PIK3CA Exon Research Articles

KRAS, NRAS, PIK3CA Exon 20, and BRAF Genotypes in Synchronous and Metachronous Primary Colorectal Cancers : Diagnostic and Therapeutic Implications

Targeted therapy of advanced colorectal carcinoma (CRC) necessitates KRAS genotyping. Because we were interested in diagnostic and therapeutic consequences, we studied the KRAS, NRAS, PIK3CA exon 20, and BRAF genotypes in synchronous and metachronous primary CRCs; in addition, we studied their available metastases. We studied 21 patients with 43 synchronous and 2 metachronous adenocarcinomas of the colorectum (n = 20) and stomach (n = 1). Five patients had liver metastases and one had a distant lymph node metastasis. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed by Sanger sequencing and pyrosequencing. Fifty-seven different neoplastic lesions were genotyped, showing 18 (31.6%) KRAS, 2 (3.5%) NRAS, and 7 (12.3%) BRAF mutations, distributed among 10 (47.6%), 1 (4.8%), and 5 (23.8%) of the patients. An identical genotype of all synchronous primary CRCs was found only in 7 (35%) of the patients; the remainder had dissimilar genotypes in various combinations. Interestingly, a single patient had an unknown KRAS genotype (c.37_39dupGGC). Six patients with 13 primary carcinomas had distant metastases. In three of these patients, the metastasis shared the genotype only with one of the primary tumors, because the other primary tumors had another genotype. Synchronous and metachronous primary CRCs of the same patient have variable KRAS, NRAS, and BRAF genotypes. When metastases occur in these patients, the genotype has diagnostic and therapeutic implications and should be determined from the simultaneous or metachronous distant metastases.

Abstract 3799: PIK3CA and BRAF mutational status and association with clinicopathological features in Iranian colonrectal cancer patients

Abstract Introduction:Colorectal cancer (CRC) is one of the most important malignancies worldwide and it is the third most common cancer amongst Males and the fourth amongst females in Iranian cancer patients. Somatic mutations of PIK3CA were described in CRCs with a frequency ranging from 13.6% to 32%.In CRCs, 3 mutational hot spots were described (E542K, E545K, and H1047R), covering about 80% of all PIK3CA mutations. Mutations of the B-RAF have been identified in colorectal cancer with a ranging from 5% to 22 %.in CRC, All of the mutations of B-RAF have been found in exons 11 and 15, which encode the kinase domain of this protein.V600E substitution in exon 15 covering about 90% of all B-raf mutations.The aim of this study was to identify PIK3CA gene mutations in exons 9 and 20 and B-Raf gene mutations in exon 11 and 15 among Iranian CRC patients, and to consider whether they are linked with the clinicopathological parameters. Materials and Methods: Patients and tissue specimens; Tissue samples were obtained from 27 consecutive patients with CRC (10 men and 17 women; age range, 32-88 years; with median age of 61.3 years). Histopathological examinations were performed, and all tumors were confirmed as adenocarcinoma.Mutational analysis of PIK3CA in tumor samples: Fresh tumors, containing at least 70% of neoplastic cells and their adjacents were extracted for genomic DNA using the QIAamp Mini kit (Qiagen in accordance with the manufacturer 'sinstructions. We searched for PIK3CA mutations in exons 9 and 20.PIK3CA exon 9 includes codons 542 and 545, PIK3CA exon 20 and B-Raf mutations in exons 11 and 15 where the large majority of mutations occur in these genes. The PI3KCA and B-Raf mutational analyses were made by means of PCR sequencing. Results and conclusion: 21 (77.77%) patients had adenocarcinomas and 6 (22.23%) had mucinous adenocarcinomas. patients had stage I, II and III or IV disease with14.76%, 44.44%and 40.8% respectively and histological grade I, II and III with 66.6%,25.92% and 7.4%. respectively. Among the 27 samples analyzed (21were primary tumors and 6were metastases). The frequency of mutations in PIK3CA were present in 11.1% (3 of27) of the samples (11.1% mutation in exon 9(c.1633G>A E545K and no mutation in exon 20). The frequency of mutation in B-Raf were present in 3.7%(1 of 27) of the samples(3.7% mutation in exon 15;V600E and no mutation in exon 9).The presence of PIK3CA and B-Raf mutation were not a significance associated with gender, histological grade, age, or cancer stage in patients with adenocarcinoma of the CRC(P>0.05).Although many study have been shown in CRCs, 3 mutational hot spots for PIK3CA (E542K, E545K, and H1047R) and 2 mutational hot spots for B-RAf, our study indicated mutations were just in exon 9(E542K) and exon 15(V600E) and more samples should be investigate in exon 9,20 and other exons of PIK3CA gene and exons of B-RAF gene among Iranian colorectal cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3799. doi:10.1158/1538-7445.AM2011-3799

