA and AKT2 mutations in a variety of cancers indicates their important involvement in carcinogenesis. Therefore, we investigated their mutation frequencies in gastric cancer (GC) in China. In our study, we selected hot-spot related exons 9, 18 and 20 of PIK3CA and kinase domain exons 6–14 of AKT2 genes were screened in 10 GC cell lines, 100 advanced primary GC and matched normal tissues. Denaturing high performance liquid chromatography (DHPLC) and DNA sequencing were used to analyze the mutations in the two genes. Two point mutations in the PIK3CA gene were identified in 4 of 10 GC cell lines and in 4 of 100 GC primary tumors. Two polymorphisms in AKT2 were detected in 19 of 100 GC primary tumors. One point mutation in AKT2 was detected in 1 of 10 GC cell lines and 3 of 100 GC primary tumors but no hot spot variation was detected. Our results indicate that PIK3CA and AKT2 mutations occurred at low frequency in GC, and suggest that the PIK3CA/AKT2 pathway might engage other events during gastric carcinogenesis.

Coexisting KRAS and PIK3CA exon 20 mutations as a potential poor-prognosis factor in metastatic colorectal cancer (mCRC).

3591 Background: Molecular screening and biomarker enrichment strategies in mCRC clinical trials have demonstrated to have an impact in treatment benefit and prognosis. The data obtained from these...

Detection of Cancer DNA in Plasma of Patients with Early-Stage Breast Cancer

Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection. In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR. Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery. This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients.

Molecular patterns in deficient mismatch repair colorectal tumours: results from a French prospective multicentric biological and genetic study.

Background:To test the prognostic value of tumour protein and genetic markers in colorectal cancer (CRC) and examine whether deficient mismatch repair (dMMR) tumours had a distinct profile relative to proficient mismatch repair (pMMR) tumours.Methods:This prospective multicentric study involved 251 stage I–III CRC patients. Analysed biomarkers were EGFR (binding assay), VEGFA, thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) expressions, MMR status, mutations of KRAS (codons 12–13), BRAF (V600E), PIK3CA (exons 9 and 20), APC (exon 15) and P53 (exons 4–9), CpG island methylation phenotype status, ploidy, S-phase, LOH.Results:The only significant predictor of relapse-free survival (RFS) was tumour staging. Analyses restricted to stage III showed a trend towards a shorter RFS in KRAS-mutated (P=0.005), BRAF wt (P=0.009) and pMMR tumours (P=0.036). Deficient mismatch repair tumours significantly demonstrated higher TS (median 3.1 vs 1.4) and TP (median 5.8 vs 3.5) expression relative to pMMR (P<0.001) and show higher DPD expression (median 14.9 vs 7.9, P=0.027) and EGFR content (median 69 vs 38, P=0.037) relative to pMMR.Conclusions:Present data suggesting that both TS and DPD are overexpressed in dMMR tumours as compared with pMMR tumours provide a strong rationale that may explain the resistance of dMMR tumours to 5FU-based therapy.

PIK3CA mutations define favorable prognostic biomarkers in operable breast cancer: a systematic review and meta-analysis.

BackgroundMutations of the p110α catalytic subunit of phosphatidylinositol 3-kinase (PIK3CA) are among the most common genetic aberrations in human breast cancer. At present, controversy exists concerning the prognostic value of the mutations.MethodsWe performed a systematic review and meta-analysis to clarify the association between PIK3CA mutations and survival outcomes. A comprehensive, computerized literature search of PubMed, Web of Science databases, the Chinese Biomedical Literature Database, and Wangfang Data until August 27, 2013 was carried out. Eligible studies were included according to specific inclusion criteria. Pooled hazard ratio was estimated by using the fixed effects model or random effects model according to heterogeneity between studies.ResultsEight eligible studies were included in the analysis, all of which were retrospective cohort studies. The overall meta-analysis demonstrated that the PIK3CA mutations were associated with better clinical outcomes (hazard ratio 0.72; 95% confidence interval: 0.57–0.91; P=0.006). None of the single studies materially altered the original results and no evidence of publication bias was found. Further subgroup analysis of mutations in exons 9 and 20 did not show statistical significance.ConclusionPIK3CA mutations in operable primary breast cancer indicate a good prognosis. Further studies should be conducted to investigate the effect of PIK3CA mutations on clinical outcomes in different histologic types, different molecular subtypes of breast cancer, and different exons of PIK3CA.

