Janina Kulka

Claudin-1, -3 and -4 proteins and mRNA expression in benign and malignant breast lesions: a research study.

IntroductionWe compared levels of protein and mRNA expression of three members of the claudin (CLDN) family in malignant breast tumours and benign lesions.MethodsAltogether, 56 sections from 52 surgically resected breast specimens were analyzed for CLDN1, CLDN3 and CLDN4 expression by immunohistochemistry. mRNA was also analyzed using real-time PCR in 17 of the 52 cases.ResultsCLDNs were rarely observed exclusively at tight junction structures. CLDN1 was present in the membrane of normal duct cells and in some of the cell membranes from ductal carcinoma in situ, and was frequently observed in eight out of nine areas of apocrine metaplasia, whereas invasive tumours were negative for CLDN1 or it was present in a scattered distribution among such tumour cells (in 36/39 malignant tumours). CLDN3 was present in 49 of the 56 sections and CLDN4 was present in all 56 tissue sections. However, CLDN4 was highly positive in normal epithelial cells and was decreased or absent in 17 out of 21 ductal carcinoma grade 1, in special types of breast carcinoma (mucinous, papillary, tubular) and in areas of apocrine metaplasia. CLDN1 mRNA was downregulated by 12-fold in the sample (tumour) group as compared with the control group using GAPDH as the reference gene. CLDN3 and CLDN4 mRNA exhibited no difference in expression between invasive tumours and surrounding tissue.ConclusionsThe significant loss of CLDN1 protein in breast cancer cells suggests that CLDN1 may play a role in invasion and metastasis. The loss of CLDN4 expression in areas of apocrine metaplasia and in the majority of grade 1 invasive carcinomas also suggests a particular role for this protein in mammary glandular cell differentiation and carcinogenesis.

Pathological work-up of sentinel lymph nodes in breast cancer. Review of current data to be considered for the formulation of guidelines.

Controversies and inconsistencies regarding the pathological work-up of sentinel lymph nodes (SNs) led the European Working Group for Breast Screening Pathology (EWGBSP) to review published data and current evidence that can promote the formulation of European guidelines for the pathological work-up of SNs. After an evaluation of the accuracy of SN biopsy as a staging procedure, the yields of different sectioning methods and the immunohistochemical detection of metastatic cells are reviewed. Currently published data do not allow the significance of micrometastases or isolated tumour cells to be established, but it is suggested that approximately 18% of the cases may be associated with further nodal (non-SN) metastases, i.e. approximately 2% of all patients initially staged by SN biopsy. The methods for the intraoperative and molecular assessment of SNs are also surveyed.

Discrepancies in current practice of pathological evaluation of sentinel lymph nodes in breast cancer. Results of a questionnaire based survey by the European Working Group for Breast Screening Pathology.

Aims: To evaluate aspects of the current practice of sentinel lymph node (SLN) pathology in breast cancer via a questionnaire based survey, to recognise major issues that the European guidelines for mammography screening should address in the next revision. Methods: A questionnaire was circulated by mail or electronically by the authors in their respective countries. Replies from pathology units dealing with SLN specimens were evaluated further. Results: Of the 382 respondents, 240 European pathology units were dealing with SLN specimens. Sixty per cent of these units carried out intraoperative assessment, most commonly consisting of frozen sections. Most units slice larger SLNs into pieces and only 12% assess these slices on a single haematoxylin and eosin (HE) stained slide. Seventy one per cent of the units routinely use immunohistochemistry in all cases negative by HE. The terms micrometastasis, submicrometastasis, and isolated tumour cells (ITCs) are used in 93%, 22%, and 71% of units, respectively, but have a rather heterogeneous interpretation. Molecular SLN staging was reported by only 10 units (4%). Most institutions have their own guidelines for SLN processing, but some countries also have well recognised national guidelines. Conclusions: Pathological examination of SLNs throughout Europe varies considerably and is not standardised. The European guidelines should focus on standardising examination. They should recommend techniques that identify metastases > 2 mm as a minimum standard. Uniform reporting of additional findings may also be important, because micrometastases and ITCs may in the future be shown to have clinical relevance.

Variations in sentinel node isolated tumour cells/micrometastasis and non-sentinel node involvement rates according to different interpretations of the TNM definitions

Breast cancers with nodal isolated tumour cells (ITC) and micrometastases are categorised as node-negative and node-positive, respectively, in the tumour node metastasis (TNM) classification. Two recently published interpretations of the TNM definitions were applied to cases of low-volume sentinel lymph node (SLN) involvement and their corresponding non-SLNs for reclassification as micrometastasis or ITC. Of the 517 cases reviewed, 82 had ITC and 435 had micrometastasis on the basis of one classification, and the number of ITC increased to 207 with 310 micrometastases on the basis of the other. Approximately 24% of the cases were discordantly categorised. The rates of non-SLN metastases associated with SLN ITCs were 8.5% and 13.5%, respectively. Although the second interpretation of low-volume nodal stage categories has better reproducibility, it may underestimate the rate of non-SLN involvement. The TNM definitions of low-volume nodal metastases need to be better formulated and supplemented with visual information in the form of multiple sample images.