Abstract 4410: Initiative for Molecular Profile and Advanced Cancer Therapy (IMPACT): A personalized medicine Phase I clinical trials program at MD Anderson Cancer Center

Abstract Introduction: Ongoing clinical trials are based on targeting specific pathways in addition to the tumor histology. We examined the results of mutational analyses performed in patients with advanced cancer seen in the Phase I clinic at The University of Texas MD Anderson Cancer Center. Methods: Mutational analysis was mostly performed in the Clinical Laboratory Improvement Amendments (CLIA)-certified pathology laboratory of MD Anderson. DNA was extracted from microdissected paraffin-embedded tumor samples, and analysis was performed on specific exons, depending on the test ordered, for the following genes: PIK3CA (exon 9: codons 532-554; exon 20: codons 1011-1062); BRAF (exon 15: codons 595 to 600); KRAS and NRAS (codons 12, 13, and 61); EGFR (exons 18 to 21 of the kinase domain); KIT (exons 9, 11, 13, and 17); and RET (exon 10: codons 609, 611, 618, and 620; codon 634 of exon 11; codon 918 of exon 16). The loss of the tumor suppressor nuclear protein, PTEN, was determined using immunohistochemical staining. Results. Tumor mutational analysis was ordered for 952 patients. Overall, 103 patients did not have adequate tissue. Of the remaining 849 patients, 354 (41.69%) had ≥ 1 mutation and 495 did not have a mutation. More women (45%) than men (38%) had a mutation (p = 0.02), but age was not associated with the presence of mutations (p=0.76). Of the patients with mutations, 313 had 1 mutation, 38 had 2 mutations, and 3 had 3 mutations. The distribution of mutations by diagnosis is shown in the Table. The total distribution of mutations in our patient population was as follows: BRAF, 19.01%; KRAS, 18.74%; PIK3CA, 9.85%; NRAS, 8.17%; EGFR, 3.38%; KIT, 2.13%; and PTEN loss, 21.20%. Conclusion: Testing for PIK3CA, KRAS, NRAS, BRAF, EGFR, KIT, and RET mutations and PTEN loss in 849 patients with available tissue demonstrated that molecular driver aberrations are common in advanced cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4410. doi:10.1158/1538-7445.AM2011-4410

Abstract 1287: Profile-related evidence to determine individualized cancer therapy (PREDICT): Preliminary results of the Personalized Phase I Clinical Trials program at MD Anderson Cancer Center

Abstract Introduction: To assess the outcomes of molecularly targeted therapy, we analyzed the results of mutational analyses in patients seen in our Phase I clinic and assessed patient survival by the number of mutations in patients treated with and without matched targeted therapy. Methods: Mutational analysis was mostly performed in the CLIA-certified pathology laboratory. DNA was extracted from microdissected paraffin-embedded tumor samples. The following genes were analyzed: PIK3CA (exon 9: codons 532-554; exon 20: codons 1011-1062); BRAF (exon 15: codons 595 to 600); KRAS and NRAS (codons 12, 13, 61); EGFR (exons 18 to 21, kinase domain); KIT (exons 9, 11, 13, 17); and RET (exon 10: codons 609, 611, 618, 620; exon 11: codon 634; exon 16: codon 918). The loss of the tumor suppressor nuclear protein (PTEN) was determined by immunohistochemistry. The allocation of patients to clinical trials varied according to protocol availability; eligibility; patient's prior response to therapy, toxicity and preference; and physician's choice. Patients whose tumor had a mutation were preferably treated with a matched targeted agent. Survival was compared between patients treated with and without matched targeted therapy. Results. Of 849 patients who had tissue available, 354 (41.7%) had ≥ 1 mutation (1, n=313; 2, n=38; 3, n=3) (Table 1). The median survival of patients with 0 vs. 1 vs. 2-3 mutations was 9.4 vs. 11.7 vs. 7.3 months, respectively (p =0.02; Table 2). In 278 evaluable patients with 1 mutation, the 2-yr survival rates were 42% and 17% (p = 0.02) for patients treated with and without matched targeted therapy. Conclusion: In patients with 1 mutation, treatment with matched targeted therapy was associated with superior survival compared to treatment without matching. Patients with 1 mutation had longer survival than those with no or >1 mutation, perhaps because most of them were treated with a matched targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1287. doi:10.1158/1538-7445.AM2011-1287

