Zoltan Szollosi

Transformed dermatofibrosarcoma protuberans: a clinicopathological study of eight cases

Background: Fibrosarcomatous (FS) or malignant fibrous histiocytomatous (MFH) transformation of dermatofibrosarcoma protuberans (DFSP) is a rare, but well known, entity. DFSPs with sarcomatous areas have questionable biological behaviour. Several studies suggest that they have a higher risk for local recurrence and distant metastases than ordinary DFSPs. One recent study described no difference in the behaviour of conventional and transformed DFSP. Aims: To investigate the biological behaviour of a series of transformed DFSPs. Methods: Eight transformed DFSPs were analysed clinicopathologically. Follow up ranged from four to 36 years. Results: The tumours involved the trunk (six cases) and lower extremity (two cases) and measured 3.5–8 cm (median, 4). Sarcomatous change presented de novo in all cases. The type of sarcomatous change was FS (five cases) and MFH (three cases). The estimated proportion of sarcomatous area in the tumour was 25–70% (median, 43.37%). Mitotic counts ranged from nine to 16 mitotic figures/10 high power fields in the FS and MFH areas (median, 12), and from one to three in the DFSP areas. Six patients were treated by wide local excision with histopathologically negative margins and two were treated by simple surgical excision with positive margins. Three patients developed recurrences and one developed metastasis during follow up. Of those treated by wide local excision, one developed recurrence. All tumours expressed CD34 in the DFSP component, but only three in the sarcomatous area. Conclusions: Although DFSP containing sarcoma may be a more aggressive tumour, its behaviour can be influenced by surgical treatment.

Analysis of EGFR Gene Amplification, Protein Over-expression and Tyrosine Kinase Domain Mutation in Recurrent Glioblastoma

Gefitinib and erlotinib are both selective EGFR tyrosine kinase inhibitors (EGFR-TKIs) that have produced responses in a small subgroup of lung cancer patients. The strongest evidence for a role of EGFR in the biology of glioblastoma stems from clinical trials in which 15–20% of recurrent glioblastoma patients experienced significant tumour regression in response to these small-molecule EGFR kinase inhibitors. We examined the protein-kinase domain of the EGFR gene, EGFR protein expression and EGFR gene amplification in 20 cases of recurrent GBMs. EGFR protein over-expression was found in 65% of cases. EGFR protein over-expression was associated with EGFR gene amplification in 35% of cases, and with high polysomy in 15% of cases. No mutations were found in the TK domain of the EGFR gene. Our results confirm that mutations in the kinase domain are absent in recurrent GBM, and this might be a preponderant factor in the lack of major clinical responses of TKIs in GBM, recent studies have suggested that responsiveness to EGFR kinase inhibitors was strongly associated with coexpression of EGFRvIII and PTEN. Further prospective validation of EGFRvIII and PTEN as predictors of the clinical response to EGFR kinase inhibitors in recurrent GBM is strongly anticipated.

Transformed dermatofibrosarcoma protuberans: real time polymerase chain reaction detection of COL1A1–PDGFB fusion transcripts in sarcomatous areas

Background: Recent cytogenetic studies have shown that reciprocal translocation t (17;22)(q22;q13) and a supernumerary ring chromosome derived from the translocation r(17;22) are highly characteristic of dermatofibrosarcoma protuberans (DFSP). The chromosomal rearrangements fuse the collagen type Iα1 (COL1A1) and the platelet-derived growth factor B-chain (PDGFB) genes. The COL1A1–PDGFB fusion transcript has been shown not only in conventional DFSP but also in a small series of DFSP with fibrosarcomatons areas (DFSP-FS) using reverse transcriptase-based conventional polymerase chain reaction. Nothing is known about the status of the COL1A1–PDGFB chimaeric gene in the pleomorphic areas of DFSP-PleoSarc (formerly known as DFSP-malignant fibrous sarcoma). Aims: To show the COL1A1–PDGFB fusion transcript in transformed malignant fibrous histiocytoma. Method: A real-time polymerase chain reaction assay for the COL1A1–PDGFB fusion transcript in a series of DFSP containing sarcoma was conducted to determine whether the chimaeric gene could be identified in both components of DFSP-FS and DFSP-PleoSarc. Eight cases were analysed. Results: In seven cases, transcriptable RNA was detected, and in these cases, translocations were found between COL1A1 and PDGFB genes involving exons 27, 32, 34, 40 and 47 of the COL1A1 gene and exon 2 of the PDGFB gene. Conclusions: From a diagnostic aspect, this assay can be particularly useful in confirming the diagnosis of sarcomatous DFSP. On the other hand, the COL1A1–PDGFB fusion gene was shown in three cases of DFSP containing pleomorphic sarcoma, which supports the theory of the common histogenesis.