Expression of tight junction protein claudin-4 in Basal-Like breast carcinomas

Claudins (CLDN) are tight junction proteins which contribute to the paracellular transport and ionic permeability of various epithelia. In recent years they came into focus for their suggested role in carcinogenesis and possible role in cancer therapy. According to our previous studies, in breast tissue CLDN4 is also related to the level of cellular differentiation. Thirty-eight estrogen (ER) and progesterone receptor (PgR) negative, HER2/neu negative, but cytokeratin 5/6 positive basal-like—mainly grade 3—breast carcinomas were compared with twenty-one grade 1, twenty-five grade 2 and twenty grade 3 non-basal-like invasive breast carcinomas, in respect to their CLDN4 expression. The immunohistochemical reactions were evaluated both semiquantitatively and by morphometrical analysis. Statistically significant difference (p = 0.001) was observable regarding CLDN4 expression in the basal-like group as compared to grade 1 and 2 cancers. Further, CLDN4 expression was significantly higher (p = 0.017) in the basal-like compared with the non-basal-like grade 3 carcinomas. Our results suggest that basal-like carcinomas are a subset of breast cancer with high level of CLDN4 protein expression. The finding is in accordance with our former observation that CLDN4 is indeed related to cellular differentiation. This observation may be seen as a further proof that basal-like carcinomas represent a separable group amongst grade 3 breast carcinomas.

MicroRNA expression profiling in benign (sporadic and hereditary) and recurring adrenal pheochromocytomas

MicroRNAs are involved in the pathogenesis of several tumors, however, there have been no data on microRNA expression in pheochromocytomas to date. The objective of our study was to perform microRNA expression profiling in sporadic and hereditary benign, and recurring adrenomedullary tumors. Furthermore, the applicability of formalin-fixed paraffin-embedded tissue samples for the analysis of microRNA expression in pheochromocytomas was examined. MicroRNA expression data of three matched frozen and formalin-fixed paraffin-embedded samples were correlated. A total of 21 formalin-fixed paraffin-embedded samples (sporadic benign, multiple endocrine neoplasia 2, von Hippel-Lindau disease, sporadic recurring) were subjected to microRNA expression profiling using microarrays. MicroRNAs with significant differences in expression were validated and sample sizes were extended including tumors from neurofibromatosis type 1 patients by real-time quantitative reverse-transcription PCR (n=33). MicroRNA target prediction was carried out by TargetScan and MicroCosm Targets. Pathway analysis of targets was performed by Ingenuity Pathway Analysis and DIANA mirPath. Furthermore, microRNA expression profiles of a malignant pheochromocytoma and a pair of primary and recurrent tumors were studied by TaqMan Human MicroRNA Cards. MicroRNA expression correlated well between frozen and formalin-fixed paraffin-embedded samples (70–92%). Microarray analysis revealed 16 significantly differentially expressed microRNAs. Five of these were validated by real-time RT-PCR. miR-139-3p, miR-541 and miR-765 were significantly differentially expressed between sporadic benign and von Hippel-Lindau-related pheochromocytomas. Significantly higher expression of miR-885-5p and miR-1225-3p was found in multiple endocrine neoplasia type 2 and sporadic recurring pheochromocytomas, respectively. Pathway analysis revealed the possible involvement of Notch- and G-protein-coupled receptor signaling in tumor recurrence. MicroRNA expression profiles in the primary recurrent and recurring malignant comparisons have been similar. In conclusion, we have proved that formalin-fixed paraffin-embedded samples can be used for the analysis of microRNA expression in pheochromocytomas. MicroRNA expression patterns differ between various sporadic, hereditary and recurring tumors and miR-1225-3p may be useful for identifying recurring pheochromocytomas.