A Unique Spectrum of Somatic PIK3CA (p110α) Mutations Within Primary Endometrial Carcinomas

The goal of this study was to comprehensively define the incidence of mutations in all exons of PIK3CA in both endometrioid endometrial cancer (EEC) and nonendometrioid endometrial cancer (NEEC). We resequenced all coding exons of PIK3CA and PTEN, and exons 1 and 2 of KRAS, from 108 primary endometrial tumors. Somatic mutations were confirmed by sequencing matched normal DNAs. The biochemical properties of a subset of novel PIK3CA mutations were determined by exogenously expressing wild type and mutant constructs in U2OS cells and measuring levels of AKT(Ser473) phosphorylation. Somatic PIK3CA mutations were detected in 52.4% of 42 EECs and 33.3% of 66 NEECs. Half (29 of 58) of all nonsynonymous PIK3CA mutations were in exons 1-7 and half were in exons 9 and 20. The exons 1-7 mutations localized to the ABD, ABD-RBD linker and C2 domains of p110α. Within these regions, Arg88, Arg93, Gly106, Lys111, Glu365, and Glu453, were recurrently mutated; Arg88, Arg93, and Lys111 formed mutation hotspots. The p110α-R93W, -G106R, -G106V, -K111E, -delP449-L455, and -E453K mutants led to increased levels of phospho-AKT(Ser473) compared to wild-type p110α. Overall, 62% of exons 1-7 PIK3CA mutants and 64% of exons 9-20 PIK3CA mutants were activating; 72% of exon 1-7 mutations have not previously been reported in endometrial cancer. Our study identified a new subgroup of endometrial cancer patients with activating mutations in the amino-terminal domains of p110α; these patients might be appropriate for consideration in clinical trials of targeted therapies directed against the PI3K pathway.

Impact of KRAS, BRAF, and PIK3CA mutations, PTEN, AREG, and EREG expression, and skin rash in metastatic colorectal cancer patients treated with cetuximab-containing salvage treatment.

445 Background: To investigate the predictive significance of KRAS exon 2, BRAF V600E, PIK3CA exon 9 and 20 mutational status, AREG- EREG mRNA expression, PTEN protein expression and skin rash in patients with metastatic colorectal cancer (mCRC) treated with cetuximab containing salvage chemotherapy. Methods: Primary tumors from 112 mCRC patients were analyzed. The worst skin toxicity during treatment was recorded. Results: KRAS, BRAF and PIK3CA mutations were present in 37 (33%), 8 (7.2%) and 11 (9.8%) cases, respectively, PTEN was lost in 21 (19.8%) cases, AREG and EREG were overexpressed in 48 (45%) and 51 (49%) cases. In the whole study population, time to tumor progression (TTP) and overall survival (OS) was significantly lower in patients with KRAS (p=0.001 and p=0.026, respectively) or BRAF (p=0.001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p=0.018 and p=0.013, respectively) or EREG (p=0.002 and p=0.004, respectively) and in those with grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). In KRAS wt patients TTP and OS was significantly lower in patients with BRAF (p=0.0001 and p<0.0001, respectively) mutant tumors, downregulation of AREG (p=0.021 and p=0.004, respectively) or EREG (p=0.0001 and p<0.0001, respectively) and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively). TTP was significantly lower in patients with PIK3CA mutations (p=0.01) or lost PTEN (p=0.002). Multivariate analysis revealed KRAS (hazard ratio [HR] 4.3, p<0.0001), BRAF mutation (HR: 5.1, p<0.0001), EREG low mRNA expression (HR: 1.6, p=0.021) and absence of severe/moderate skin rash (HR: 4.0, p<0.0001) as independent prognostic factors for decreased TTP. Similarly, KRAS (HR 2.9, p=0.01), BRAF mutation (HR: 3.0, p=0.001), EREG low mRNA expression (HR: 1.7, p=0.021), absecence of severe/moderate skin rash (HR: 3.7, p<0.0001) and the presence of undifferantited tumours (HR: 2.2, p=0.001) were revealed as independent prognostic factors for decreased OS. Conclusions: These results underscore the potential of advanced CRCs genetic profiling in order to identify patients with different treatment response. No significant financial relationships to disclose.

