No abstract

Endocrine therapy is important for management of patients with estrogen receptor (ER)-positive breast cancer; however, positive ER staining does not reliably predict therapy response. We assessed the potential to improve prediction of response to endocrine treatment of a novel test that quantifies functional ER pathway activity from mRNA levels of ER pathway-specific target genes. ER pathway activity was assessed on datasets from three neoadjuvant-treated ER-positive breast cancer patient cohorts: Edinburgh: 3-month letrozole, 55 pre-/2-week/posttreatment matched samples; TEAM IIa: 3-to 6-month exemestane, 49 pre-/28 posttreatment paired samples; and NEWEST: 16-week fulvestrant, 39 pretreatment samples. ER target gene mRNA levels were measured in fresh-frozen tissue (Edinburgh, NEWEST) with Affymetrix microarrays, and in formalin-fixed paraffin-embedded samples (TEAM IIa) with qRT-PCR. Approximately one third of ER-positive patients had a functionally inactive ER pathway activity score (ERPAS), which was associated with a nonresponding status. Quantitative ERPAS decreased significantly upon therapy (P < 0.001 Edinburgh and TEAM IIa). Responders had a higher pretreatment ERPAS and a larger 2-week decrease in activity (P ¼ 0.02 Edinburgh). Progressive disease was associated with low baseline ERPAS (P ¼ 0.03 TEAM IIa; P ¼ 0.02 NEWEST), which did not decrease further during treatment (P ¼ 0.003 TEAM IIa). In contrast, the staining-based ER Allred score was not significantly associated with therapy response (P ¼ 0.2). The ERPAS identified a subgroup of ER-positive patients with a functionally inactive ER pathway associated with primary endocrine resistance. Results confirm the potential of measuring functional ER pathway activity to improve prediction of response and resistance to endocrine therapy.

Folate receptor alpha (FRα) is known to be upregulated in a variety of cancers, including non-small cell lung cancer (NSCLC) and breast cancer. To ensure reliable implementation of diagnostic- and therapeutic agents, concordance of FRα expression between biopsy, primary tumor and metastases is important. Using immunohistochemistry (Mab 26B3.F2) these concordances were investigated in 60 NSCLC and 40 breast cancer patients. False positivity of FRα expression on breast and lung cancer biopsies was limited to less than 5%. In NSCLC, FRα expression was shown in 21/34 adenocarcinomas and 4/26 squamous cell carcinomas (SCC). Concordance of FRα expression between biopsy and primary tumor was achieved in respectively 83% and 91% of adenocarcinomas and SCCs. Approximately 80% of all local and distant metastases of NSCLC patients showed concordant FRα expression as their corresponding primary tumor. In breast cancer, FRα positivity was shown in 12/40 biopsies, 20/40 lumpectomies and 6/20 LN metastases, with concordance of 68% between biopsy and primary tumor and 60% between primary tumor and LN metastases. In conclusion, this study shows high concordance rates of FRα expression between biopsies and metastases compared to primary NSCLC and breast cancers, underscoring the applicability of FRα-targeted agents in these patients.

Circulating tumor cells (CTC) are detected by the CellSearch System in 20% to 25% of patients with primary breast cancer (pBC). To improve CTC detection, we investigated melanoma cell adhesion molecule (MCAM) as enrichment marker next to epithelial cell adhesion molecule (EpCAM) and tested the clinical relevance of MCAM-positive CTCs in patients with HER2-negative stage II/III pBC starting neoadjuvant chemotherapy (NAC) in the NEOZOTAC trial. Using the CellSearch System, EpCAM-positive and MCAM-positive CTCs were separately enriched from 7.5 mL blood, at baseline and after the first NAC cycle. Circulating endothelial cells (CEC) were measured using flow cytometry. Primary objective was to improve the CTC detection rate to !40% combining EpCAM/MCAM. Correlations of CTC and CEC counts and pathologic complete response (pCR) were also explored. At baseline, we detected EpCAM-positive and MCAM-positive CTCs in 12 of 68 (18%) and 8 of 68 (12%) patients, respectively. After one cycle, this was 7 of 44 (16%) and 7 of 44 (16%) patients, respectively. The detection rate improved from 18% at baseline and 16% after one cycle with EpCAM to 25% (P ¼ 0.08) and 30% (P ¼ 0.02), respectively, with EpCAM/ MCAM. No patients with MCAM-positive CTCs versus 23% of patients without MCAM-positive CTCs at baseline achieved pCR (P ¼ 0.13). EpCAM-positive CTCs and CEC counts were not correlated to pCR. Combined EpCAM/MCAM CellSearch enrichment thus increased the CTC detection rate in stage II/III pBC. We found no associations of CTC and CEC counts with pCR to NAC. The clinical relevance of MCAM-positive CTCs deserves further study.

Introduction The tumor‐associated stroma is of importance for tumor progression and is generally accepted to have a significant influence on patient prognosis. However, little is known regarding specific features of tumor‐associated stromal tissues and response to (neoadjuvant) chemotherapy. This study investigated the predictive value of extracellular matrix organization on response to chemotherapy in patients treated in the NEOZOTAC trial. Methods Stromal organisation was analyzed via a simple method using image analysis software on hematoxylin and eosin (H&E)‐stained slides from primary tumor biopsies collected as part of the NEOZOTAC trial. Heidenhain's AZAN trichrome‐stained slides were also analyzed for comparison of collagen evaluation. Sections were stained for phospho‐Smad2 (pS2) in order to determine the relationship of TGF‐β signaling with stromal organization. Results A statistically significant relationship was observed between stroma consisting of organised collagen and pathological response to neoadjuvant chemotherapy (Odds Ratio 0.276, 95%CI 0.124–0.614, P = 0.002). This parameter was also related to ER‐status (P = 0.003), clinical tumor ‐status (P = 0.041), nodal status (P = 0.029) and pS2 status (P = 0.025). Correlation between stromal organisation determined on H&E‐stained and AZAN‐stained tissue sections was high (Pearson's correlation coefficient = 0.806). Conclusion Intratumoral stromal organisation determined using pre‐treatment breast cancer biopsies was related to pathological response to chemotherapy. This parameter might play a role in the management of breast cancer for identifying those patients that are likely to benefit from neoadjuvant chemotherapy.