Barbara Mayer

De-novo expression of CD44 and survival in gastric cancer

We have examined the cell surface molecule CD44, which is attracting interest because of reports that isoforms are associated with metastasis. The prognostic value of CD44 expression has yet to be assessed for a solid tumour. Benign (59) and malignant (primary 61, metastatic 59) gastric tissues were examined with antibodies directed at epitopes common to known CD44 isoforms. Normal mucosa was CD44 negative. In atrophic gastritis and intestinal metaplasia expression was restricted to the epithelial cells of the basal glands and was positively correlated with an increased leucocyte infiltrate and with the expression of HLA DR by mucosal cells. These observations suggest a role for chronic inflammation in the induction of CD44 expression on benign mucosa. No such association was observed between inflammatory infiltrate and CD44 expression on gastric tumours. CD44 expression, observed in only 49% of primary tumours, was associated with distant metastases at time of diagnosis and, among 31 curatively resected patients, with tumour recurrence (p = 0.0014) and increased mortality (p = 0.001) during follow-up averaging 17 months. When we used an antibody directed against the CD44 variant exon 9v, we found a good correlation between the expression of total CD44 and of exon 9v containing isoforms, and 9v expression in primary tumours was significantly and positively associated with tumour recurrence and mortality.

Expression of the CD6 T lymphocyte differentiation antigen in normal human brain

Antigens shared by the immune and central nervous systems (CNS) have been described repeatedly. The present study reports the expression of the CD6 lymphocyte differentiation antigen in normal human brain evidenced by immunohistochemistry and Northern blot analysis. A panel of various anti-CD6 monoclonal antibodies (mabs) tested on serial cryostat sections identified CD6-positive cells randomly scattered in parenchyma of all examined brain areas. Northern blot analysis with a highly sensitive cRNA probe revealed a 3.1 kb CD6-specific mRNA in various brain regions, especially in basal ganglia and cortex cerebellum. Staining with mabs raised against different hematopoietic cell types, as well as hybridization with probes specific for the beta- and gamma-T cell receptor (TCR) chains support the notion that CD6 is expressed by original brain cells. The nature of the CD6-positive cell type and possible functions of shared antigens in immune and nervous systems are discussed.

Impact of the spheroid model complexity on drug response

Pharmaceutical investigators are searching for preclinical models closely resembling the original cancer and predicting clinical outcome. This study compares drug response of three in vitro 3D-drug screening models with different complexity. Tumor cell line spheroids were generated from the cell lines Caco-2, DLD-1, COLO 205, HT-29 and HCT-116, and treated with clinically relevant combination therapies, namely 5-FU/oxaliplatin (FO), 5-FU/irinotecan (FI) and the molecular drugs Cetuximab, Trastuzumab, Vorinostat and Everolimus. Treatment results were compared with spheroids originated from tumor cell lines (Caco-2, DLD-1) co-cultured with stromal cells (PBMCs, cancer-associated fibroblasts of colorectal origin) and spheroids directly prepared from colon cancer tissues. Different microenvironment compositions altered the tumor cell line spheroid response patterns. Adding PBMCs increased resistance to FO treatment by 10-15% in Caco-2 and DLD-1 spheroids but decreased resistance to FI by 16% in DLD-1 spheroids. Fibroblast co-cultures decreased resistance to FI in Caco-2 spheroids by 38% but had no impact on FO. Treatment of colon cancer tissue spheroids revealed three distinct response pattern subgroups not detectable in 3D cell lines models. The cancer tissue spheroid model mimics both tumor characteristics and the stromal microenvironment and therefore is an invaluable screening model for pharmaceutical drug development.

Estrogen Receptor Expression Profile of Disseminated Epithelial Tumor Cells in Bone Marrow of Breast Cancer Patients

The estrogen receptor (ER) status in primary breast cancer represents an important prognostic factor and has a profound impact on therapeutic decisions. However, ER expression profile on disseminated breast cancer cells is largely unknown, although these cells are one of the main target structures in adjuvant therapy after local curative resection (R0) achieved in most breast cancer patients. Thus, the present pilot study was designed to evaluate the ER expression profile on disseminated epithelial cells in bone marrow, one of the preferential organs for manifestation of distant metastases in breast cancer. Using the alkaline phosphatase anti-alkaline phosphatase-immunogold double staining procedure, in a panel of 17 breast cancer patients, epithelial cells (mab CK2) detected in bone marrow were analyzed for ER expression (mab 1D5) and compared with ER expression in the corresponding primary tumors. Whereas eleven of the 17 patients (64.7%) were ER-positive in primary carcinomas, only two patients (11.8%) revealed ER-positive epithelial cells in bone marrow. In addition, one of these two patients demonstrated a heterogeneous ER expression pattern, with both ER-positive and ER-negative epithelial cells in bone marrow. Although in both of these cases the ER-positive epithelial cells in bone marrow derived from ER-positive primary tumors, in this small patient cohort none of the prognostic relevant clinical and pathological factors tested, i.e., TNM-classification, grading, and ER status in primary breast cancer, correlated with the ER status in bone marrow. The striking discrepancy between ER expression in primary breast cancers and the corresponding disseminated epithelial cells in bone marrow suggests either the selective dissemination of ER-negative tumor cells into the bone marrow or a negative impact of the bone marrow microenvironment on epithelial ER expression. This phenomenon might influence therapeutic effects of antihormonal treatment.