Prognostic significance of claudin expression changes in breast cancer with regional lymph node metastasis

Adherent and tight junction molecules have been described to contribute to carcinogenesis and tumor progression. Additionally, the group of claudin-low tumors have recently been identified as a molecular subgroup of breast carcinoma. In our study, we examined the expression pattern of claudins, beta-catenin and E-cadherin in invasive ductal (IDCs) and lobular (ILCs) carcinomas and their corresponding lymph node metastases (LNMs). Tissue microarrays of 97 breast samples (60 invasive ductal carcinomas, 37 invasive lobular carcinomas) and their corresponding LNMs have been analyzed immunohistochemically for claudin-1, -2, -3, -4, -5, -7, beta-catenin and E-cadherin expression. The stained slides were digitalized with a slide scanner and the reactions were evaluated semiquantitatively. When compared to LNMs, in the IDC group beta-catenin and claudin-2, -3, -4 and -7 protein expression showed different pattern while claudin-1, -2, -3, -4 and -7 were differently expressed in the ILC group. Lymph node metastases developed a notable increase of claudin-5 expression in both groups. Decrease or loss of claudin-1 and expression of claudin-4 in lymph node metastases correlated with reduced disease-free survival in our patients. According to our observations, the expression of epithelial junctional molecules, especially claudins, is different in primary breast carcinomas compared to their lymph node metastases as demonstrated by immunohistochemistry. Loss of claudin junctional molecules might contribute to tumor progression, and certain claudin expression pattern might be of prognostic relevance.

Leiomyosarcomas of great vessels.

Sarcomas of the great vessels are rare. Altogether 400 such cases have been described in the aorta, the pulmonary artery, and inferior vena cava. The clinical symptoms are generally related to embolic phenomena, aneurysm formation, and widespread metastases, especially to bones. With improved diagnostic modalities more cases are diagnosed and treated surgically. Resection of the tumor may prolong the patient’s life. In this paper authors present two cases of such rare sarcomas. In our first case a tumor has developed in the thoracic aorta with symptoms of imminent aortic dissection. The tumorous nature of the lesion was revealed only histologically, since neither the operation, nor macroscopic picture gave any clue to its tumorous nature. The second case was a male patient with a huge retroperitoneal tumor arising from the inferior vena cava, which was clinically suspected to be a carcinomaarising in the adrenal gland.

Distribution pattern of the Ki67 labelling index in breast cancer and its implications for choosing cut-off values

The Ki67 labelling index (LI - proportion of staining cells) is widely used to reflect proliferation in breast carcinomas. Several cut-off values have been suggested to distinguish between tumours with low and high proliferative activity. The aim of the current study was to evaluate the distribution of Ki67 LIs in breast carcinomas diagnosed at different institutions by different pathologists using the method reflecting their daily practice. Pathologists using Ki67 were asked to provide data (including the LI, type of the specimen, receptor status, grade) on 100 consecutively stained cases, as well as details of their evaluation. A full dataset of 1709 carcinomas was collected from 19 departments. The median Ki67 LI was 17% for all tumours and 14% for oestrogen receptor-positive and HER2-negative carcinomas. Tumours with higher mitotic counts were associated with higher Ki67 LIs. Ki67 LIs tended to cluster around values ending with 5 or 0 both in cases where the values were obtained by counting the proportion of stained tumour cell nuclei and those where the values were obtained by estimation. On the basis of the distribution pattern described, some currently used Ki67 LI cut off values are not realistic, and it is proposed to select more realistic values ending with 0 or 5.

Detection of HER-2/neu gene amplification in breast carcinomas using quantitative real-time PCR - A comparison with immunohistochemical and FISH results

The aim of our study was to evaluate the value of quantitative real-time-PCR (qPCR) in the determination of HER-2/neu amplification status of human breast carcinomas by comparing qPCR, FISH and immunohistochemistry results from the same samples. A total of 210 breast carcinomas were examined. Ready-to-use CB11 antibody was applied to detect HER-2/neu oncoprotein expression. In 76 out of 210 cases FISH was performed, and 162 cases were investigated with qPCR. Seventy-five tumors were 2+ or 3+ positive with immunohistochemistry, while 135 samples were either completely negative or 1+. In 45 cases results from all three methods were available. Out of these, in twenty negative and sixteen positive cases both FISH and qPCR led to similar results. The mean qPCR amplification ratio in the concordant positive cases was 5.424 while in the qPCR+/FISH-group the mean ratio was 2.765. Out of 121 samples with scores of 0 or 1+ immunohistochemical result, analyzed also with qPCR, 26 showed HER-2/neu gene amplification. In these cases the mean amplification ratio was 2.53. Comparison of FISH and qPCR together with immunohistochemistry shows that qPCR is more sensitive to detect HER-2/neu gene amplification in tumors scored as 2+ with immunohistochemistry, but the diagnostic cut-off ratio should be defined above 2.7 to avoid high number of false positive cases. Amongst the immunohistochemistry score 2+ cases, 10 of 18 showed gene amplification by qPCR while 10 of 26 by FISH. In conclusion, a well calibrated HER-2/neu qPCR assay may serve as useful alternative to FISH in breast cancer patients.