Low PTEN levels and <Emphasis Type="Italic">PIK3CA</Emphasis> mutations predict resistance to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2 over-expressing breast cancer

Purpose

Aberrant activation of the PI3K pathway has been implicated in resistance to HER2-targeted therapy, but results of clinical trials are confounded by the co-administration of chemotherapy. We investigated the effect of perturbations of this pathway in breast cancers from patients treated with neoadjuvant anti-HER2-targeted therapy without chemotherapy.

Patients and methods

Baseline tumor samples from patients with HER2-positive breast cancer enrolled in TBCRC006 (NCT00548184), a 12-week neoadjuvant clinical trial with lapatinib plus trastuzumab [plus endocrine therapy for estrogen receptor (ER)-positive tumors], were assessed for PTEN status by immunohistochemistry and PIK3CA mutations by sequencing. Results were correlated with pathologic complete response (pCR).

Results

Of 64 evaluable patients, PTEN immunohistochemistry and PIK3CA mutation analysis were performed for 59 and 46 patients, respectively. PTEN status (dichotomized by H-score median) was correlated with pCR (32% in high PTEN vs. 9% in low PTEN, p = 0.04). PIK3CA mutations were identified in 14/46 tumors at baseline (30%) and did not correlate with ER or PTEN status. One patient whose tumor harbored a PIK3CA mutation achieved pCR (p = 0.14). When considered together (43 cases), 1/25 cases (4%) with a PIK3CA mutation and/or low PTEN expression levels had a pCR compared to 7/18 cases (39%) with wild-type PI3KCA and high PTEN expression levels (p = 0.006).

Conclusion

PI3K pathway activation is associated with resistance to lapatinib and trastuzumab in breast cancers, without chemotherapy. Further studies are warranted to investigate how to use these biomarkers to identify upfront patients who may respond to anti-HER2 alone, without chemotherapy.

     

Breast cancer risk models: a comprehensive overview of existing models,validation, and clinical applications

Purpose

Inflammatory breast cancer is an aggressive form of breast cancer that shows distinct clinical features from non-inflammatory breast cancer. Genomic understanding of inflammatory breast cancer will shed light on biological targets for this disease. Our objective was to identify targeted hotspot mutations using multiplex genome sequencing in inflammatory breast cancer and compare the findings with those for patients with non-inflammatory breast cancer to further recognize novel targets.

Methods

We studied 400 patients with metastatic breast cancer who had somatic hotspot mutation testing using a 46- or 50-gene multiplex platform from March 2012 to December 2014. Among this population, 24 patients had inflammatory breast cancer and 376 patients had non-inflammatory breast cancer. We tested a total of 26 samples from 24 patients with inflammatory breast cancer.

Results

The average number of mutations per patient was higher in inflammatory breast cancer than in non-inflammatory breast cancer (1.23 vs. 0.65, respectively). Identified somatic mutations in inflammatory breast cancer were TP53 (n = 18, 75%), PIK3CA (n = 10, 41.7%), and ERBB2 (n = 4, 16.7%). TP53 and ERBB2 mutations were significantly more prevalent in inflammatory breast cancer than in non-inflammatory breast cancer (P < 0.01). All patients with ERBB2 mutations had hormone receptor (HR)+ primary tumors.

Conclusions

TP53, PIK3CA, and ERBB2 were detected as three major somatic mutations in metastatic inflammatory breast cancer patients. While the inflammatory breast cancer TP53 and PIK3CA mutations mirrored previously reported data for metastatic non-inflammatory breast cancer, this is the first report of higher frequency of ERBB2 mutation in inflammatory breast cancer, especially in the HR+ subtype. Once validated in a larger cohort of inflammatory breast cancer patients, this novel finding could lead to development of treatments for HR+ inflammatory breast cancer.

     

<Emphasis Type="Italic">PIK3CA</Emphasis> mutations and <Emphasis Type="Italic">TP53</Emphasis> alterations cooperate to increase cancerous phenotypes and tumor heterogeneity

Background/purpose

The combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies.

Experimental design/Methods

We used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies.

Results

Compared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic.

Conclusions

This cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.

