Axillary reverse mapping in breast cancer surgery - functional study.

The aim of the study was to map the lymphatic drainage of the upper extremity that traverses the axilla and elucidate its relationship with the lymphatic drainage of the breast. In 79 breast cancer patients indicated to the axillary lymph node dissection for category cN1, cN2, Technetium-99m (particle size <80 nm) was applied prior to surgery at two injection sites between the second and third metacarpophalangeal joints to visualize upper extremity lymphatics. During the surgery, the axilla was anatomically divided into 6 quadrants. A C-Trak® device was used for the intraoperative detection of radioactivity. After verifying activity, the nodes were resected and their position was recorded. Active nodes were sent separately according to topographic localizations for microscopic examination. All affected nodes (both macro- and micrometastases) were recorded as positive. The location, involvement and radioactivity, and the number of lymph nodes obtained were analyzed. In total, 1,109 lymph nodes were removed and examined. Radioactive nodes were found in all 79 patients. A total of 230 radioactive nodes were found. 21 nodes were both radioactive and metastatically affected. Results show that part of the lymph from the upper extremity flows through the nodes in the central part of the axilla and mixes with the lymph from the breast. This suggests that lymphatic drainage of the upper limb cannot be functionally separated from lymphatic drainage of the breast. The results also explain the possible mechanical cause of arm lymphedema after sentinel lymph node biopsy.

Apoptosis - associated genes and their role in predicting responses to neoadjuvant breast cancer treatment.

BACKGROUND: Neoadjuvant chemotherapy is used in the treatment of breast carcinoma because it substantially reduces the size of the primary tumor and lymph node metastases. The present study investigated biomarkers that can predict a pathologic response to the therapy. MATERIAL/METHODS: The role of apoptosis in regression of the tumors after neoadjuvant chemotherapy was determined by TUNEL and anti-active caspase 3 assay. The transcriptional profile of 84 key apoptosis genes was evaluated in both pre-therapeutically obtained tumor tissue by core needle biopsy and in specimens removed by final surgery, using a pathway-specific real-time PCR assay. Obtained data were analyzed by hierarchical cluster analysis and correlation analysis. The immunohistochemical profile of each tumor was determined using the standard ABC method. RESULTS: On the basis of a hierarchical cluster analysis of 13 significantly changed genes, we divided patients into good and poor prognosis groups, which correlate well with progression-free survival. In the good prognosis group, we found a statistically significant down-regulation of the expression of MCL1 and IGF1R genes after neoadjuvant treatment. We also found a statistically significant overexpression of BCL2L10, BCL2AF1, CASP8, CASP10, CASP14, CIDEB, FADD, HRK, TNFRSF25, TNFSF8 and CD70 genes. In contrast, we found up-regulation of IGF1R after the treatment in the group with poor prognosis. CONCLUSIONS: Gene expression profiling using real-time PCR assay is a valuable research tool for the investigation of molecular markers, which reflect tumor biology and treatment response.

Two-dimensional electrophoretic comparison of metastatic and non-metastatic human breast tumors using in vitro cultured epithelial cells derived from the cancer tissues.

BACKGROUND: Breast carcinomas represent a heterogeneous group of tumors diverse in behavior, outcome, and response to therapy. Identification of proteins resembling the tumor biology can improve the diagnosis, prediction, treatment selection, and targeting of therapy. Since the beginning of the post-genomic era, the focus of molecular biology gradually moved from genomes to proteins and proteomes and to their functionality. Proteomics can potentially capture dynamic changes in protein expression integrating both genetic and epigenetic influences. METHODS: We prepared primary cultures of epithelial cells from 23 breast cancer tissue samples and performed comparative proteomic analysis. Seven patients developed distant metastases within three-year follow-up. These samples were included into a metastase-positive group, the others formed a metastase-negative group. Two-dimensional electrophoretical (2-DE) gels in pH range 4-7 were prepared. Spot densities in 2-DE protein maps were subjected to statistical analyses (R/maanova package) and data-mining analysis (GUHA). For identification of proteins in selected spots, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed. RESULTS: Three protein spots were significantly altered between the metastatic and non-metastatic groups. The correlations were proven at the 0.05 significance level. Nucleophosmin was increased in the group with metastases. The levels of 2,3-trans-enoyl-CoA isomerase and glutathione peroxidase 1 were decreased. CONCLUSION: We have performed an extensive proteomic study of mammary epithelial cells from breast cancer patients. We have found differentially expressed proteins between the samples from metastase-positive and metastase-negative patient groups.

Expression pattern of adaptor protein PAG: correlation between secondary lymphatic follicle and histogenetically related malignant lymphomas.

