Jozsef Toth

Novel Ganglioside Antigen Identified by B Cells in Human Medullary Breast Carcinomas: The Proof of Principle Concerning the Tumor-Infiltrating B Lymphocytes

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region VH-JH, Vκ-Jκ, and Vλ-Jλ genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected VH and Vκ clones and tested for binding activity. Our data analysis revealed that some of the VH-JH, Vκ-Jκ, and Vλ-Jλ region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment κ minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the VH of which proved to be the overexpressed VH3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.

HER-2/neu genotype of breast cancer may change in bone metastasis.

The genotype of breast cancer (BRC) is considered to be relatively stable during tumor progression, accordingly, determination of the estrogen receptor and HER-2/neu status is currently based on the primary tumor. However, recent data suggest that the gene expression profile of the metastatic lesion can be different compared to that of the primary BRC. Accordingly, it is possible that the HER-2/neu status is different in the metastatic lesion and the primary BRC. Since the bone is the most frequent metastatic site during the progression of BRC, we have analyzed the HER-2/neu status of 48 bone metastatic BRC cases by immunohistochemistry and fluorescent in situ hybridization, and it was possible to compare it to the primary site in 23 cases. The frequency of HER-2/neu amplification of BRC in the primary tumors was found to be 17.4% compared to 10.5% in bone metastases. Half of BRC cases with HER-2/neu amplification lost this genotype in bone metastases (4/23 versus 2/23, respectively) and even in the 2 cases where HER-2/neu amplification was retained in the metastases, the copy number was found to be decreased compared to the primary tumor. Based on our data and previous reports in the literature, we suggest to perform HER-2/neu testing both on primary tumor and samples obtained from BRC metastases, at least in case of primary tumors with HER-2/neu amplification, before introduction of HER-2/neu-targeting therapy. (Pathology Oncology Research Vol 12, No 3, 149–152)

Tumor-infiltrating B cell immunoglobulin variable region gene usage in invasive ductal breast carcinoma.

A major focus of tumor immunology is to reveal the potential role and capacity of immunocompetent cells found in different solid tumor tissues. The most abundant infiltrating cells (TIL), the T lymphocytes have been investigated in details concerning T-cell receptor usage and specificity. However, B cells have hardly been investigated in this respect, although high cellular B-cell infiltration has been correlated with improved patients’ survival in some breast carcinomas. This led to our objectives to study variable region gene usage of the tumor-infiltrating B cells in different breast carcinoma types. By defining the immunoglobulin repertoire of the tumor-infiltrating B lymphocytes in the most common invasive ductal carcinoma (IDC) of the breast we compared it to the rare medullary breast carcinoma (MBC). After phenotyping infiltrating ductal carcinomas, B cells were obtained from tumor tissue by microdissection technique. Numerous rearranged TIL-B immunoglobulin heavy chain V genes (VH) were amplified, cloned, sequenced, and comparatively analyzed. Some characteristics were found for both breast carcinoma types. The immunoglobulins produced by TIL-B in ductal carcinoma are highly matured and oligoclonal. We conclude that Ig variable region gene usage reveals similar and distinguishable characteristics of TIL-B immunoglobulin repertoires, which are representative of the nature of the immune responses in invasive ductal and medullary breast carcinomas.

Challenging tumour immunological techniques that help to track cancer stem cells in malignant melanomas and other solid tumours

Aim of the study The arsenal of questions and answers about the minor cancer initiating cancer stem cell (CSC) population put responsible for cancer invasiveness and metastases, has left with an unsolved puzzle. Specific aims of a complex project were partly focused on revealing new biomarkers of cancer. We designed and set up novel techniques to facilitate the detection of cancerous cells. Materials and methods As a novel approach, we investigated B cells infiltrating breast carcinomas and melanomas (TIL-B) in terms of their tumour antigen binding potential. By developing the TIL-B phage display technology we provide here a new technology for the specific detection of highly tumour-associated antigens. Single chain Fv (scFv) antibody fragment phage ELISA, immunofluorescence (IF) FACS analysis, chamber slide technique with IF confocal laser microscopy and immunohistochemistry (IHC) in paraffin-embedded tissue sections were set up and standardized. Results We showed strong tumour-associated disialylated glycosphingolipid expression levels on various cancer cells using scFv antibody fragments, generated previously by uniquely invasive breast carcinoma TIL-B phage display library technology. Conclusions We report herein a novel strategy to obtain antibody fragments of human origin that recognise tumour-associated ganglioside antigens. Our investigations have the power to detect privileged molecules in cancer progression, invasiveness, and metastases. The technical achievements of this study are being harnessed for early diagnostics and effective cancer therapeutics.