Juliane Hannemann

Changes in Gene Expression Associated With Response to Neoadjuvant Chemotherapy in Breast Cancer

PURPOSE At present, clinically useful markers predicting response of primary breast carcinomas to either doxorubicin-cyclophosphamide (AC) or doxorubicin-docetaxel (AD) are lacking. We investigated whether gene expression profiles of the primary tumor could be used to predict treatment response to either of those chemotherapy regimens. PATIENTS AND METHODS Within a single-institution, randomized, phase II trial, patients with locally advanced breast cancer received six courses of either AC (n = 24) or AD (n = 24) neoadjuvant chemotherapy. Gene expression profiles were generated from core-needle biopsies obtained before treatment and correlated with the response of the primary tumor to the chemotherapy administered. Additionally, pretreatment gene expression profiles were compared with those in tumors remaining after chemotherapy. RESULTS Ten (20%) of 48 patients showed a (near) pathologic complete remission of the primary tumor after treatment. No gene expression pattern correlating with response could be identified for all patients or for the AC or AD groups separately. The comparison of the pretreatment biopsy and the tumor excised after chemotherapy revealed differences in gene expression in tumors that showed a partial remission but not in tumors that did not respond to chemotherapy. CONCLUSION No gene expression profile predicting the response of primary breast carcinomas to AC- or AD-based neoadjuvant chemotherapy could be detected in this interim analysis. More subtle differences in gene expression are likely to be present but can only be reliably identified by studying a larger group of patients. Response of a breast tumor to neoadjuvant chemotherapy results in alterations in gene expression.

Classification of ductal carcinoma in situ by gene expression profiling.

IntroductionDuctal carcinoma in situ (DCIS) is characterised by the intraductal proliferation of malignant epithelial cells. Several histological classification systems have been developed, but assessing the histological type/grade of DCIS lesions is still challenging, making treatment decisions based on these features difficult. To obtain insight in the molecular basis of the development of different types of DCIS and its progression to invasive breast cancer, we have studied differences in gene expression between different types of DCIS and between DCIS and invasive breast carcinomas.MethodsGene expression profiling using microarray analysis has been performed on 40 in situ and 40 invasive breast cancer cases.ResultsDCIS cases were classified as well- (n = 6), intermediately (n = 18), and poorly (n = 14) differentiated type. Of the 40 invasive breast cancer samples, five samples were grade I, 11 samples were grade II, and 24 samples were grade III. Using two-dimensional hierarchical clustering, the basal-like type, ERB-B2 type, and the luminal-type tumours originally described for invasive breast cancer could also be identified in DCIS.ConclusionUsing supervised classification, we identified a gene expression classifier of 35 genes, which differed between DCIS and invasive breast cancer; a classifier of 43 genes could be identified separating between well- and poorly differentiated DCIS samples.

Concordance of clinical and molecular breast cancer subtyping in the context of preoperative chemotherapy response

ER, PR and HER2 status in breast cancer are important markers for the selection of drug therapy. By immunohistochemistry (IHC), three major breast cancer subtypes can be distinguished: Triple negative (TNIHC), HER2+IHC and LuminalIHC (ER+IHC/HER2−IHC). By using the intrinsic gene set defined by Hu et al. five molecular subtypes (BasalmRNA, HER2+mRNA, Luminal AmRNA, Luminal BmRNA and Normal-likemRNA) can be defined. We studied the concordance between analogous subtypes and their prediction of response to neoadjuvant chemotherapy. We classified 195 breast tumors by both IHC and mRNA expression analysis of patients who received neoadjuvant treatment at the Netherlands Cancer institute for Stage II–III breast cancer between 2000 and 2007. The pathological complete remission (pCR) rate was used to assess chemotherapy response. The IHC and molecular subtypes showed high concordance with the exception of the HER2+IHC group. 60% of the HER2+IHC tumors were not classified as HER2+mRNA. The HER2+IHC/Luminal A or BmRNA group had a low response rate to a trastuzumab-chemotherapy combination with a pCR rate of 8%, while the HER2+mRNA group had a pCR rate of 54%. The Luminal AmRNA and Luminal BmRNA groups showed similar degrees of response to chemotherapy. Neither the PR status nor the endocrine responsiveness index subdivided the ER+IHC tumors accurately into Luminal AmRNA and Luminal BmRNA groups. Molecular subtyping suggests the existence of a HER2+IHC/LuminalmRNA group that responds poorly to trastuzumab-based chemotherapy. For LuminalIHC and triple negativeIHC tumors, further subdivision into molecular subgroups does not offer a clear advantage in treatment selection.