     

The Prognostic Influence of BRAF Mutation and other Molecular,Clinical and Laboratory Parameters in Stage IV Colorectal Cancer

Our aim was to evaluate the predictive and prognostic influence of BRAF mutation and other molecular, clinical and laboratory parameters in stage IV colorectal cancer (CRC). 60 patients were included in this retrospective analysis, and 17 variables were examined for their relation with treatment response and survival. KRAS mutation was identified in 40.3 % of cases, BRAF and PIK3CA in 8.8 % and 10.5 % respectively. 29.8 % of patients responded to treatment. Median survival time was 14.3 months. Weight loss, fever, abdominal metastases, blood transfusion, hypoalbuminaimia, BRAF and PIK3CA mutations, CRP and DNA Index were associated with survival. In multivariate analysis, male patients had 3.8 times higher probability of response, increased DNA Index was inversely correlated with response and one unit raise of DNA Index augmented 6 times the probability of death. Our findings potentiate the prognostic role of BRAF, PIK3CA mutations and ploidy in advanced CRC.     

APOBEC3B is an enzymatic source of molecular alterations in esophageal squamous cell carcinoma

APOBEC3B belongs to the cytidine deaminase apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3) family of enzymes and induces C to T transitions of target DNA by cytidine deamination. Recently, several mutations in various cancers have been linked to APOBEC3B, suggesting a crucial role for this protein in carcinogenesis and cancer development. However, the significance of APOBEC3B in esophageal squamous cell carcinoma (ESCC) remains uncertain. In addition, the APOBEC3B immunoreactivity in cancer tissues is uncertain. Recently, we have demonstrated that PIK3CA mutation and the methylation level of long interspersed nucleotide element 1 (LINE-1) (a surrogate marker of global DNA methylation level) are prognostic markers and have crucial role on malignancy in ESCC patients. This study aims to clarify the impact of APOBEC3B on the clinical, pathological, and molecular features of ESCC. We evaluated APOBEC3B expression in ESCC and investigated the relationships among the immunoreactivity of APOBEC3B, clinical and pathological features, and the molecular features of ESCC (PIK3CA mutation, p53 expression, and LINE-1 methylation level). The immunoreactivity and mRNA level of APOBEC3B were significantly higher in cancer tissues than in noncancerous esophageal mucosae (P = 0.050). APOBEC3B expression was significantly correlated with PIK3CA mutation (P = 0.013), particularly with C to T transitions of PIK3CA (P = 0.041). Moreover, a high expression of APOBEC3B was significantly associated with LINE-1 hypomethylation (P = 0.027). Given the crucial roles of PIK3CA mutation and LINE-1 methylation levels, our findings might provide new insights into the biological mechanisms of ESCC tumorigenesis and progression.     

Genetic landscape of meningioma

Meningioma is the most common intracranial tumor, arising from arachnoid cells of the meninges. Monosomy 22 and inactivating mutations of NF2 are well-known genetic alterations of meningiomas. More recently, mutations in TRAF7, AKT1, KLF4, SMO, and PIK3CA were identified by next-generation sequencing. We here reviewed 553 meningiomas for the mutational patterns of the six genes. NF2 aberration was observed in 55 % of meningiomas. Mutations of TRAF7, AKT1, KLF4, PIK3CA, and SMO were identified in 20, 9, 9, 4.5, and 3 % of cases, respectively. Altogether, 80 % of cases harbored at least one of the genetic alterations in these genes. NF2 alterations and mutations of the other genes were mutually exclusive with a few exceptions. Clinicopathologically, tumors with mutations in TRAF7/AKT1 and SMO shared specific features: they were located in the anterior fossa, median middle fossa, or anterior calvarium, and most of them were meningothelial or transitional meningiomas. TRAF7/KLF4 type meningiomas showed different characteristics in that they occurred in the lateral middle fossa and median posterior fossa as well as anterior fossa and median middle fossa, and contained a secretory meningioma component. We also discuss the mutational hotspots of these genes and other genetic/cytogenetic alterations contributing to tumorigenesis or progression of meningiomas.     