Double-Blind, Placebo-Controlled, Multicenter, Randomized, Phase IIB Neoadjuvant Study of Letrozole-Lapatinib in Postmenopausal Hormone Receptor–Positive, Human Epidermal Growth Factor Receptor 2–Negative, Operable Breast Cancer

This is a randomized, double-blind, placebo-controlled study aimed to evaluate the clinical and biologic effects of letrozole plus lapatinib or placebo as neoadjuvant therapy in hormone receptor (HR) -positive/human epidermal growth factor receptor 2 (HER2) -negative operable breast cancer. Ninety-two postmenopausal women with stage II to IIIA primary breast cancer were randomly assigned to preoperative therapy consisting of 6 months of letrozole 2.5 mg orally daily plus lapatinib 1,500 mg orally daily or placebo. Surgery was performed within 2 weeks from the last study medication. Clinical response was assessed by ultrasonography. Pre- and post-treatment samples were evaluated for selected biomarkers. Fresh-frozen tissue samples were collected for genomic analyses. Numerically similar clinical response rates (partial + complete response) were observed (70% for letrozole-lapatinib and 63% for letrozole-placebo). Toxicities were generally mild and manageable. A significant decrease in Ki-67 and pAKT expression from baseline to surgery was observed in both arms. Overall, 34 patients (37%) had a mutation in PIK3CA exon 9 or 20. In the letrozole-lapatinib arm, the probability of achieving a clinical response was significantly higher in the presence of PIK3CA mutation (objective response rate, 93% v 63% in PIK3CA wild type; P = .040). The combination of letrozole-lapatinib in early breast cancer was feasible, with expected and manageable toxicities. In unselected estrogen receptor-positive/HER2-negative patients, letrozole-lapatinib and letrozole-placebo resulted in a similar overall clinical response rate and similar effect on Ki-67 and pAKT. Our secondary end point findings of a significant correlation between PIK3CA mutation and response to letrozole-lapatinib in HR-positive/HER2-negative early breast cancer must now be independently confirmed.

Comparative genomic hybridisation array and DNA sequencing to direct treatment of metastatic breast cancer: a multicentre, prospective trial (SAFIR01/UNICANCER)

Breast cancer is characterised by genomic alterations. We did a multicentre molecular screening study to identify abnormalities in individual patients with the aim of providing targeted therapy matched to individuals' genomic alterations. From June 16, 2011, to July 30, 2012, we recruited patients who had breast cancer with a metastasis accessible for biopsy in 18 centres in France. Comparative genomic hybridisation (CGH) array and Sanger sequencing on PIK3CA (exon 10 and 21) and AKT1 (exon 4) were used to assess metastatic biopsy samples in five centres. Therapeutic targets were decided on the basis of identified genomic alterations. The primary objective was to include 30% of patients in clinical trials testing a targeted therapy and, therefore, the primary outcome was the proportion of patients to whom a targeted therapy could be offered. For the primary endpoint, the analyses were done on the overall population registered for the trial. This trial is registered with ClinicalTrials.gov, number NCT01414933. 423 patients were included, and biopsy samples were obtained from 407 (metastatic breast cancer was not found in four). CGH array and Sanger sequencing were feasible in 283 (67%) and 297 (70%) patients, respectively. A targetable genomic alteration was identified in 195 (46%) patients, most frequently in PIK3CA (74 [25%] of 297 identified genomic alterations), CCND1 (53 [19%]), and FGFR1 (36 [13%]). 117 (39%) of 297 patients with genomic tests available presented with rare genomic alterations (defined as occurring in less than 5% of the general population), including AKT1 mutations, and EGFR, MDM2, FGFR2, AKT2, IGF1R, and MET high-level amplifications. Therapy could be personalised in 55 (13%) of 423 patients. Of the 43 patients who were assessable and received targeted therapy, four (9%) had an objective response, and nine others (21%) had stable disease for more than 16 weeks. Serious (grade 3 or higher) adverse events related to biopsy were reported in four (1%) of enrolled patients, including pneumothorax (grade 3, one patient), pain (grade 3, one patient), haematoma (grade 3, one patient), and haemorrhagic shock (grade 3, one patient). Personalisation of medicine for metastatic breast cancer is feasible, including for rare genomic alterations. French National Cancer Institute, Breast Cancer Research Foundation, Odyssea, Operation Parrains Chercheurs.

