Astrid Bosma

PIK3CA Mutations Are Associated With Decreased Benefit to Neoadjuvant Human Epidermal Growth Factor Receptor 2–Targeted Therapies in Breast Cancer

PURPOSE We investigated whether mutations in the gene encoding the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (PIK3CA) correlates with response to neoadjuvant human epidermal growth factor receptor 2 (HER2) -targeted therapies in patients with breast cancer. PATIENTS AND METHODS Baseline tissue biopsies were available from patients with HER2-positive early breast cancer who were enrolled onto the Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimization trial (NeoALTTO). Activating mutations in PIK3CA were identified using mass spectrometry-based genotyping. RESULTS PIK3CA mutations were identified in 23% of HER2-positive breast tumors, and these mutations were associated with poorer outcome in all of the treatment arms. Patients treated with a combination of trastuzumab and lapatinib who had wild-type PIK3CA obtained a total pathologic complete response (pCR) rate of 53.1%, which decreased to 28.6% in patients with tumors that carried PIK3CA activating mutations (P = .012). CONCLUSION Activating mutations in PIK3CA predicted poor pCR in patients with HER2-positive breast cancer treated with neoadjuvant therapies that target HER2. Consequently, the combination of anti-HER2 agents and PI3K inhibitors is being investigated.

Identification of recurrent FGFR3 fusion genes in lung cancer through kinome‐centred RNA sequencing

Oncogenic fusion genes that involve kinases have proven to be effective targets for therapy in a wide range of cancers. Unfortunately, the diagnostic approaches required to identify these events are struggling to keep pace with the diverse array of genetic alterations that occur in cancer. Diagnostic screening in solid tumours is particularly challenging, as many fusion genes occur with a low frequency. To overcome these limitations, we developed a capture enrichment strategy to enable high‐throughput transcript sequencing of the human kinome. This approach provides a global overview of kinase fusion events, irrespective of the identity of the fusion partner. To demonstrate the utility of this system, we profiled 100 non‐small cell lung cancers and identified numerous genetic alterations impacting fibroblast growth factor receptor 3 (FGFR3) in lung squamous cell carcinoma and a novel ALK fusion partner in lung adenocarcinoma.

Marker genes for circulating tumour cells predict survival in metastasized breast cancer patients

We investigated the prognostic significance of circulating breast cancer cells in peripheral blood detected by quantitative RT–PCR of marker genes in patients with advanced breast cancer. Blood samples from 94 breast cancer patients with metastatic disease (M1) were examined for circulating tumour cells by studying the mRNA expression of CK19, p1B, PS2 and EGP2 by real-time PCR. Using a score function, developed for predicting circulating tumour cells by quadratic discriminant analysis (QDA), the four expression levels were combined into a single discriminant value. Tumour cells were present in 24 out of 94 (31%) of the patients. In 77% (72 out of 94) of the patients distant metastatic disease was localised in the bone. In 36% (26 out of 72) of the patients with bone metastases at the time of blood sampling, a positive QDA for the four genes was found, in contrast to only 14% (three out of 22) without bone involvement. Overall survival rates by Kaplan–Meier revealed no prognostic effect for the presence of bone metastases (P=0.93). However, patients with a positive QDA value did have a progression-free survival at 1 year of 3% and overall survival at 2 years of 17%, against 22 and 36% for patients with a negative QDA value (P=0.015 and 0.0053, respectively). Breast cancer patients with metastatic disease have a significantly worse progression-free and overall survival when circulating tumour cells can be detected in their peripheral blood.

Detection of metastases in sentinel lymph nodes of breast cancer patients by multiple mRNA markers.

Disseminated breast tumour cells in sentinel lymph nodes (SNs) were evaluated by quantitative real-time PCR and the sensitivity of this assay was compared to the routine histological analysis. First, several candidate marker genes were tested for their specificity in axillary lymph nodes (ALN) of 50 breast cancer patients and 43 women without breast cancer. The marker gene panel selected, designed to detect the mRNA of CK19, p1B, EGP2 and SBEM, was subsequently applied to detect metastases in 70 SNs that were free of metastases as determined by standard histological evaluation. Remarkably, seven negative SNs showed increased marker gene expression, suggesting the presence of (micro) metastases. Four of these seven SNs positive by real-time PCR proved to contain tumour deposits after careful review of the slides or further sectioning of the paraffin-embedded material. In three PCR positive SNs, however, no tumour cells were found by haematoxylin and eosin staining (H&E) and immunohistologically analysis. The quantitative real-time PCR assay with multiple mRNA markers for the detection of disseminated breast cancer cells in SNs thus resulted in an upstaging of SNs containing metastastic disease of 10% compared to the routine histological analysis. The application of this technique may be of clinical relevance, as it is suggested that micrometastatic disease in SNs are associated with further nodal non-SN metastases in breast cancer.