Comparison of <Emphasis Type="Italic">KRAS</Emphasis> mutation status between primary tumor and metastasis in Chinese colorectal cancer patients

Detection of KRAS mutation status is a routine clinical procedure for predicting response to anti-EGFR therapy in colorectal cancer (CRC) patients. Previous studies showed high concordance of KRAS mutation status in primary lesion and corresponding metastatic sites in CRC. However, the data were mostly from Caucasians. The aim of this study is to compare KRAS mutation and other molecules mutation status between primary tumor and corresponding metastatic lesion in Chinese patients with CRC. In this retrospective study, Chinese CRC patients with paired samples of primary tumor and metastatic site were detected for KRAS codon 12 and 13 with quantitative real-time PCR, or detected for OncoCarta? panel of 19 genes with MassARRAY^® technique, including KRAS, BRAF, NRAS and PIK3CA et al. Forty-eight paired CRC samples were analyzed for KRAS codon 12 and 13 using quantitative real-time PCR. Ten paired samples were analyzed by 19 genes OncoCarta? Panel with MassARRAY^® technique. KRAS mutation was found in 15 (25.9 %) primary tumors and 18 (31.0 %) metastases. The discordance of KRAS was observed in 11 (19.0 %) patients. Alteration of mutation points in primary site with mutant KRAS was not observed. In the 10 patients with multiple gene detection, PIK3CA mutation showed concordant mutation status in primary tumor and metastatic site, whereas discordance in BRAF, NRAS and AKT1 was detected. A concordance rate of 81.0 % was detected in KRAS mutation between primary tumor and metastatic lesion in Chinese patients with CRC. Discordance of BRAF, NRAS and AKT1 mutation status in primary tumor and metastases was observed.     

Hotspot mutations in PIK3CA associate with first-line treatment outcome for aromatase inhibitors but not for tamoxifen

PIK3CA mutations occur frequently in breast cancer, predominantly in exons 9 and 20. The aim of this retrospective study is to evaluate the PIK3CA mutation status for its relationship with prognosis and first-line endocrine therapy outcome. PIK3CA exon 9 and 20 were evaluated for mutations in 1,352 primary breast cancer specimens by SnaPshot multiplex analyses. The mutation status was studied for their relationship with metastasis-free survival (MFS) in 342 untreated lymph node-negative (LNN) patients and to time to progression (TTP) in estrogen receptor (ER)-positive patients with metastatic disease treated with first-line tamoxifen (N = 447) or aromatase inhibitors (AIs; N = 84). We detected in 423 patients hotspot mutations for PIK3CA (31 %). Mutations in exon 20 were detected in 251 patients (59 %), with H1047L and H1047R mutations in 37 (15 %) and 214 (85 %) cases, respectively. Mutations in PIK3CA exon 9 were discovered in 173 patients (41 %), with E542K and E545K mutations in 57 (32 %) and 104 (60 %) cases as most prevalent ones. Evaluation of the untreated LNN patients for prognosis showed no relationship between MFS and PIK3CA mutations, neither for exon 9 [HR = 1.04 (95 % CI 0.57–1.89), P = 0.90] nor for exon 20 [HR = 0.98 (95 % CI 0.63–1.54); P = 0.94] when compared to wild-type. The PIK3CA mutation status was also not associated with treatment outcome after first-line tamoxifen. On the other hand, patients treated with first-line AIs showed a longer TTP when having a PIK3CA mutation in exon 9 [HR = 0.40 (95 % CI 0.17–0.95); P = 0.038] or exon 20 [HR = 0.50 (95 % CI 0.27–0.91); P = 0.024] compared to wild-types, both significant in uni- and multivariate analysis including traditional predictive factors. All results remained when only HER2-negative patients were evaluated for each cohort. PIK3CA mutations in ER-positive tumors were significantly associated with a favorable outcome after first-line AIs, which needs further confirmation in other datasets. Mutations were not associated with prognosis in untreated LNN patients nor predictive outcome after first-line tamoxifen therapy in advanced disease patients.     

<Emphasis Type="Italic">APOBEC3B</Emphasis> high expression status is associated with aggressive phenotype in Japanese breast cancers

Background

The members of AID/APOBEC protein family possess cytidine deaminase activity that converts cytidine residue to uridine on DNA and RNA. Recent studies have shown the possible influence of APOBEC3B (A3B) as DNA mutators of breast cancer genome. However, the clinical significance of A3B expression in Japanese breast cancer has not been studied in detail.

Methods

Ninety-three primary breast cancer tissues (74 estrogen-receptor (ER) positive, 3 ER and HER2 positive, 6 HER2 positive, and 10 triple negative) including 37 tumor-normal pairs were assessed for A3B mRNA expression using quantitative real-time RT-PCR. We analyzed the relation between A3B expression, mutation analysis of TP53 and PIK3CA by direct sequencing, polymorphic A3B deletion allele and human papillomavirus (HPV) infection in tumors.