A novel deleterious PTEN mutation in a patient with early-onset bilateral breast cancer

BackgroundAn early age at Breast Cancer (BC) onset may be a hallmark of inherited predisposition, but BRCA1/2 mutations are only found in a minority of younger BC patients. Among the others, a fraction may carry mutations in rarer BC genes, such as TP53, STK11, CDH1 and PTEN. As the identification of women harboring such mutations allows for targeted risk-management, the knowledge of associated manifestations and an accurate clinical and family history evaluation are warranted.Case presentationWe describe the case of a woman who developed an infiltrating ductal carcinoma of the right breast at the age of 32, a contralateral BC at age 36 and another BC of the right breast at 40. When she was 39 years-old, during a dermatological examination, mucocutaneous features suggestive of Cowden Syndrome, a disorder associated to germ-line PTEN mutations, were noticed. PTEN genetic testing revealed the novel c.71A > T (p.Asp24Val) mutation, whose deleterious effect, suggested by conservation data and in silico tools, was definitely demonstrated by the incapacity of mutant PTEN to inhibit Akt phosphorylation when used to complement PTEN-null cells. In BC tissue, despite the absence of LOH or somatic mutations of PTEN, Akt phosphorylation was markedly increased in comparison to normal tissue, thus implying additional somatic events into the deregulation of the PI3K/Akt/mTOR pathway and, presumably, into carcinogenesis. Hence, known oncogenic mutations in PIK3CA (exons 10 and 21) and AKT1 (exon 2) were screened in tumor DNA with negative results, which suggests that the responsible somatic event(s) is a different, uncommon one.ConclusionThis case stresses the importance of clinical/genetic assessment of early-onset BC patients in order to identify mutation carriers, who are at high risk of new events, so requiring tailored management. Moreover, it revealed a novel PTEN mutation with pathogenic effect, pointing out, however, the need for further efforts to elucidate the molecular steps of PTEN-associated carcinogenesis.

PIK3CA mutations, phosphatase and tensin homolog, human epidermal growth factor receptor 2, and insulin-like growth factor 1 receptor and adjuvant tamoxifen resistance in postmenopausal breast cancer patients