Sessile Serrated Adenoma With Early Neoplastic Progression

Sessile serrated adenoma (SSA), also referred to as sessile serrated polyp, has been proposed as a precursor lesion to microsatellite unstable carcinoma. However, the mechanism of stepwise progression from SSA to early invasive carcinoma has been unclear. The purpose of this study was to elucidate the histologic characteristics and possible role of p53, β-catenin, BRAF, KRAS, and PIK3CA in the development and progression of SSA. We analyzed 12 cases of SSA with neoplastic progression (SSAN), including 7 cases of intraepithelial high-grade dysplasia (HGD) and 5 cases of submucosal invasive carcinoma, and compared them with 53 SSAs and 66 hyperplastic polyps (HPs) by immunohistochemistry and gene mutation analysis. Histologically, 75% (9 of 12) of SSANs showed tubular or tubulovillous growth patterns rather than serrated ones in the HGD/intramucosal carcinoma component. All 5 SSANs with invasive carcinoma lost their serrated structure and developed increased extracellular mucin in their submucosal carcinoma component, a consistent feature of mucinous adenocarcinoma. Nuclear accumulations of β-catenin and p53 were observed in 50% (6 of 12) and 41.7% (5 of 12) of SSANs, respectively, and were exclusively present in HGD/carcinoma areas. By contrast, neither nuclear β-catenin nor p53 expressions were seen in HPs or SSAs (P<0.0001). BRAF mutations (V600E) were observed in 45.8% (11 of 24) of HPs, 60.9% (14 of 23) of SSAs, and 63.6% (7 of 11) of SSANs, and were equally found in both SSA and carcinoma/HGD areas of the individual SSANs. KRAS exon 1 mutations were uncommon in all 3 groups (4.2%, 4.4%, and 0%, respectively). No mutations of PIK3CA exon 9 or exon 20 were found in any cases that were examined. These findings suggest that BRAF mutations may be associated with the pathogenesis of SSA, but progression to HGD or early invasive carcinoma may be associated with other factors, such as alterations of p53 and β-catenin. In addition, our histologic observations suggest a possible close association between SSAN and mucinous adenocarcinoma.

Molecular Markers for the Prediction of Anti-EGFR Monoclonal Antibody Treatment Efficacy in Metastatic Colorectal Cancer

The implementation of individualized targeted therapy for metastatic colorectal cancer (mCRC), in addition to standard chemotherapeutic regimens, currently is a topic under debate. Approximately 35% - 45% of mCRC patients exhibit mutated KRAS, which is considered to be an independent predictor of poor response to treatment with epidermal growth factor receptor (EGFR) monoclonal antibody. However, only about 50% of patients with wild-type KRAS respond to anti-EGFR therapy. Two major EGFR-dependent signaling pathways, RAS-RAF-MAPK and PI3K-PTEN-AKT, may be involved in the poor response to anti-EGFR. Increased EGFR gene copy number as detected by fluorescence in situ hybridization, but not increased EGFR protein expression, correlates with efficacy of anti-EGFR treatment. The identification of mutations in BRAF and PIK3CA (exon 20) and deletions in PTEN also may help clinicians screen for anti-EGFR resistance in mCRC patients with wild-type KRAS. To guide health professionals through the realm of individualized targeted therapies for mCRC, we review recent progress on identifying negative predictors and prognostic markers of anti-EGFR treatment efficacy.