Results

A3B mRNA was overexpressed in tumors compared with normal tissue. Patients with high A3B expression were associated with subtype and progression of lymph node metastasis and pathological nuclear grade. However, the expression was not related to any other clinicopathological factors, including mutation of TP53 and PIK3CA, polymorphic A3B deletion allele, HPV infection and survival time.

Conclusion

The expression of A3B in breast cancer was higher than in non-cancerous tissues and was related to the lymph node metastasis and nuclear grade, which are reliable aggressive phenotype markers in breast cancer. Evaluation of A3B expression in tumor may be a marker for breast cancer with malignant potential.

     

PTEN loss is associated with a poor response to trastuzumab in HER2-overexpressing gastroesophageal adenocarcinoma

Background

Although trastuzumab improves the outcome of patients with human epidermal growth factor receptor 2 (HER2)-overexpressing gastric or gastroesophageal junction adenocarcinoma (collectively referred to as “gastroesophageal adenocarcinoma”; GEA), no clinical response is observed in a substantial population of patients. A predictive biomarker of trastuzumab response is required. The aim of this study was to evaluate whether the hyperactivation of the downstream phosphatidylinositol 3-kinase pathway, due to phosphatase and tensin homolog (PTEN) loss or PIK3CA mutations, could provide trastuzumab resistance in GEA.

Methods

Expression of HER2 and PTEN, and PIK3CA gene mutations were screened in 264 surgically resected GEA specimens. The effects of PTEN knockdown on the response to trastuzumab on cell viability, HER2 downstream signaling, apoptosis, and cell cycle were evaluated in HER2-overexpressing NCI-N87 gastric adenocarcinoma and OE19 esophageal adenocarcinoma cell lines. Inhibition of xenograft tumor growth by trastuzumab was investigated in OE19 cells with or without PTEN knockdown. The PTEN expression and objective response were analyzed in 23 GEA patients who received trastuzumab-based therapy.

Results

PTEN loss was identified in 34.5 % of HER2-overexpressing GEA patients, whereas PIK3CA mutations were rare (5.6 %). Trastuzumab-mediated growth suppression, apoptosis, and G₁ cell cycle arrest were inhibited by PTEN knockdown through Akt activation in NCI-N87 and OE19 cells. PTEN knockdown impaired the antiproliferative effect of trastuzumab in OE19 xenograft models. A clinical response was observed in 50 % of PTEN-positive tumors (9 of 18) but in no tumors with PTEN loss (none of 5).

Conclusions

PTEN loss was frequently found in HER2-overexpressing tumors, and was associated with a poor response to trastuzumab-based therapy in patients with GEA.

     

PIK3CA mutation in Chinese patients with lung squamous cell carcinoma

Objective

To investigate PIK3CA mutation in Chinese patients with lung squamous cell carcinoma (LSCC) and explore their relationship with clinicopathological profiles.

Methods

Tumor samples from 123 cases of LSCC were included in this study. PIK3CA mutations in exon 9 and 20 were screened by pyrosequencing and confirmed by clone sequencing or amplification refractory mutation system (ARMS). Denaturing performance liquid chromatography (DHPLC) was employed for evaluation of EGFR mutation in exon 19, 21 and KRAS mutation.

Results

PIK3CA mutations were found in 3 (2.4%) patients. The mutation type included E545K, E452Q and H1047R. Of these three patients, one coupled with EGFR mutation, and the other two coupled with PIK3CA amplification. All the three patients shared the same clinicopathologic characteristics: male, less than 60 years old, had smoke history, stage III and carried wild-type KRAS.

Conclusions

The frequency of PIK3CA mutation is low in Chinese patients with LSCC. The mutational status of PIK3CA is not mutually exclusive to EGFR mutation.Key Words: Lung squamous cell carcinoma (LSCC), PIK3CA mutation, EGFR mutation, KRAS mutation     

Revisiting breast cancer patients who previously tested negative for <Emphasis Type="Italic">BRCA</Emphasis> mutations using a 12-gene panel

Purpose

BRCA mutations contribute to about 20% of all hereditary breast cancers. With full-genome sequencing as the emerging standard for genetic testing, other breast cancer susceptibility genes have been identified and may collectively contribute to up to 30% of all hereditary breast cancers. We re-assessed women who had previously tested negative for a BRCA mutation when outdated techniques were used, and discuss the implications of identifying a mutation several years after initial genetic testing.