IntroductionInhibitors of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway can overcome endocrine resistance in estrogen receptor (ER) α-positive breast cancer, but companion diagnostics indicating PI3K/AKT/mTOR activation and consequently endocrine resistance are lacking. PIK3CA mutations frequently occur in ERα-positive breast cancer and result in PI3K/AKT/mTOR activation in vitro. Nevertheless, the prognostic and treatment-predictive value of these mutations in ERα-positive breast cancer is contradictive. We tested the clinical validity of PIK3CA mutations and other canonic pathway drivers to predict intrinsic resistance to adjuvant tamoxifen. In addition, we tested the association between these drivers and downstream activated proteins.MethodsPrimary tumors from 563 ERα-positive postmenopausal patients, randomized between adjuvant tamoxifen (1 to 3 years) versus observation were recollected. PIK3CA hotspot mutations in exon 9 and exon 20 were assessed with Sequenom Mass Spectometry. Immunohistochemistry was performed for human epidermal growth factor receptor 2 (HER2), phosphatase and tensin homolog (PTEN), and insulin-like growth factor 1 receptor (IGF-1R). We tested the association between these molecular alterations and downstream activated proteins (like phospho-protein kinase B (p-AKT), phospho-mammalian target of rapamycin (p-mTOR), p-ERK1/2, and p-p70S6K). Recurrence-free interval improvement with tamoxifen versus control was assessed according to the presence or absence of canonic pathway drivers, by using Cox proportional hazard models, including a test for interaction.ResultsPIK3CA mutations (both exon 9 and exon 20) were associated with low tumor grade. An enrichment of PIK3CA exon 20 mutations was observed in progesterone receptor- positive tumors. PIK3CA exon 20 mutations were not associated with downstream-activated proteins. No significant interaction between PIK3CA mutations or any of the other canonic pathway drivers and tamoxifen-treatment benefit was found.ConclusionPIK3CA mutations do not have clinical validity to predict intrinsic resistance to adjuvant tamoxifen and may therefore be unsuitable as companion diagnostic for PI3K/AKT/mTOR inhibitors in ERα- positive, postmenopausal, early breast cancer patients.

Molecular and clinical prognostic factors in metastatic colorectal cancer (CRC) patients (pts): A retrospective study.

511 Background: Strong evidence is emerging about the usefulness of mutational profiling for CRC pts. This study aimed to evaluate the overall survival in three molecular groups, taking as reference the all-wild type category: (1) BRAF mutated; (2) KRAS mutated codons 12-13 only; (3) any of KRAS codons 61-146, PIK3CA exon 9-20 or NRAS cod 12-13-61 mutations. Also clinical variables were investigated as potential prognostic factors. Methods: Data of 194 consecutive pts treated for metastatic colorectal cancer (mCRC) at our University Hospital in Udine, Italy, between Jan 2004 and Jan 2013 were reviewed. Point mutations were detected by pyrosequencing platform PyroMark Q96 ID instrument for KRAS/NRAS codons 12, 13, 61, and 146, BRAF exon 15, and PIK3CA exons 9 and 20. Clinical and molecular prognostic factors were identified using the Cox proportional hazards model. Results: The all wild-type population consisted of 76 pts (39%). 62 cases (32%) harboured mutations in KRAS codons 12-13. BRAF V600E mutation was found in 10 (5.2%) samples. Mutations in KRAS 61-146, PIK3CA and NRAS codons were detected in 9 (4.6%), 32 (16.5%) and 6 (3.1%) pts, respectively. All factors significant in univariate analysis were subjected to multivariate analysis (see Table). The all-wild type category had the longest survival (27.7 months). Patients carrying BRAF mutations reported an overall survival of 7.6 months and those with KRAS 12-13 mutation 16.7 months. Conclusions: This study reinforces the prognostic value of a full mutational molecular profile and points out some prognostic clinical factors in CRC. The influence of clinical variables such as right colon cancer, primary tumour not resected, exposure to bevacizumab and lines of chemotherapy need further investigation. [Table: see text]

Mutations within the EGFR signaling pathway: Influence on efficacy in FIRE-3—A randomized phase III study of FOLFIRI plus cetuximab or bevacizumab as first-line treatment for wild-type (WT) KRAS (exon 2) metastatic colorectal cancer (mCRC) patients.