Abstract P1-11-04: A Phase II Preoperative Trial of Concurrent Trastuzumab and Paclitaxel without Anthracycline in HER2-Positive Operable Breast Cancer

Abstract Background Concurrent trastuzumab with paclitaxel/fluorouracil, epirubicin, and cyclophosphamide (P/FEC) chemotherapy as neoadjuvant treatment revealed the high pathologic complete response (pCR) rate of 54.5% (Buzdar, 2007). Deregulation of the phosphatidylinositol 3-kinase (PI3K) pathway either through loss of PTEN or mutation of the catalytic subunit alpha of PI3K (PIK3CA) might confer resistance to trastuzumab. The objective of this study was to determine the efficacy of concurrent administration of trastuzumab and paclitaxel wtihout anthracycline as preoperative chemotherapy. The correlation between deregulation of PI3K and resistance to trastuzumab was also investigated. Patients &amp; methods Patients with HER2-positive, operable breast cancer received 12 cycles of weekly paclitaxel (80 mg/m2) and trastuzumab (4mg/kg loading dose then 2 mg/kg) for 12 weeks before surgery. PTEN status was evaluated by immunohistochemistry. PTEN staining intensity scores was recorded on an integer scale from 0 to 2+ (0; no staining, 1+; reduced staining, and 2+; equal staining as compared to the internal control). Sequencing of PIK3CA exons 9 and 20 was done by PCR amplification and direct sequencing. pCR was defined as no residual invasive carcinoma in the breast. Results Twenty-eight patients were enrolled and assessable for clinical and pathologic responses. The overall response rate was 92.9%, including a complete response in 13 patients and a partial response in 13 patients. The pCR rate was 53.6% (15/28). Twenty-three patients (82.1%) underwent breast concerving surgery. Progesteron receptor (PgR) status was significantly correlated with pCR (p=0.025). Eight of 24 patients (33.3%) were scored PTEN negative. PIK3CA mutations were identified in 4 of 13 patients (30.8%). There was no significant difference in pCR rate and PTEN loss/PIK3CA mutation. Correlation of PgR status and pCR Conclusions These data indicate that the combination of trastuzumab and paclitaxel without anthracycline is effective preoperative chemotherapy with high pCR rate. PTEN loss and/or PIK3CA mutation were not useful predictors of resistance to trastuzumab. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-11-04.

230 Essential role of fibroblast growth factor receptor 2 (FGFR2) in tumorigenesis of human cancers harboring FGFR2 amplification demonstrated by a functional blocking antibody

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.

76 Design and functional analysis of a novel hybrid TPR peptide targeting Hsp90

The phosphatidylinositol-3-kinases PI3K/AKT pathway regulates many growth and survival mechanisms in the cell, and it has been implicated in development and progression of many human cancers, including multiple myeloma. Recently, many reports have revealed that the PIK3CA gene which encodes the p110 catalytic subunit of PI3K kinase is mutated in many human malignancies. To investigate the oncogenic role of PI3K/AKT pathway in multiple myeloma we sequenced three hot exons: exons 9 and 20 of PIK3CA gene and exon 3 of AKT1 gene in 27 multiple myeloma patients.Our results indicate the absence of the four hot spot mutations E542K, E545K, H1047R and E17K in all studied cases. These findings suggest that PI3K/AKT mutations may not play a major role in multiple myeloma.

Therapeutic implications of resistance to molecular therapies in metastatic colorectal cancer

Metastatic colorectal cancer (mCRC) patients carrying KRAS mutated tumors do not benefit from epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies. Indeed, the mutational status of KRAS is currently a validated predictive biomarker employed to select mCRC patients for EGFR targeted drugs. When patients fail standard 5-fluorouracil-, oxaliplatin-, irinotecan- and bevacizumab-based therapies, EGFR-targeted salvage therapy can be prescribed only for those individuals with KRAS wild-type cancer. Thus, clinicians are now facing the urgent issue of better understanding the biology of KRAS mutant disease, in order to devise novel effective therapies in such defined genetic setting. In addition to KRAS, recent data point out that BRAF and PIK3CA exon 20 mutations hamper response to EGFR-targeted treatment in mCRC, potentially excluding from treatment also patients with these molecular alterations in their tumor. This review will focus on current knowledge regarding the molecular landscape of mCRC including and beyond KRAS, and will summarize novel rationally-developed combinatorial regimens that are being evaluated in early clinical trials.

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis

Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12 weeks versus 24 weeks (hazard ratio [HR] 1.98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00, 0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population. Belgian Federation against Cancer (Stichting tegen Kanker).

Activation of the PI3K pathway and prognostic information in epithelial ovarian cancer.