Methods

We evaluated the prevalence of mutations in 12 breast cancer susceptibility genes (including BRCA1 and BRCA2) in 190 breast cancer patients with a strong family history of breast cancer. These women had previously tested negative for mutations in the large coding exons of BRCA1 and BRCA2 using the protein truncation test (PTT) between the years of 1996 and 2013.

Results

We identified pathogenic mutations in 17 of 190 (9%) women. Six mutations were detected in BRCA1 (n = 2) and BRCA2 (n = 4). Eleven mutations were found in other breast cancer susceptibility genes including CHEK2 (n = 5), PALB2 (n = 2), BLM (n = 2), ATM (n = 1) and TP53 (n = 1).

Conclusion

Among 190 breast cancer patients with a family history of the disease, and who previously received a negative result for BRCA mutations using the PTT, 17 (9%) women were found to carry a high-risk pathogenic mutation in a breast cancer susceptibility gene. Six of these women were BRCA mutation carriers who were missed previously. These findings support the rationale for updated genetic testing in patients who tested BRCA mutation negative using outdated techniques.

     

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients

The frequently altered phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is involved in the regulation of cellular processes required for breast carcinogenesis. The aim of the project was to develop a method to identify hotspot mutations in the PIK3CA gene in circulating tumor cells (CTCs) of metastatic breast cancer (metBC) patients.From 44 enrolled CTC-positive metBC patients a total number of 57 peripheral blood samples were analysed by CellSearch^®. Genomic DNA of enriched CTCs was isolated, amplified and analyzed for PIK3CA mutations in exons 9 and 20 which lead to E542K, E545K or H1047R amino acid changes and result in increased PI3K activity. The mutations were detected by using SNaPshot-methodology comprising PCR amplification and single nucleotide primer extension.SNaPshot analysis was established using genomic DNA from different breast cancer cell lines and then successfully transferred to investigate blood samples and single cells. Overall, twelve hotspot mutations in either exon 9/E545K (6/12, 50%) or exon 20/H1047R (6/12, 50%) could be determined within 9 out of 57 (15.8%) blood samples from 7 out of 44 (15.9%) patients; CTC counts ranged from 1 to 9748. PIK3CA variants E542K, E545G and E545A were not detected.Analysing the PIK3CA genotype of CTCs has clinical relevance with respect to drug resistance, e.g. against HER2-targeted therapy. The herein described approach including SNaPshot technology provides a simple method to characterize hotspot mutations within CTCs enriched from peripheral blood and can be easily adopted for analysing further therapeutically relevant SNPs.     

Phosphatidylinositol-3-kinase and AKT1 mutations occur early in breast carcinoma

Mutationally activated protein kinases are appealing therapeutic targets in breast carcinoma. Mutations in phosphatidylinositol-3-kinase (PI3KCA) have been described in 8–40% of invasive breast carcinomas, and AKT1 mutations have been characterized in 1–8% of breast carcinomas. However, there is little data on these mutations in breast precursor lesions. To further delineate the molecular evolution of breast tumorigenesis, samples of invasive breast carcinoma with an accompanying in situ component were macro dissected from formalin-fixed paraffin embedded tissue and screened for mutations in PIK3CA exons 7, 9, 20, and AKT1 exon 2. Laser capture micro dissection (LCM) was performed on mutation-positive carcinomas to directly compare the genotypes of separated invasive and in situ tumor cells. Among 81 cases of invasive carcinoma, there were eight mutations in PIK3CA exon 20 (7 H1047R, 1 H1047L) and four mutations in exon 9 (2 E545K, 1 E542K, 1 E545G), totaling 12/81 (14.8%). In 11 cases examined, paired LCM in situ tumor showed the identical PIK3CA mutation in invasive and in situ carcinoma. Likewise, 3 of 78 (3.8%) invasive carcinomas showed an AKT1 E17K mutation, and this mutation was identified in matching in situ carcinoma in both informative cases. Mutational status did not correlate with clinical parameters including hormone receptor status, grade, and lymph node status. The complete concordance of PIK3CA and AKT1 mutations in matched samples of invasive and in situ tumor indicates that these mutations occur early in breast cancer development and has implications with regard to therapeutics targeted to the PI3 kinase pathway.     