445 Background: The FIRE-3 study (AIO KRK-0306) was designed as a randomized multicenter trial to compare the efficacy of FOLFIRI plus cetuximab (cet) to FOLFIRI plus bevacizumab (bev) as first-line treatment in KRAS WT mCRC patients. FOLFIRI plus cet as first-line treatment of KRAS WT mCRC patients resulted in comparable overall response rates (ORR) and progression free survival (PFS) when compared to FOLFIRI plus bev. Overall survival (OS) was significantly longer in the FOLFIRI plus cet arm. Methods: In a preplanned analysis, the effect of mutations within the EGFR dependent pathway were investigated. Next to mutations within KRAS (exon 2, 3, 4), NRAS (exon 2, 3, 4) and BRAF (V600E), mutations within PIK3CA (exon 9 and 20) and Akt were investigated and their impact on ORR, PFS and OS within the FIRE-3 population was evaluated. The analysis of all mutations was carried out employing pyrosequencing. Results: The ITT population consisted of 592 KRAS WT (exon 2) patients. The current analysis includes 488 cases (82.4%) with tumor tissue available. In 407 pts sequencing of all RAS mutations was possible. The ORR within the WT RAS patient group was higher in the FOLFIRI plus cet arm (65.5% vs 59.6%; Fisher´s p: 0.157). HRs (cet; bev) for pts with WT RASwere 0.93 (95% CI, 0.74-1.17; p = 0.54) for PFS and 0.70 (95% CI, 0.53-0.92; p = 0.01) for OS. PIK3CA mutation did not influence PFS nor OS when compared to the RAS wt population. Conclusions: ORR and OS were increased in patients with cet plus FOLFIRI as compared to bev plus FOLFIRI in patients without RAS mutations. Exclusion of patients with RAS mutations identifies a population which is more likely to benefit from cetuximab. Clinical trial information: NCT00433927.

Associations between clinical characteristics and oncogene expression in patients with non-small cell lung cancer.

More than 40 oncogenes associated with non-small cell lung cancer (NSCLC) have been identified with varied gene expression. The correlations between specific clinical characteristics and oncogene expression in NSCLC patients were examined. From October 2011 to September 2012, a total of 60 patients with NSCLC (male:female, 34:24; mean age, 59.5 ± 10.6 years; age range, 31-81 years) were diagnosed and evaluated for treatment with radical resection at a single facility. Eligible patients exhibiting tumor node metastasis (TNM) stage I-III NSCLC confirmed by post-surgical pathology were included. mRNA expression was detected by branched DNA-liquidchip technology (bDNA-LCT) and mutations were detected at EGFR exons 18, 19, 20, and 21, KRAS exons 2 and 3, BRAF and PIK3CA exons 9 and 20. Correlations between gene expression at mutations and clinical characteristics of gender, age, histological type, degree of differentiation, smoking status, immunohistochemical (IHC) evaluation of TTF-1, TNM staging, and discrete age ("nage") were examined. Significant associations were observed between IHC staining for TTF-1 and histological type (P = 0.00001) and with BRAC1, TYMS, RRM1, and TUBB3 expression (P = 0.0187, 0.0051, 0.024, and 0.0238, respectively). Significant cross-correlations were observed between TYMS, BRAC1, TOP2A, STMN1, TUBB3, and RRM1 expression (P < 0.05), but not between EGFR exon 21, KRAS exon 2, and PIK3CA exon 9 expression and any other mutation expression (P > 0.05). Relationships between clinical characteristics and oncogene expression in NSCLC, particularly those of TTF-1 level and smoking status, may be useful indicators of prognosis and development of anti-cancer drug resistance.

Systematic review/Meta-analysis Anti-epidermal growth factor receptor monoclonal antibody-based therapy for metastatic colorectal cancer: a meta-analysis of the effect of PIK3CA mutations in KRAS wild-type patients

IntroductionWe conducted a meta-analysis to dissect the association between PIK3CA mutations (exon 9 and exon 20) and resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs) in KRAS wild-type metastatic colorectal cancer (mCRC) patients.Material and methodsIn 11 previously published studies, 864 cancer patients were treated with cetuximab or panitumumab-based therapy. Primary outcomes included objective response (complete response + partial response vs. stable disease + progressive disease), progression-free survival (PFS), and overall survival (OS). We calculated the odds ratio (OR) or hazard ratio (HR) with 95% confidence intervals (CIs) to estimate the risk or hazard. We found consistent and clinically substantial risk or hazard for objective response, PFS, and OS in the cetuximab or panitumumab-treated mCRC patients.ResultsPIK3CA mutations as a whole were associated with reduced response and poor PFS and OS in KRAS wild-type mCRC patients (objective response: OR = 0.42 and 95% CI 0.23–0.75; PFS: HR = 1.54 and 95% CI 1.13–2.09; and OS: HR = 1.4 and 95% CI 1.02–1.91). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with an OR of 0.21 (95% CI 0.05–0.93).ConclusionsPIK3CA mutations as a whole might be useful prognostic factors for assessing clinical outcomes of anti-EGFR MoAb-based chemotherapies in KRAS wild-type mCRC patients. In particular, PIK3CA exon 20 mutations were significantly associated with lack of response.