10610 Background: The PI3K/AKT/mTOR signaling pathway is a central network in a number of cancers, including EOC. Identifying associations between key molecules and clinical outcomes may help expose and develop therapeutic targets in this cancer. Methods: Clinical data, tissue microarrays (TMAs) and genomic DNA from 194 EOCs (100 serous, 58 endometrioid, and 34 clear cell) were obtained from the Australian Ovarian Cancer Study. These were analyzed for mutations in PIK3CA, AKT1, KRAS and BRAF using high resolution melt analysis and confirmed by sequencing. IHC for phosphorylated proteins (pAKT, pS6, p4EBP1) was performed on the TMAs and considered overexpressed if above the median value for the cohort. Statistical analyses included chi-squared and cox-multivariate tests and the Kaplan-Meier method for survival. Results: PIK3CA (exon 9 and 20) and KRAS (exon 2) mutations were most frequent in endometrioid (12% PIK3CA, 12% KRAS) and clear cell (8% PIK3CA, 8% KRAS) EOCs. Few mutations in these genes were found in serous subtypes (1%) and no AKT1 (exon 4) or BRAF (exon 15) mutations were found. Coexisting mutations in PIK3CA and KRAS occurred in two endometrioid tumors. There was a trend for endometrioid EOCs to have increased pAKT expression relative to other subtypes (p = 0.058). Furthermore, increased pAKT was associated with a significant improvement in survival of the whole group (HR 0.6 [p = 0.05]). This association was still evident but not statistically significant in a multivariate survival model accounting for tumor subtype (p = 0.175). There was a strong correlation between pS6 and p4EBP1 that are downstream of mTORC1 (p = < 0.01). Increased expression of p4EBP1 (p = 0.01) and pS6 (p = 0.02) was seen in serous and clear cell subtypes respectively, but did not affect clinical outcome. Conclusions: Mutations in genes and high expression of phosphoproteins related to the PI3K pathway are associated with specific subtypes of EOC and may contribute to differences in clinical outcome. Endometrioid EOC had a higher frequency of mutations in the pathway and higher expression of pAKT suggesting investigation of targeting PI3K or AKT is warranted. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Pfizer

Markers for EGFR pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab

Background Anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC) treatment are only effective in patients with KRAS wild type tumours. Here we assess the predictive value of other potential relevant markers involved in the epidermal growth factor receptor (EGFR) signalling pathways for response to cetuximab-based treatment. Materials and methods Formalin-fixed paraffin-embedded colorectal cancer tissue of the primary tumour was obtained from 559 mCRC patients treated with chemotherapy and bevacizumab with or without cetuximab (phase III CAIRO2 study). DNA was isolated for mutation analysis of BRAF (V600E) , KRAS (codon 12 and 13) and PIK3CA (exon 9 and 20). Tissue microarray’s (TMA’s) were constructed for the assessment of EGFR and HER2 gene copy number (GCN), and EGFR and PTEN protein expression. The results of these markers, individually or in combination, were correlated with progression-free survival (PFS) and overall survival (OS) in the subgroup of patients with a KRAS wild type tumour treated in the cetuximab-arm. KRAS wild type patients treated without cetuximab were used as a control group. Results A total of 208 tumours (39.4%) contained a KRAS mutation, 8.7% a BRAF mutation and 9.9% a PIK3CA mutation. Loss of PTEN expression and the presence EGFR protein expression were observed in 42.0% and 61.7% of the samples, respectively. An increased EGFR GCN was observed in 15.3% of the samples, and 11.5% of the evaluable samples contained an increased HER2 GCN. In KRAS wild type patients treated with cetuximab a BRAF mutation was significantly and independently associated with PFS and OS. In patients treated without cetuximab the PFS and OS were also associated with the BRAF genotype. No prognostic or predictive value was observed for any of the other markers when tested individually or in combination. Conclusions BRAF genotype is correlated with PFS and OS in KRAS wild type mCRC patients, which is independent of cetuximab treatment. PIK3CA mutation, loss of PTEN expression, EGFR GCN and HER2 GCN have no predictive value for response to treatment with cetuximab, neither individually nor in combination with other markers.