No common denominator for breast cancer lymph node metastasis

The axillary lymph node status is the most powerful prognostic factor for breast cancer patients to date. The molecular mechanisms that control lymph node metastasis, however, remain poorly understood. To define patterns of genes or gene regulatory pathways that drive breast cancer lymph node metastasis, we compared the gene expression profiles of 15 primary breast carcinomas and their matching lymph node metastases using microarrays. In general, primary breast carcinomas and lymph node metastases do not differ at the transcriptional level by a common subset of genes. No classifier or single gene discriminating the group of primary tumours from those of the lymph node metastases could be identified. Also, in a series of 295 breast tumours, no classifier predicting lymph node metastasis could be developed. However, subtle differences in the expression of genes involved in extracellular-matrix organisation and growth factor signalling are detected in individual pairs of matching primary and metastatic tumours. Surprisingly, however, different sets of these genes are either up- or downregulated in lymph node metastases. Our data suggest that breast carcinomas do not use a shared gene set to accomplish lymph node metastasis.

Comparison of immunocytochemistry, reverse transcriptase polymerase chain reaction, and nucleic acid sequence-based amplification for the detection of circulating breast cancer cells.

Detection of tumor cells in blood and bone marrow is increasingly used for the staging of patients with breast cancer and to evaluate the presence of tumor cells in peripheral blood progenitor cell collections to be used after high–dose therapy. We evaluated the sensitivity and specificity of three different methods for detection of tumor cells among non–tumor tissue. An immunocytochemical assay using antibodies directed against epitopes of the cytokeratin–19 (CK19) protein and two RNA–based methods: reverse transcriptase polymerase chain reaction (RT–PCR) and Nucleic Acid Sequence–Based Amplification (NASBA) for the same target gene were tested. With all the three methods, false–positive results were observed when peripheral blood mononuclear cells (PBMC) of healthy volunteers were tested. There was no concordance between the RNA–based assays and the immunocytochemical assay. The false–positive results in the RNA–based assays may be due to ‘illegitimate expression’ of epithelial genes in normal PBMC. The false–positive results in the immunocytochemical assay resulted from background staining of monocytes and granulocytes. This study demonstrates that CK19 is not a suitable target to detect the presence of breast tumor tells in PBMC. To reliably detect circulating tumor cells with RNA methods, the selection of suitable target genes is required, which are highly expressed in tumors but not at all in normal cells of blood and bone marrow. Genes with such characteristics may be identifiable with novel differential display techniques.

The prognostic significance of tumour cell detection in the peripheral blood versus the bone marrow in 733 early-stage breast cancer patients

IntroductionThe detection of circulating tumour cells (CTCs) in the peripheral blood and disseminated tumour cells (DTCs) in the bone marrow are promising prognostic tools for risk stratification in early breast cancer. There is, however, a need for further validation of these techniques in larger patient cohorts with adequate follow-up periods.MethodsWe assayed CTCs and DTCs at primary surgery in 733 stage I or II breast cancer patients with a median follow-up time of 7.6 years. CTCs were detected in samples of peripheral blood mononuclear cells previously stored in liquid-nitrogen using a previously-developed multi-marker quantitative PCR (QPCR)-based assay. DTCs were detected in bone marrow samples by immunocytochemical analysis using anti-cytokeratin antibodies.ResultsCTCs were detected in 7.9% of patients, while DTCs were found in 11.7%. Both CTC and DTC positivity predicted poor metastasis-free survival (MFS) and breast cancer-specific survival (BCSS); MFS hazard ratio (HR) = 2.4 (P < 0.001)/1.9 (P = 0.006), and BCSS HR = 2.5 (P < 0.001)/2.3 (P = 0.01), for CTC/DTC status, respectively). Multivariate analyses demonstrated that CTC status was an independent prognostic variable for both MFS and BCSS. CTC status also identified a subset of patients with significantly poorer outcome among low-risk node negative patients that did not receive adjuvant systemic therapy (MFS HR 2.3 (P = 0.039), BCSS HR 2.9 (P = 0.017)). Using both tests provided increased prognostic information and indicated different relevance within biologically dissimilar breast cancer subtypes.ConclusionsThese results support the use of CTC analysis in early breast cancer to generate clinically useful prognostic information.