Abstract P1-04-04: Towards methodology of detecting PIK3CA mutations on single cell level by massive parallel sequencing

Abstract Background: The oncogene PIK3CA encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase. Gain-of-function mutations occur in 20 – 40% of the diagnosed breast cancer cases and may predict response to PI3K/AKT/mTOR inhibitors. We have developed a methodology to determine the PIK3CA exon 9 and 20 mutational status on single cell level, allowing analysis of low-input patient material such as circulating tumor cells (CTC). Methods: Single and groups of cultured breast cancer cells (BT20, MDA-MB-361 and MCF7) were isolated and recovered from spiked blood samples using the CellSearch and DEPArray platform respectively, and subjected to whole-genome amplification. Additionally, Formalin-fixed, paraffin-embedded (FFPE) tissue sections from 3 breast cancer patients were collected, of which two were mutant (MT) and one carried the wild-type (WT) genotype, as previously determined by external laboratories. Targeted PIK3CA PCR for exon 9 and 20 with unique multiplex identifiers (MIDs) allowed mixing of 32 samples and differentiation in the sequence data. Massive parallel sequencing was carried out using Titanium Amplicon chemistry (Lib-A) on a 454 GS Junior and data were analyzed using the amplicon variant analyzer (AVA) software. Results: A throughput of 120.464 high-quality reads with a mean coverage depth of 987 reads (range 288 – 5979) allowed the reliable assessment of variants, which were concordant with NCBI SNP/COSMIC databases and external laboratories. In BT20, the variants and frequencies were the missense P539R (48,83%) and H1047R (45,36%) mutations. Missense E545K mutation was detected in MDA-MB-361 (33,33%) and MCF7 (70,83%). Above mentioned mutations were detected in 73,3% and 100% of analyzed single and groups of tumor cells, respectively. Inability to determine the mutational status in some single cells was due to loss of amplicon during purification. In the MT FFPE tissue sections, two variants were detected and were concordant with previously obtained results. The variants and frequencies detected were E542K (14,12%) and E545K (21,38%), and correlated well with the tumor cell content of the actual sample (40% and 50%, respectively). Additionally, variant analysis showed 2 different SNPs in exon 20, which are classified as the synonymous mutations T1025T and A995A. In the WT sample no PIK3CA mutations were detected, suggesting a 100% specificity on the obtained 454 data. Conclusions: Our study delivers a semiautomated workflow using next-generation sequencing for the assessment of PIK3CA gain-of-function mutations on FFPE tissue sections and on single DEPArray purified cells from CellSearch pre-enriched whole blood samples. We are now implementing this workflow in the analysis of circulating tumor cells from patients with metastatic breast cancer. At the time of submitting, a cohort of 40 patients with luminal breast cancer was selected to assess the PIK3CA mutational status in the primary tumor, CTC and (if present) metastatic compartment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-04.

Effectors of Epidermal Growth Factor Receptor Pathway: The Genetic Profiling of KRAS, BRAF, PIK3CA, NRAS Mutations in Colorectal Cancer Characteristics and Personalized Medicine