Abstract 2152: Genome-wide single nucleotide polymorphism arrays in endometrial carcinomas associate extensive chromosomal instability with poor prognosis and unveil prevalent alterations in Ras/PI3-kinase pathway

Abstract [Objectives]: Endometrial cancer is one of the tumor types, in which either chromosomal instability (CIN) or microsatellite instability (MSI) may occur, and is known to possess mutations frequently in Ras-PI3K (phosphatidylinositol 3’-kinase) pathway. In this study, we attempted to clarify prognostic impact of genomic instability, determined by CIN and MSI status, and figure out the relationship between alterations in the Ras-PI3K pathway genes and the status of genomic instability. [Materials and Methods]: Under the approval of institutional ethical committees, we analyzed allele-specific chromosomal copy number alterations by SNP typing arrays in 31 endometrioid endometrial adenocarcinomas with paired DNA (25 by the 50K and six by the 250K probes). We also screened 25 of the 31 samples for MSI status with five microsatellite markers, and 22 samples for mutations in PIK3CA (exon 9 and 20), PTEN (exons 1-8) and K-Ras (exon1 and 2). Survival curves were constructed using the Kaplan-Meier method and compared with a log-rank test. [Results]: We detected five or more copy number changes (classified as CIN-extensive) in nine (29%), one to four changes (CIN-intermediate) in 17 (55%) and no changes (CIN-negative) in five (16%) tumors. Positive MSI was detected in ten (40%) of the 25 tumors and was less common in CIN-extensive tumors (14%), compared with CIN-intermediate/negative tumors (50%). CIN-extensive was significantly poor prognostic (p=0.0034), and the prognosis was shown to be independent from any other clinicpopathological characteristics by multivariate analysis. In contrast, positive MSI showed a trend to be a favorable prognostic factor (p=0.069), in spite of the association with vascular invasion (p=0.049). SNP array analysis unveiled copy number neutral LOH at 54 loci in 13 tumors (42%), including four at the locus of PTEN, and homozygous deletions at five regions in three tumors, including the locus of PTEN and NF1. Totally we detected eight (26%) tumors with PTEN deletions, and four (13%) with NF1 deletions. We identified mutations of PTEN, PIK3CA and K-Ras in 16 (73%), 6 (27%) and 2 (9%) out of 22 tumors, respectively. Seven (78%) of the nine CIN-extensive tumors harbor deletions at the loci of PTEN and/or NF1, with less frequent PTEN mutations (22%). On the other hand, PTEN mutations were detected in all the ten MSI-positive tumors, five of which coexist with PIK3CA and/or K-Ras mutations. We detected no deletions of NF1 and only one (10%) LOH of PTEN in the ten MSI-positive tumors. [Conclusion]: Our results demonstrated that the degree of CIN is a useful biomarker for prognosis in endometrial carcinomas. In addition, genomic alterations in the Ras-PI3K pathway are remarkably widespread in endometrial carcinomas, regardless of the type of genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2152.

Mutational and Immunohistochemical Study of the PI3K/Akt Pathway in Papillary Thyroid Carcinoma in Greece

PI3K/Akt signaling pathway plays critical role in many cell processes. There is indication that enhanced activation of PI3K/Akt cascade is implicated in thyroid tumors. Aim of this study was to evaluate the mutational status and expression of PI3K/Akt pathway mediators in papillary thyroid carcinoma in Greece. We evaluated the presence of mutations in PIK3CA (exons 9 and 20), AKT1 (exons 6-11), AKT2 (exons 6-11), AKT3 (exons 5-10), PTEN (exons 3-8), and PDPK1 (exons 4-10) genes in 83 papillary thyroid carcinomas by DNA sequencing. The expression levels of phospho-Akt and insulin-like growth factor I receptor (IGF-IR) were evaluated by immunohistochemistry. PIK3CA mutations were found in three samples. The analysis of AKT1 revealed one silent mutation in exon 9 (G726A) in 16 samples. One specimen carried an AKT3 mutation. One missense mutation was found in one sample in PTEN. No mutations were found in AKT2 and PDPK1. Increased levels of phosphorylated total Akt and IGF-IR were identified in some papillary cancers. Our findings indicate that PI3K/Akt signaling pathway is activated in some papillary tumors. However, mutations in genes coding most mediators of the pathway have not been proven to be the major modus of enhanced activation. These data suggest a potential role for PI3K/Akt-mediated signaling in papillary thyroid tumors.