Sensitive methods for screening of the <Emphasis Type="Italic">MEK1</Emphasis> gene mutations in patients with central nervous system metastases of non-small cell lung cancer

Background

The mitogen-activated protein kinases 1 and 2 (MEK1, MEK2) are fundamental partners in the RAS–RAF–MEK–ERK pathway that is involved in regulation of cell proliferation, differentiation and survival. Downregulation of the MEK cascades has been implicated in acquiring of the malignant phenotype in various cancers. Somatic mutations in MEK1 gene (substitutions K57N, Q56P, D67N) were described in <1 % of non-small cell lung cancer (NSCLC) and they were more commonly reported in adenocarcinoma patients with current or former smoking status.

Materials and methods

In the following study, we assessed the MEK1 gene mutations in 145 FFPE tissue samples from central nervous system (CNS) metastases of NSCLC using HRM-PCR and ASP-qPCR techniques. The studied group was heterogeneous in terms of histopathology and smoking status. The prevalence of the MEK1 gene mutation was correlated with the occurrence of mutations in KRAS, EGFR, DDR2, PIK3CA, NRAS, HER2, AKT1 and PTEN genes.

Results

Using HRM and ASP-qPCR methods we identified one (0.7 %; 1/145) MEK1 substitution (Q56P) in CNS metastases of NSCLC. The mutation was identified in a single, 50-year-old, current smoking men with adenocarcinoma (1.25 %; 1/80 of all adenocarcinomas).

Conclusions

According to the current knowledge, the incidence of MEK1 gene mutation in CNS metastatic lesion of NSCLC is the first such report worldwide. The analysis of gene profile in cancer patients may extend the scope of molecularly targeted therapies used both in patients with primary and metastatic tumors of NSCLC.

     

CpG island methylator phenotype is associated with the efficacy of sequential oxaliplatin- and irinotecan-based chemotherapy and EGFR-related gene mutation in Japanese patients with metastatic colorectal cancer

Background

The CpG island methylator phenotype (CIMP) with multiple promoter methylated loci has been observed in a subset of human colorectal cancer (CRC) cases. CIMP status, which is closely associated with specific clinicopathological and molecular characteristics, is considered a potential predictive biomarker for efficacy of cancer treatment. However, the relationship between the effect of standard chemotherapy, including cytotoxic drugs and anti-epidermal growth factor receptor (EGFR) antibodies, and CIMP status has not been elucidated.

Methods

In 125 metastatic colorectal cancer (mCRC) patients, we investigated how clinical outcome of chemotherapy was related to CIMP status as detected by methylation-specific PCR (MSP) and to genetic status in five EGFR-related genes (KRAS, BRAF, PIK3CA, NRAS, and AKT1) as detected by direct sequencing.

Results

CIMP-positive status was significantly associated with proximal tumor location and peritoneum metastasis (all P values <0.05). The progression-free survival of patients with CIMP-positive tumors receiving sequential therapy with FOLFOX as the first-line treatment followed by irinotecan-based therapy as the second-line treatment (median = 6.6 months) was inferior to that of such patients receiving the reverse sequence (median = 15.2 months; P = 0.043). Furthermore, CIMP-positive tumors showed higher mutation frequencies for the five EGFR-related genes (74.1 %) than the CIMP-negative tumors did (50.0 %). Among the KRAS wild-type tumors, CIMP-positive tumors were associated with a worse clinical outcome than CIMP-negative tumors following anti-EGFR antibody therapy.

Conclusion

Sequential FOLFOX followed by an irinotecan-based regimen is unfavorable in patients with CIMP-positive tumors. High frequencies of mutation in EGFR-related genes in CIMP-positive tumors may cause the lower response to anti-EGFR antibody therapy seen in patients with wild-type KRAS and CIMP-positive tumors.

     

PIK3CA mutation impact on survival in breast cancer patients and in ERα, PR and ERBB2-based subgroups

Introduction

PIK3CA is the oncogene showing the highest frequency of gain-of-function mutations in breast cancer, but the prognostic value of PIK3CA mutation status is controversial.

Methods

We investigated the prognostic significance of PIK3CA mutation status in a series of 452 patients with unilateral invasive primary breast cancer and known long-term outcome (median follow-up 10 years).

Results

PIK3CA mutations were identified in 151 tumors (33.4%). The frequency of PIK3CA mutations differed markedly according to hormone receptor (estrogen receptor alpha [ER??] and progesterone receptor [PR]) and ERBB2 status, ranging from 12.5% in the triple-negative subgroup (ER-/PR-/ERBB2-) to 41.1% in the HR+/ERBB2- subgroup. PIK3CA mutation was associated with significantly longer metastasis-free survival in the overall population (P = 0.0056), and especially in the PR-positive and ERBB2-positive subgroups. In Cox multivariate regression analysis, the prognostic significance of PIK3CA mutation status persisted only in the ERBB2-positive subgroup.