De novo expression of the cell adhesion molecule E-selectin on gastric cancer endothelium

Background and aims: Angiogenesis and the molecular phenotype of the tumor vasculature determine tumor growth and metastasis. Patients/Methods: In a series of 58 gastric cancer patients, vascular density and the antigenic profile of endothelial cells in normal, inflamed and malignant gastric tissues were compared using immunohistochemistry. Results: In both benign gastric mucosa and primary gastric cancer vascular density was inflammation-independent. However, increased vascularity in primary tumors was positively associated with a high tumor cell density suggesting tumor-induced angiogenesis (P=0.00001). P-selectin was expressed in most of the gastric mucosa samples on a small fraction of vessels and increased in the presence of moderate to strong leukocyte infiltrate. VCAM-1 positive mucosal vessels were rare and showed no association with inflammation. E-selectin and the EN 7/44 antigen defining budding vessels were absent on normal and inflamed endothelium. In contrast, in primary gastric cancer de novo expression of both E-selectin and the EN 7/44 antigen was observed. E-selectin positive vessels were preferentially found in vascular-rich tumor areas (P=0.0043) independently of leukocyte infiltration. Upregulation of VCAM-1 on tumor-associated endothelium was closely related to inflammation (P=0.019), while P-selectin expression resembled that in benign mucosa. Conclusions: Differentially expressed vascular molecules may influence the functional characteristics of extravasating leukocytes and represent new targets in anti-gastric cancer therapy.

High expression of a Lewis(x)-related epitope in gastric carcinomas indicates metastatic potential and poor prognosis

BACKGROUND & AIMS The acquisition of metastatic potential is accompanied by phenotypic changes. The aim of this study was to identify those changes that may lead to the development of new antimetastatic strategies in gastric cancer. METHODS A new murine monoclonal antibody showing differential reactivity with benign and malignant gastric tissues was isolated. The expression pattern of the recognized 2B4 antigen was determined with immunohistochemistry, and the antigen was analyzed by immunoprecipitation and enzyme digestion. Its prognostic impact in gastric cancer was tested in univariate and multivariate analyses. RESULTS In gastric mucosa, 2B4 expression was significantly reduced on mucosal glands in the presence of an inflammatory infiltrate and could be modulated in vitro by exposure to interferon alfa and gamma and phorbol esters. Twenty-eight percent of the primary gastric carcinomas showed high levels of 2B4. This correlated significantly with clinicopathological parameters of advanced disease (tumor size of > 50 mm, M1 stage, and UICC stage IIIB/IV). In multivariate analysis, high 2B4 expression was found to be a new, independent parameter of poor prognosis. The 2B4 monoclonal antibody was shown to react with the trisaccharide Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc, i.e., Lewis(x). CONCLUSIONS High levels of the Lewis(x)-related epitope defined by MAb 2B4 in primary gastric carcinomas is an independent parameter of poor survival.

Bringing 3D tumor models to the clinic – predictive value for personalized medicine

Current decision‐guiding algorithms in cancer drug treatment are based on decades of research and numerous clinical trials. For the majority of patients, this data is successfully applied for a systemic disease management. For a number of patients however, treatment stratification according to clinically based risk criteria will not be sufficient. The most effective treatment options are ideally identified prior to the start of clinical drug therapy. This review will discuss the implementation of three‐dimensional (3D) cell culture models as a preclinical testing paradigm for the efficacy of clinical cancer treatment. Patient tumor‐derived cells in 3D cultures duplicate the individual tumor microenvironment with a minimum of confounding factors. Clinical implementation of such personalized tumor models requires a high quality of methodological and clinical validation comparable to other biomarkers. A non‐systematic literature search demonstrated the small number of prospective studies that have been conducted in this area of research. This may explain the current reluctance of many physicians and insurance providers in implementing this type of assay into the clinical diagnostic routine despite potential benefit for patients. Achieving valid and reproducible results with a high level of evidence is central in improving the acceptance of preclinical 3D tumor models.

The Rhesus D-negative phenotype is an independent predictor of poor prognosis in curatively (RO) resected gastric cancer patients.

Among gastric cancer patients, the Rhesus D-negative phenotype correlated with increased tumour recurrence [all patients, n = 83, P = 0.026; curatively (R0) resected patients, n = 51, P = 0.093] and reduced overall survival time (all patients, log-rank P = 0.0028; R0 patients, log-rank P = 0.0003) and was identified in multivariate analysis as the most important independent prognostic marker in the R0 patient group (relative risk 9.1, P = 0.0013).