Dynamic Contrast-Enhanced MRI for Prediction of Breast Cancer Response to Neoadjuvant Chemotherapy: Initial Results

OBJECTIVE The aim of this study was to establish changes in contrast-enhanced MRI of breast cancer during neoadjuvant chemotherapy that are indicative of pathology outcome. MATERIALS AND METHODS In 54 patients with breast cancer, dynamic contrast-enhanced MRI was performed before chemotherapy and after two chemotherapy cycles. Imaging was correlated with final histopathology. Multivariate analysis with cross-validation was performed on MRI features describing kinetics and morphology of contrast uptake in the early and late phases of enhancement. Receiver operating characteristic (ROC) analysis was used to develop a guideline that switches patients at high risk for incomplete remission to a different chemotherapy regimen while maintaining first-line therapy in 95% of patients who are not at risk (i.e., high specificity). RESULTS Change in largest diameter of late enhancement during chemotherapy was the single most predictive MRI characteristic for tumor response in multivariate analysis (A(z) [area under the ROC curve] = 0.73, p < 0.00001). Insufficient (< 25%) decrease in largest diameter of late enhancement during chemotherapy was most indicative of residual tumor at final pathology. Using this criterion, the fraction of unfavorable responders indicated by MRI was 41% (22/54). Approximately half (44%, 14/32) of the patients who showed favorable response at MRI achieved complete remission at pathology. Conversely, 95% (21/22) of patients who showed unfavorable response at MRI had residual tumor at pathology. CONCLUSION Reduction of less than 25% in largest diameter of late enhancement during neoadjuvant chemotherapy shows the potential to predict residual tumor after therapy with high specificity.

Changes in Keratin Expression during Metastatic Progression of Breast Cancer: Impact on the Detection of Circulating Tumor Cells

Purpose: Circulating tumor cells (CTC) might function as early markers for breast cancer metastasis or monitoring therapy efficacy. Enrichment and identification of CTCs are based on epithelial markers that might be modulated during epithelial–mesenchymal transition. Little is known about the expression of keratins in CTCs and whether all CTCs can be detected with antibodies directed against a limited panel of keratins. Experimental Design: Protein expression of keratin 2, 4–10, 13–16, 18, and 19 were assessed by a cocktail of antibodies (C11, AE1, AE3, and K7) and keratin antibodies C11 and A45-B/B3 alone in 11 breast cancer cell lines and 50 primary breast carcinomas and their lymph node metastases. Furthermore, CTCs were assessed in blood of 70 metastatic breast cancer patients. Results: Claudin-low cell lines did not show expression of normal breast epithelial keratins but were positive for K14 and K16, detected by the cocktail only. Primary breast carcinomas showed changes in keratin expression during metastatic progression to the lymph nodes. In 35 of 70 patients CTCs were identified, of which 83%, 40%, and 57% were identified by the cocktail, C11 and A45-B/B3, respectively. Identification of CTCs by the cocktail was associated with shorter survival (P < 0.01). In silico analyses revealed association between KRT16 expression and shorter relapse-free survival in metastatic breast cancer. Conclusion: Breast cancer cells show a complex pattern of keratin expression with potential biologic relevance. Individual keratin antibodies recognizing only a limited set of keratins inherit the risk to miss biologically relevant CTCs in cancer patients, and antibody cocktails including these keratins are therefore recommended. Clin Cancer Res; 18(4); 993–1003. ©2012 AACR.