Mutations in KRAS oncogene are recognized biomarkers that predict lack of response to anti- epidermal growth factor receptor (EGFR) antibody therapies. However, some patients with KRAS wild-type tumors still do not respond, so other downstream mutations in BRAF, PIK3CA and NRAS should be investigated. Herein we used direct sequencing to analyze mutation status for 676 patients in KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons12, 13 and 61). Clinicopathological characteristics associations were analyzed together with overall survival (OS) of metastatic colorectal cancer patients (mCRC). We found 35.9% (242/674) tumors harbored a KRAS mutation, 6.96% (47/675) harbored a BRAF mutation, 9.9% (62/625) harbored a PIK3CA mutation and 4.19% (26/621) harbored a NRAS mutation. KRAS mutation coexisted with BRAF, PIK3CA and NRAS mutation, PIK3CA exon9 mutation appeared more frequently in KRAS mutant tumors (P = 0.027) while NRAS mutation almost existed in KRAS wild-types (P<0.001). Female patients and older group harbored a higher KRAS mutation (P = 0.018 and P = 0.031, respectively); BRAF (V600E) mutation showed a higher frequency in colon cancer and poor differentiation tumors (P = 0.020 and P = 0.030, respectively); proximal tumors appeared a higher PIK3CA mutation (P<0.001) and distant metastatic tumors shared a higher NRAS mutation (P = 0.010). However, in this study no significant result was found between OS and gene mutation in mCRC group. To our knowledge, the first large-scale retrospective study on comprehensive genetic profile which associated with anti-EGFR MoAbs treatment selection in East Asian CRC population, appeared a specific genotype distribution picture, and the results provided a better understanding between clinicopathological characteristics and gene mutations in CRC patients.

Human Papillomavirus-Associated Adenocarcinoma of the Base of Tongue: Potentially Actionable Genetic Changes

While human papillomavirus (HPV)-positive squamous and adenosquamous carcinomas of the oropharynx have been well characterized, HPV-associated pure adenocarcinomas are exceptionally rare. Herein we report the clinicopathologic features of one such HPV-associated adenocarcinoma of the base of tongue (BOT). A 70year-old male presented with a 2.8cm base of tongue mass and lymphadenopathy. Immunohistochemically, the adenocarcinoma was p63 negative and p16 positive. HPV positivity was shown by in situ hybridization. Features of salivary type tumor or metastasis from a distant primary were absent. IonTorrent™ semiconductor sequencing analysis for 739 cancer-associated mutations in 46 actionable cancer genes was performed and PIK3CA exon 9 (p.E545K) and MET exon 2 (p.E168D) mutations were identified. No PIK3CA or MET amplification was identified by fluorescence in situ hybridization. A re-review of archival HPV-positive oropharyngeal squamous cell carcinomas (n=89, 1983-2013) showed no additional cases of adenocarcinoma. The clinical follow-up for the three previously reported cases of HPV-associated adenocarcinoma of the BOT was updated. All previously reported cases were tested and were negative for PIK3CA exon 9 and 20 and MET exon 2 mutations. These findings offer a molecular basis for potential therapeutic use of PIK3CA inhibitors in a subset of patients with HPV-associated adenocarcinoma of BOT.

PIK3CA missense mutation is associated with unfavorable outcome in grade 3 endometrioid carcinoma but not in serous endometrial carcinoma

ObjectiveTo evaluate the outcome association of PIK3CA mutational status within histological types of rigorously classified high-grade endometrial carcinomas. MethodsWe assessed PIK3CA mutational status in exon 9 and exon 20 hot spots by Sanger sequencing of DNA derived from formalin fixed paraffin embedded tissue of 57 grade 3 endometrioid, 26 serous, 11 clear cell and 5 dedifferentiated carcinomas. We correlated PIK3CA mutation status with clinicopathological and other molecular parameters. Univariate and multivariate disease specific survival analysis was performed using Kaplan–Meier and Cox regression analyses. ResultsPIK3CA exon 9 or exon 20 missense mutations were identified in 20 of 99 (20%) high-grade endometrial carcinomas without significant difference across histological types (p=0.22). Presence of PIK3CA exon 9 or exon 20 missense mutations was associated with shorter disease specific survival within grade 3 endometrioid (p=0.0029) but not endometrial serous (p=0.57) carcinoma based on univariate analysis. Within grade 3 endometrioid carcinoma, PIK3CA exon 9 or exon 20 missense mutations were more commonly observed in cases that were deficient for mismatch repair protein expression (p=0.0058) and showed loss of ARID1A expression (p=0.037). ConclusionsPIK3CA exon 9 or exon 20 missense mutations are present across all histological types of high-grade endometrial carcinomas but a significant outcome association is only seen in grade 3 endometrioid carcinoma, suggesting a greater biological importance in this tumor type.