Abstract P3-06-13: Prospective cohort study using the breast cancer spheroid model as a predictor for response to neoadjuvant therapy – The SpheroNEO study

(1) PURPOSE The aim of the prospective trial was to determine if cell survival in a breast cancer spheroid model following cytostatic treatment in vitro predicts treatment response in breast cancer patients receiving equivalent neoadjuvant therapy. (2) PATIENTS AND METHODS Three-dimensional spheroids were directly generated from fresh tumor biopsy samples of 78 patients eligible for neoadjuvant therapy. Cell survival in vitro , as well baseline clinical and pathological characteristics were correlated with the outcome following treatment of each patient to determine the factor(s) most highly associated with pathological complete response (pCR i.e.ypT0/ypN0) at surgery. (3) RESULTS Cell survival after treatment in the breast cancer spheroid model proved to be a sensitive and specific predictor for pCR in individual breast cancer patients. A mean cell survival of 21.8% was found in the breast cancer spheroid model for 22 patients with pCR versus 63.8% in 56 patients without pCR ( P = .001). A receiver operator characteristic analysis determined an area under the ROC curve of 0.86 (95% CI: 0.77 to 0.96) for cell survival compared to classic factors i.e. negative hormone receptor and positive Her2/neu status, and age ≤ 50 years at primary diagnosis (AUC = 0.80, 95% CI: 0.70 to 0.90). A cutoff of 35% cell survival was proposed, which grouped patients according to likelihood for pCR. Out of the 32 patients with values below this threshold, 21 patients (65.6%) and one patient (2.2%) with a cell survival greater than 35% achieved pCR respectively; (sensitivity 95.5% (95% CI: 0.86 to 1.00); specificity 80.4% (95% CI: 0.70 to 0.91)).The specificity was improved to 81% (95% CI: 58 to 95%) if the patient was treated per-protocol. A positive correlation was also found between cell survival in vitro and residual tumor size ( P = .021), indicating the possibility of the model to predict degree of response. (4) CONCLUSION The breast cancer spheroid model is a valuable predictor for treatment outcome in patients undergoing neoadjuvant chemotherapy for primary breast cancer. Preclinical selection of the most efficient drugs is a prerequisite to improve pCR. Citation Format: Kathrin ML Halfter, Nina Ditsch, Hans-Christian Kolberg, Holger Fischer, Tanja Hauzenberger, Franz Edler von Koch, Ingo Bauerfeind, Gunter von Minckwitz, Ilona Funke, Alexander Crispin, Barbara Mayer. Prospective cohort study using the breast cancer spheroid model as a predictor for response to neoadjuvant therapy – The SpheroNEO study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-13.

Targeting Pleural Carcinoma Cells with the Anti-EpCAM BiTE Antibody MT110 by Stimulating Microenvironment T Cells.

Rationale: Despite therapy, most patients with advanced breast cancer develop malignant pleural effusion (MPE) associated with short survival. Strikingly, the metastatic cancer cells are frequently hormon receptor negative and show a reduced Her2/neu expression compared to the corresponding primary tumors. Thus, new treatment strategies are urgently needed.Objectives: In the present study, the efficacy of the bispecific single-chain antibody MT110 targeting both the epithelial antigen EpCAM (CD326) and the T cell antigen CD3 was investigated ex vivo with MPE samples of breast cancer patients.Methods: Target antigen expression of MPE cells was analyzed by immunohistochemistry. Percentage of redirected target cell lysis by MT110 was determined by double staining of cells with 7-amino actinomycin and an anti-EpCAM antibody using FACS analysis. Activation of autologous CD4+ and CD8+ cells in response to MT110 was studied by expression of CD25 and granzyme B using FACS staining and different cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) using ELISA analysis.Results: EpCAM+ cells were found in 14 out of 18 (78%) MPE samples from metastatic breast cancer patients. The fraction of EpCAM+ pleural carcinoma cells varied between 30% and 100% (mean 78%). CD3-positive cells were detected in all MPE samples ranging from 60% to 93% (mean 80%). Seven effusion samples were analyzed for MT110 treatment and revealed a dose-dependent and specific redirected lysis of EpCAM+ cells after 48h and 72h with 10 ng/ml (48h, 72h: p=0.03, respectively) and 1000 ng/ml (48h, p=0.03; 72h, p=0.016). After 72 h, 57% ± 29.5% (mean ± SD) of EpCAM+ cells were killed using 1000 ng/ml MT110. The ratio between effector (E) and target (T) cells revealed no influence on the extent of the redircted specific lysis. Antibody treatment increased the fraction of CD25/CD4 cells after 48h (p=0.03) and 72h (p=0.016). Similar, CD25 expression on CD8 cells was stimulated after 48h (p=0.03) and 72h (p=0.016) using 1000 ng/ml MT110. Cytokine analysis revealed a strong TH1 immune response detecting an increased TNFα (p=0.016) and IFNγ (p=0.03) secretion. Conclusion: Single-agent therapy with MT110 is capable of activating unstimulated autologous T cells for efficient and specific redirected lysis of EpCAM+ tumor cells in MPE from breast cancer patients. MPE revealed interindividual differences regarding the extent of specific lysis and the stimulation of the microenvironmental T cells. Thus, treatment with the bispecific antibody MT110 might by an option in the management of a subcohort of metastatic breast cancer patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4133.