An α‐E‐catenin (CTNNA1) mutation in hereditary diffuse gastric cancer

Diffuse gastric cancers typically present as late‐stage tumours and, as a result, the 5 year survival rate is poor. Some gastric cancers are hereditary and these tend to be of the diffuse type; 30–40% of hereditary diffuse gastric cancers (HDGCs) can be explained by defective germline alleles of E‐cadherin (CDH1), but for the remaining families the factors driving susceptibility remain unknown. We had access to a large HDGC pedigree with no obvious mutation in CDH1, and applied exome sequencing to identify new genes involved in gastric cancer. We identified a germline truncating allele of α‐E‐catenin (CTNNA1) that was present in two family members with invasive diffuse gastric cancer and four in which intramucosal signet ring cells were detected as part of endoscopic surveillance. The remaining CTNNA1 allele was silenced in the two diffuse gastric cancers from the family that were available for screening, and this was also true for signet ring cells identified in endoscopic biopsies. Since α‐E‐catenin functions in the same complex as E‐cadherin, our results call attention to the broader signalling network surrounding these proteins in HDGC. We also detected somatic mutations in one tumour and found substantial overlap with genes mutated in sporadic gastric cancer, including PIK3CA, ARID1A, MED12 and MED23.

Integration of genomic, transcriptomic and proteomic data identifies two biologically distinct subtypes of invasive lobular breast cancer

Invasive lobular carcinoma (ILC) is the second most frequently occurring histological breast cancer subtype after invasive ductal carcinoma (IDC), accounting for around 10% of all breast cancers. The molecular processes that drive the development of ILC are still largely unknown. We have performed a comprehensive genomic, transcriptomic and proteomic analysis of a large ILC patient cohort and present here an integrated molecular portrait of ILC. Mutations in CDH1 and in the PI3K pathway are the most frequent molecular alterations in ILC. We identified two main subtypes of ILCs: (i) an immune related subtype with mRNA up-regulation of PD-L1, PD-1 and CTLA-4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone related subtype, associated with Epithelial to Mesenchymal Transition (EMT), and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using the somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization may help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies.

Insulin-Like Growth Factor (IGF)-System mRNA Quantities in Normal and Tumor Breast Tissue of Women with Sporadic and Familial Breast Cancer Risk

The insulin-like growth factor (IGF)-system plays a role in breast cancer susceptibility as well as in growth and progression of breast carcinomas. So far, findings have been based on serum IGF-I levels and semi-quantitative assessment of IGF-system expression levels in model systems and human tissue. Quantitative data on mRNA expression in different types of human breast tissue are lacking. Breast tissue samples (n= 83) were available from 72 women. Messenger RNA expression of IGF-I, IGF-II, and their receptors (IGF-1R and IGF-2R) was assessed by real-time RT-PCR. We found a large variation in mRNA levels. Expression of each gene was significantly higher in normal tissue than in tumor tissue (median for normal and tumor tissue, respectively (arbitrary units); IGF-I: 25.2 and 1.4; IGF-II: 5.9 and 0.6; IGF-1R: 0.18 and 0.07; IGF-2R: 1.8 and 0.9; p < 0.0001, Mann–Whitney test). Interestingly, in tumor tissue from patients with a strong family history of breast cancer, expression of both receptors was higher than in sporadic patients (IGF-1R: 0.13 and 0.05, p= 0.04; IGF-2R: 1.1 and 0.8, p= 0.04). For cancer-free controls, expression of IGF-II and IGF-2R in normal breast tissue was also higher in women with a family history of breast cancer than in women without such a family history (IGF-II: 7.2 and 1.5, p= 0.02; IGF-2R: 2.6 and 1.5, p= 0.09). Our study quantitatively shows that mRNA expression levels of IGF-system components in the breast are generally higher in normal tissue compared with tumor tissue, and higher in tissue from women with a family history of breast cancer. A basis has therefore been created for studies aimed at understanding IGF as a breast cancer risk factor, the relationship between IGF-systems in serum and tissues, and effects of lifestyle factors on the IGF-system.