Molecular subtypes of breast cancer and amplification of topoisomerase IIα: Predictive role in dose intensive adjuvant chemotherapy

Benefit from chemotherapy treatment in breast cancer patients is determined by the molecular make-up of the tumour. In a retrospective analysis, we determined the molecular subtypes of breast cancer originally defined by expression microarrays by immunohistochemistry in tumours of patients who took part in a randomised study of adjuvant high-dose chemotherapy in breast cancer. In addition, the topoisomerase IIα (TOP2A) amplification status was determined by fluorescence in situ hybridisation and chromogenic in situ hybridisation. 411 of the 753 tumours (55%) were classified as luminal-like, 137 (18%) as basal-like and 205 (27%) as human epithelial receptor type 2 (HER2) amplified. The basal-like tumours were defined as having no expression of ER and HER2; 98 of them did express epidermal growth factor receptor and/or cytokeratin 5/6. The luminal-like tumours had a significantly better recurrence free and overall survival than the other two groups. From the 194 HER2-positive tumours, 47 (24%) were shown to harbour an amplification of TOP2A. Patients with an HER2-amplified tumour randomised to the high-dose therapy arm did worse than those in the conventional treatment arm, possibly caused by the lower cumulative anthracycline dose in the high-dose arm. The tumours with a TOP2A amplification contributed hardly to this difference, suggesting that TOP2A amplification is not the cause of the steep dose–response curve for anthracyclines in breast cancer. Possibly, the difference of the cumulative dose of only 25% between the treatment arms was insufficient to yield a survival difference.

Indicators of homologous recombination deficiency in breast cancer and association with response to neoadjuvant chemotherapy

BACKGROUND Tumors with homologous recombination deficiency (HRD), such as BRCA1-associated breast cancers, are not able to reliably repair DNA double-strand breaks (DSBs) and are therefore highly sensitive to both DSB-inducing chemotherapy and poly (ADP-ribose) polymerase inhibitors. We have studied markers that may indicate the presence of HRD in HER2-negative breast cancers and related them to neoadjuvant chemotherapy response. PATIENTS AND METHODS Array comparative genomic hybridization (aCGH), BRCA1 promoter methylation, BRCA1 messenger RNA (mRNA) expression and EMSY amplification were assessed in 163 HER2-negative pretreatment biopsies from patients scheduled for neoadjuvant chemotherapy. RESULTS Features of BRCA1 dysfunction were frequent in triple-negative (TN) tumors: a BRCA1-like aCGH pattern, promoter methylation and reduced mRNA expression were observed in, respectively, 57%, 25% and 36% of the TN tumors. In ER+ tumors, a BRCA2-like aCGH pattern and the amplification of the BRCA2 inhibiting gene EMSY were frequently observed (43% and 13%, respectively) and this BRCA2-like profile was associated with a better response to neoadjuvant chemotherapy. CONCLUSIONS Abnormalities associated with BRCA1 inactivation are present in about half of the TN breast cancers but were not predictive of chemotherapy response. In ER+/HER2- tumors, a BRCA2-like aCGH pattern was predictive of chemotherapy response. These findings should be confirmed in independent series.

BRCA1 Loss Preexisting in Small Subpopulations of Prostate Cancer Is Associated with Advanced Disease and Metastatic Spread to Lymph Nodes and Peripheral Blood

Purpose: A preliminary study performed on a small cohort of multifocal prostate cancer (PCa) detected BRCA1 allelic imbalances among circulating tumor cells (CTC). The present analysis was aimed to elucidate the biological and clinical roles of BRCA1 losses in metastatic spread and tumor progression in PCa patients. Experimental Design: To map molecular progression in PCa outgrowth, we used fluorescence in situ hybridization analysis of primary tumors and lymph node sections, and CTCs from peripheral blood. Results: We found that 14% of 133 tested patients carried monoallelic BRCA1 loss in at least one tumor focus. Extended molecular analysis of chr17q revealed that this aberration was often a part of larger cytogenetic rearrangement involving chr17q21 accompanied by allelic imbalance of the tumor suppressor gene PTEN and lack of BRCA1 promoter methylation. The BRCA1 losses correlated with advanced T stage (P < 0.05), invasion to pelvic lymph nodes (P < 0.05), as well as biochemical recurrence (P < 0.01). Their prevalence was twice as high within 62 lymph node metastases (LNM) as in primary tumors (27%, P < 0.01). The analysis of 11 matched primary PCa-LNM pairs confirmed the suspected transmission of genetic abnormalities between these two sites. In four of seven patients with metastatic disease, BRCA1 losses appeared in a minute fraction of cytokeratin- and vimentin-positive CTCs. Conclusions: Small subpopulations of PCa cells bearing BRCA1 losses might be one confounding factor initiating tumor dissemination and might provide an early indicator of shortened disease-free survival. Clin Cancer Res; 16(13); 3340–8. ©2010 AACR.