Rare Mutations in the PIK3CA Gene Contribute to Aggressive Endometrial Cancer

The molecular basis of endometrial cancer (EC), a common gynecologic malignancy, often includes mutational activation of the PIK3CA and KRAS genes. We aimed to determine the distribution of mutations in the two genes depending on patient clinocopathological characteristics. We sequenced exon 1 of the KRAS gene and exons 9 and 20 of the PIK3CA gene in 108 consecutive EC tumor samples. PIK3CA mutations were present in 24 of the 108 (22.2%) cases and KRAS mutations in 18 of the 108 (16.7%) cases. PIK3CA mutations occurred more frequently in KRAS-mutated samples (7/18, 38.9%; p = 0.06) than in KRAS wild type (17/90, 18.9%) and showed a very high frequency in metastatic tumors (4/9, 44.4%; p = 0.1) and in samples displaying serous differentiation-serous and mixed endometrioid/serous tumors (6/12, 50.0%; p = 0.021)-where KRAS mutations were rare (11.1% and 16.7%, respectively) and did not exist independently of a PIK3CA mutation. Non-hotspot (i.e., non-E542K, -E545K, and -H1047R) mutations in the PIK3CA gene showed higher frequency in metastatic cases (3/9, 33.3%; p = 0.05). Tumors displaying serous differentiation showed a particular pattern-they harbored exclusively mutations in PIK3CA exon 20 (5/12, 41.7%; p = 0.005) and most of these were non-hotspot (4/12, 33.3%; p = 0.02). In all other comparisons exons 9 and 20 mutation distribution did not differ. These results suggest the need for further exploration of the significance of PIK3CA mutations in respect to aggressive EC.

High prevalence of PIK3CA/AKT pathway mutations in papillary neoplasms of the breast

Papillary lesions of the breast have an uncertain relationship to the histogenesis of breast carcinoma, and are thus diagnostically and managerially challenging. Molecular genetic studies have provided evidence that ductal carcinoma in situ and even atypical ductal hyperplasia are precursors of invasive carcinoma. However, papillary lesions have been seldom studied. We screened papillary breast neoplasms for activating point mutations in PIK3CA, AKT1, and RAS protein-family members, which are common in invasive ductal carcinomas. DNA extracts were prepared from sections of 89 papillary lesions, including 61 benign papillomas (28 without significant hyperplasia; 33 with moderate to florid hyperplasia), 11 papillomas with atypical ductal hyperplasia, 7 papillomas with carcinoma in situ, and 10 papillary carcinomas. Extracts were screened for PIK3CA and AKT1 mutations using mass spectrometry; cases that were negative were further screened for mutations in AKT2, BRAF, CDK, EGFR, ERBB2, KRAS, NRAS, and HRAS. Mutations were confirmed by sequencing or HPLC assay. A total of 55 of 89 papillary neoplasms harbored mutations (62%), predominantly in AKT1 (E17K, 27 cases) and PIK3CA (exon 20 >exon 9, 27 cases). Papillomas had more mutations in AKT1 (54%) than in PIK3CA (21%), whereas papillomas with hyperplasia had more PIK3CA (42%) than AKT1 (15%) mutations, as did papillomas with atypical ductal hyperplasia (PIK3CA 45%, AKT1 27%, and NRAS 9%). Among seven papillomas with carcinoma in situ, three had AKT1 mutations. The 10 papillary carcinomas showed an overall lower frequency of mutations, including 1 with an AKT1 mutation (in a tumor arising from a papilloma), 1 with an NRAS gene mutation (Q61H), and 2 with PIK3CA mutations (1 overlapping with the NRAS Q61H). These findings indicate that approximately two-thirds of papillomas are driven by mutations in the PI3CA/AKT pathway. Some papillary carcinomas may arise from these lesions, but others may have different molecular origins.

Hotspot mutations of PIK3CA and AKT1 genes are absent in multiple myeloma

The phosphatidylinositol-3-kinases PI3K/AKT pathway regulates many growth and survival mechanisms in the cell, and it has been implicated in development and progression of many human cancers, including multiple myeloma. Recently, many reports have revealed that the PIK3CA gene which encodes the p110 catalytic subunit of PI3K kinase is mutated in many human malignancies. To investigate the oncogenic role of PI3K/AKT pathway in multiple myeloma we sequenced three hot exons: exons 9 and 20 of PIK3CA gene and exon 3 of AKT1 gene in 27 multiple myeloma patients. Our results indicate the absence of the four hot spot mutations E542K, E545K, H1047R and E17K in all studied cases. These findings suggest that PI3K/AKT mutations may not play a major role in multiple myeloma.