Transmembrane adaptor protein PAG, also known as Csk-binding protein (Cbp), which binds and activates the cytoplasmic tyrosine kinase Csk, the major negative regulator of Src-family kinases, was found to be expressed in germinal centers of lymphoid follicles as well as in follicular, but not mantle cell lymphomas. Expression of PAG may reflect its role in regulation of proliferation and differentiation of germinal center B-cells. From the routine histopathology point of view, PAG might be a new positive marker of follicular lymphoma and a negative marker of mantle cell lymphoma.

Renal epithelioid angiomyolipoma--a close mimic of renal cell carcinoma. Report of a case and review of the literature.

The epithelioid variant of angiomyolipoma (EAML) is a rare tumor of unpredictable behavior that is composed of epithelioid, spindle, and giant cells and contains no or only a minimal amount of lipomatous tissue. The picture can lead to an erroneous diagnosis of renal cell carcinoma or sarcoma. We report on a case of EAML in the kidney of a 47-year-old female without any signs of tuberous sclerosis and review the literature. Grossly, a well-demarcated, spheroid, largely hemorrhagic tumor measuring 4.2 cm in diameter occupied the central third of the kidney. Histologically, it was solid, highly cellular, with occasional microcysts, composed of medium to large epithelioid cells with clear or oxyphilic cytoplasm, short spindle cells, and numerous giant multinucleated cells. After extensive sampling, adult-appearing fat tissue was found to present as rare foci of microscopic dimensions. Immunohistochemically, the tumor cells showed positive reactions with antibodies against HMB-45, melan A, CD-68, muscle-specific actin, and, rarely, smooth muscle actin. Cytokeratins and epithelial membrane antigen were negative. The EAML is a variant growing in a carcinoma-like pattern that can lead to an erroneous diagnosis of renal cell carcinoma. An extensive sampling and HMB-45 and CD-68 positivity combined with cytokeratin negativity are of paramount importance for the correct diagnosis. As a sporadic renal tumor it followed a benign course in most of the reported cases.

Microvessel density of mantle cell lymphoma. A retrospective study of its prognostic role and the correlation with the Ki-67 and the mantle cell lymphoma international prognostic index in 177 cases.

The clinical course and therapy of mantle cell lymphoma (MCL) are heterogeneous and often unsatisfactory. Prognostic factors are needed to stratify the patients. Microvessel density (MVD) has prognostic significance in some malignancies. There is little information about the vasculature of MCL, although some antiangiogenic drugs are in use. We studied MVD using systematic uniform random sampling and unbiased counting frames in immunohistochemical reactions with anti-CD34 antibody in pre-therapeutic extramedullary MCL samples of 177 patients. We analyzed the relationship of MVD to overall survival (OS) and progression-free survival (PFS), as well as to proliferative activity (Ki-67), mantle cell lymphoma prognostic index (MIPI), morphological variant, pattern of growth, and localization. MVD varied widely: range 54.6-503.6 vessels/mm(2), median 158.2 vessels/mm(2). Higher MVD was associated with bone marrow infiltration at the time of diagnosis (P = 0.001). High MVD was associated with significantly worse OS (P = 0.04) only in patients treated with non-intensive (conventional) therapy. MVD correlated positively with MIPI scores but not with the proliferation, morphological variant, growth pattern, or localization. Univariate analysis identified a prognostic influence of morphological variant, MIPI, and proliferative activity on OS and PFS and a prognostic influence of bone marrow infiltration at the time of diagnosis on PFS. Multivariate analysis showed prognostic influence of MIPI and proliferative activity on OS and PFS only. In conclusion, this is the first clinicopathological study of MVD of MCL with long-term follow-up showing negative prognostic trends of high MVD in MCL and positive correlation of MVD and MIPI.

Optimized protocol for gene expression analysis in formalin-fixed, paraffin-embedded tissue using real-time quantitative polymerase chain reaction.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common tissue specimen available after microscopic examination. Molecular methods, such as polymerase chain reaction (PCR) and gene expression examination, serve as a source of diagnostic and prognostic information but require high-quality RNA. However, the increasing application of RNA extracted from FFPE tissue frequently results in very small and degraded quantities of nucleic acid. This study targets gene expression analysis from FFPE specimens using real-time quantitative PCR. The whole protocol consists of several steps, that is, RNA extraction and its quality control, reverse transcription, and fluorescence detection during real-time quantitative PCR. We compared several methods in each step, chose the most effective, and with that combination we successfully examined 95% (62 from 65) FFPE samples for our genes of interest. We reached the best results with RNA isolation by using a commercial kit, carefully interpreted UV spectrophotometric values, and meticulously chose reverse transcriptase and TaqMan fluorescence detection. Our protocol improves the utility of FFPE tissue for molecular profiling studies.