Conclusions

This study confirms the high prevalence of PIK3CA mutations in breast cancer. PIK3CA mutation is an emerging tumor marker which might become used in treatment-choosing process. The independent prognostic value of PIK3CA mutation status in ERBB2-positive breast cancer patients should be now confirmed in larger series of patients included in randomized prospective ERBB2-based clinical trials.     

Mutational analysis of triple-negative breast cancers within the International Breast Cancer Study Group (IBCSG) Trial 22-00

Purpose

We investigated the occurrence and the prognostic and predictive relationship of a selected number of somatic mutations in triple-negative breast cancer (TNBC) patients having known clinical outcomes treated within the IBCSG Trial 22-00.

Methods

A matched case–control sampling selected patients enrolled in the IBCSG Trial 22-00 who had TNBC tumors, based on local assessment. Cases had invasive breast cancer recurrence (at local, regional, or distant site) according to the protocol definition. Matched controls had not recurred. Mutational analysis was performed with OncoCarta panel v1.0 using Mass Array System. The panel includes 19 genes belonging to different functional pathways as PI3K pathway, receptor tyrosine kinase, and cell cycle-metabolic group. Conditional logistic regression assessed the association of mutation status with breast cancer recurrence.

Results

Mutation assessment was successful for 135 patients (49 cases, 86 controls). A total of 37 (27.4%) of the 135 patients had at least one mutation in the selected genes. PIK3CA was the most common mutated gene (18/135; 13.3%), followed by BRAF, KIT and PDGFRA (each 4/135, 3.0%) and AKT1 (3/135; 2.2%). TNBC patients with at least one mutation had increased odds of recurrence compared with those with wild-type tumors (odds ratio (OR) 2.28; 95% CI 0.88–5.92), though this difference was not statistically significant (p?=?0.09). We found no evidence that these mutations were predictive for the value of maintenance metronomic chemotherapy.

Conclusions

Mutations in the tested oncogenes were not associated with breast cancer recurrence in this TNBC subset of patients. The question of whether any of these mutated genes (e.g., PIK3CA) may represent a useful therapeutic target in TNBC may be answered by ongoing clinical trials and/or larger dataset analysis.

     

Clinical usefulness of KRAS, BRAF, and PIK3CA mutations as predictive markers of cetuximab efficacy in irinotecan- and oxaliplatin-refractory Japanese patients with metastatic colorectal cancer

Background

Anti-epidermal growth factor receptor (EGFR) antibodies, cetuximab, and panitumumab are established as a new treatment option for metastatic colorectal cancer (mCRC). Among activating mutations downstream of EGFR, the KRAS mutation, which is present in 30–45 % of CRC patients, has shown to be a predictive biomarker of resistance to anti-EGFR antibody therapy based on Caucasian studies.

Methods

Forty-three chemotherapy-refractory Japanese patients with mCRC were treated with cetuximab monotherapy or cetuximab plus irinotecan. KRAS, BRAF, and PIK3CA mutational status of tumors was assessed. The association between mutational status and treatment outcome was evaluated.

Results

Of 43 tumors, KRAS, BRAF, and PIK3CA mutations were identified in 12 (27.9 %), 2 (4.7 %), and 2 (4.7 %) tumors, respectively. The wild-type KRAS subgroup showed better clinical outcomes than the mutant KRAS subgroup in terms of response rate (RR) (31.3 % vs. 0 %, P = 0.034) and progression-free survival (PFS) (5.1 vs. 3.0 months, P = 0.017). No responder to treatment was shown in 16 (37.2 %) patients with tumors harboring mutations in any one of the three genes (KRAS, BRAF, and PIK3CA). The wild-type subgroup without any mutations in KRAS, BRAF, and PIK3CA had a better RR (37.0 %) and PFS (6.4 months) than did the wild-type KRAS subgroup.

Conclusion

Our data indicated that KRAS status is predictive of cetuximab response in the Japanese population. The additional analysis of BRAF and PIK3CA genes in wild-type KRAS patients could improve selection of patients who are most likely to benefit from anti-EGFR antibody therapy.