Genomic signature of BRCA1 deficiency in sporadic basal-like breast tumors.

About 10–20% of all breast carcinomas show a basal‐like phenotype, while ∼ 90% of breast tumors from BRCA1‐mutation carriers are of this subtype. There is growing evidence that BRCA1‐mutated tumors are not just a specific subset of the basal‐like tumors, but that (the majority of) basal‐like tumors show a dysfunctional BRCA1 pathway. This has major treatment implications, because emerging regimens specifically targeting DNA repair mechanisms would then be most effective against these tumors. To further understand the involvement of BRCA1 deficiency in sporadic basal‐like tumors, we investigated 41 basal‐like tumors for BRCA1 mRNA expression by quantitative real‐time polymerase chain reaction, BRCA1 promoter methylation, their genomic profile by array‐CGH, and gene expression levels by whole genome expression arrays. Array‐CGH results were compared to those of 34 proven BRCA1‐mutated tumors. Basal‐like tumors were subdivided into two equal groups: deficient and proficient in BRCA1 gene expression. The chromosomal makeup of BRCA1 deficient sporadic basal‐like tumors was similar to that of BRCA1‐mutated tumors. BRCA1 proficient sporadic basal‐like tumors were more similar to nonbasal‐like tumors. Only half of the basal‐like breast tumors are actually deficient in BRCA1 expression. Gain of chromosome arm 3q is a marker for BRCA1 deficiency in hereditary and sporadic breast tumors.

Abstract PD07-07: Indicators of Homologous Recombination Deficiency in Breast Cancer and Association with Response to Neoadjuvant Chemotherapy

Background: Tumors with homologous recombination (HR) deficiency are highly sensitive to DNA double strand break (DSB) inducing agents, such as alkylating agents and poly (ADP-ribose) polymerase (PARP)-inhibitors. BRCA1 or BRCA2- mutated tumors, which are HR deficient, have characteristic DNA gains and losses that can be assessed by an array Comparative Genomic Hybridization (aCGH) classifier, one for BRCA1 mutations and one for BRCA2 mutations. We have studied these aCGH profiles together with several other HR deficiency indicators in sporadic breast cancers and we have correlated their presence to neoadjuvant chemotherapy response. Material and Methods: A total of 163 HER2-negative pre-treatment biopsies were examined, procured from sporadic breast cancer patients scheduled to receive neoadjuvant therapy with doxorubicin and cyclophosphamide. Triple negative (TN) and estrogen receptor positive (ER+/HER2-) tumors were analyzed separately. aCGH was performed to assess BRCA1-like and BRCA2-like profiles. In addition, BRCA1 promoter methylation, BRCA1 mRNA expression and amplification of the BRCA2-inhibiting gene EMSY were analyzed. Response to neoadjuvant treatment was assessed by measuring pathological complete remission (pCR) and near pCR at the time of surgery. Results: Inactivation of BRCA1 was frequent in TN tumors: 57% of these tumors showed a BRCA1-like profile at aCGH. BRCA 1 promoter methylation and reduced BRCA1 mRNA expression were observed in 25% and 36% of the TN tumors, respectively. The BRCA1-like aCGH profile was not clearly associated with a better neoadjuvant treatment response (58% vs. 48%, p=0.47). In ER+ tumors, a BRCA2-like aCGH profile and the amplification of the BRCA2 inhibiting gene EMSY were frequently observed (43% and 13% respectively). A BRCA2-like aCGH profile was associated with a significantly higher response rate (35% vs. 14%, p=0.014). EMSY amplification and a BRCA2-like aCGH profile occurred together in only one case, suggesting mutual exclusivity. EMSY was not associated with treatment response, questioning the role of EMSY in HR deficiency. Conclusion: Alterations associated with BRCA1 inactivation are present in about half of the TN breast cancers, but were not predictive of chemotherapy response. In ER+/HER2- tumors, the BRCA2-like aCGH profile predicts sensitivity to DSB-inducing chemotherapy, and possibly as well to new targeted agents, such as the PARP inhibitors. After validation in independent series the aCGH classifiers may lead to a diagnostic test that could assist in neoadjuvant treatment selection. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD07-07.