Abstract B26: Actionable mutations in cell-free DNA in plasma of patients with advanced cancers referred for experimental targeted therapies.

Abstract Background: Actionable mutations confer a survival advantage to cancer cells and can provide therapeutic targets. Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable, low-risk, and inexpensive source of biologic material for mutation analysis. Methods: cfDNA in plasma samples from patients with advanced cancers who progressed on systemic therapy was tested for 21 mutations in BRAF (codon 600), EGFR (exons 19-21), KRAS (codons 12, 13), and PIK3CA (exons 9, 20) using the BEAMing method. Results were compared to mutation analysis results from archival primary or metastatic tumor tissue from a CLIA-certified laboratory. Results: cfDNA was extracted from 155 patients with advanced cancers. BRAF mutations were detected in 27% (33/122) plasma samples and in 30% (37/122) of archival tumor samples resulting in agreement in 88.5% cases (kappa=0.72, 95% confidence interval [CI] 0.58- 0.86). EGFR mutations were detected in 8% (6/77) plasma samples and in 6% (5/77) tumor samples resulting in agreement in 98.7% cases (kappa=0.90, 95% CI 0.71- 1.09). KRAS mutations were detected in 50% (55/110) of plasma samples and in 53% (58/110) of tumor samples, resulting in agreement in 82.7% cases (kappa=0.66, 95% CI 0.51- 0.80). PIK3CA mutations were detected in 20% (19/94) of plasma samples and in 14% (13/94) of tumor samples, resulting in concordance in 91.5% cases (kappa=0.70, 95% CI 0.51- 0.89). Of interest, 2 of 9 patients with BRAF mutations (both melanoma) in the tumor, but not in cfDNA had plasma collection at the time of progression on prior BRAF targeting therapy. Also, a patient with V600E BRAF-mutated melanoma, who previously progressed on a MEK inhibitor, displayed a G12R KRAS mutation in cfDNA and a patient with V600K BRAF-mutated melanoma with additional E542K and H1047R PIK3CA mutations in cfDNA was ultimately refractory to BRAF inhibitor treatment. Also, a V600K BRAF mutation was found in the cfDNA of a patient with colorectal cancer and a G12V KRAS mutation. In addition, 2 patients with EGFR-mutated lung adenocarcinoma (exon 19 and L858R), who progressed on erlotinib, had T790M EGFR (n=2) and E545K PIK3CA (n=1) mutations in cfDNA, plausibly explaining secondary resistance to erlotinib. A patient with Erdheim-Chester disease and a V600E BRAF mutation also had a T790M EGFR mutation in cfDNA. Finally, a patient with H1047R PIK3CA mutant ovarian cancer showed an additional G12C KRAS mutation in cfDNA and experienced a partial response followed by early progression on a PI3K inhibitor. Patient characteristics, mutation types and detailed discrepancy analysis will be presented. Conclusions: Detecting actionable mutations in cfDNA is a noninvasive alternative to mutation testing in tumor tissue with an acceptable level of concordance, and should be investigated further for selecting appropriate targeted therapies and monitoring mutation profiles. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B26. Citation Format: Filip Janku, Philipp Angenendt, Aung Naing, Gerald S. Falchook, Apostolia M. Tsimberidou, Veronica R. Holley, Siqing Fu, David S. Hong, Jennifer J. Wheler, Sarina A. Piha-Paul, Ralph G. Zinner, Daniel D. Karp, Vivek Subbiah, Kevin B. Kim, Funda Meric-Bernstam, Frank Diehl, Razelle Kurzrock. Actionable mutations in cell-free DNA in plasma of patients with advanced cancers referred for experimental targeted therapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B26.