FISH and immunohistochemical status of the hepatocyte growth factor receptor (c-Met) in 184 invasive breast tumors

In their report, Gotte and coworkers [1] analyzed the expression of c-Met in 200 patients with ductal carcinoma in situ. They concluded that c-Met could be related to angiogenic and lymphangiogenic factors in ductal carcinoma in situ. On the other hand, Greenberg and coworkers [2] studied 31 patients with ductal infiltrating carcinoma (DIC) to detect c-Met expression in their axillary fluids. They observed a correlation of c-Met expression with increasing tumor size and grade, capillary and lymphatic invasion and lymph node metastasis. We applied the fluorescent in situ hybridization (FISH) technique using the LSI D7S486/CEP7 commercial probe (Abbott Molecular Inc., Des Plaines, IL, USA), which includes the MET gene, and immunohistochemistry using c-Met monoclonal antibody clone 3D4 (Invitrogen, Carlsbad, CA, USA) to 184 archival invasive breast tumors (93 DIC and 91 lobular carcinomas). We constructed ten tissue microarrays with three replicates per sample. Pearsons chi-squared and Fishers exact test were used to analyze the results. None of the 155 breast tumors analyzed by FISH presented amplification of MET and 35 cases (22%) had a low grade of polysomy (three to five copies) of chromosome 7. Polysomy was more frequently observed in DIC (25%; P = 0.001). We tried to correlate polysomy of MET in the DIC group with grade, tumor size, lymph node status, clinical stage and expression of HER2, P53, estrogen receptor (ER) and progesterone receptor (PR). We observed that the absence of expression of PR was the unique statistically significant variable (P = 0.001). Moreover, the ER+/PR- samples presented the highest rate of polysomy (38%) compared to ER+/PR+ tumors (15%) (Table ​(Table11). Table 1 Results of IHC of c-Met and FISH of LSI D7S486/CEP7 applied to lobular and ductal carcinomas Out of 168 tumors analyzed by immunohistochemistry, 65 (38.7%) presented expression of c-Met. When histological types were compared, the DIC group also showed the highest number of c-Met-positive samples (48%; P = 0.001). From the analysis with the clinico-pathological variables, the negativity for PR was again statistically significant (P = 0.001). The ER+/PR- tumors presented more frequent expression of c-Met (68%) compared to ER+/PR+ tumors (32%) and were correlated with polysomy (P = 0.020) (Table ​(Table22). Table 2 IHC and FISH results of MET according to the status of PR receptor in DIC carcinomas We can conclude that amplification of MET in breast cancer is not a common event, as opposed to other cancer subtypes (renal, gastric and lung carcinomas). Although found in breast tumors, it seems that overexpression of c-Met is not mainly due to increassed gene copy number of MET/polysomy7. However, polysomy in the ER+/PR- group could be an important mechanism – although not the only one – responsible for the differential expression observed in this type of DIC. This c-Met overexpression and the presence of polysomy 7 could be important events to be considered with regard to the known poor response to endocrine therapies of ER+/PR- breast tumors. Lack of PR expression in ER+ tumors may be a surrogate marker of aberrant growth factor signaling [3] that could be associated with their more aggressive outcome, as has already been described [4]. Our study suggests that it would be interesting to investigate new therapeutic options for ER+/PR- DIC, which may include c-Met inhibitors.

Retrospective Analysis of the Prognostic Role of Tissue Eosinophil and Mast Cells in Hodgkin's Lymphoma

The composition of reactive cell populations, which constitute the majority of tumor load in Hodgkin’s lymphoma (HL), can influence the prognosis of the disease. Besides widely accepted and applied prognostic scores, the authors evaluate biological factors that may have a prognostic impact. Previous data indicate that the rate of eosinophils and mast cells in the reactive cell population, determined already at diagnosis, can be used for this purpose. Histological samples from 104 patients with HL with an average follow-up period of 110 (24–214) months were retrospectively analyzed. Mast cell positivity was associated with better overall survival, although this difference was only of borderline statistical significance (p=0.092). No significant difference was found in parameters like overall survival (OS, p=0.906) or event-free survival (EFS, p=0.307) of eosinophil-positive vs. -negative cases or in EFS (p=0.742) of mast cell-positive vs. -negative individuals (criterion for a positive specimen was more than 5% of appropriate cells in the reactive cell population). Looking at the effect of eosinophilia and mastocytosis together, there was no significant difference between the subgroups categorized according to the combined presence of the two cell types. It seems that tissue eosinophil and mast cell predominance have no prognostic value that could be used in clinical practice, although a tendency for correlation of mast cell positivity with overall survival could be seen. For a definitive statement, multicenter studies should be performed involving a higher number of patients suffering from HL.

Immunohistochemical Antibodies in Breast Cancer HER2 Diagnostics

Overexpression and/or gene amplification of the HER2 oncogene predicts worse prognosis and altered sensitivity to chemotherapy. Trastuzumab is capable of improving prognosis of HER2-positive breast cancer, but for the success of treatment appropriate HER2 testing is essential. Our aim was to determine the value of immunohistochemical (IHC) screening prior to fluorescence in situ hybridization (FISH). We assessed five conventional IHC assays (NCL-CB11, Pathway CB11, CBE356, CBE1, HercepTest) and the novel rabbit monoclonal antibody, RM-4B5, combined with FISH on 199 invasive breast cancer cases. Taking FISH as the endpoint, we calculated sensitivity, specificity, positive and negative predictive values (PPV, NPV) and accuracy for all IHC assays with either taking both 2+/3+ cases or only 3+ cases as IHC positives. With 2+/3+ cases HercepTest showed 100% sensitivity and NPV, while the highest specificity, PPV and accuracy was associated with RM-4B5 (97.36, 80 and 95.34%, respectively). The second highest values belonged to either NCL-CB11 or Pathway CB11. When calculating only with 3+ cases, the results were reversed with increased specificity, PPV and accuracy. Our findings suggest that improving sensitivity by using two parallel IHC reactions might be beneficial; we recommend primarily HercepTest and Pathway CB11. Nevertheless, we may consider performing FISH analysis without prior IHC screening.

Association between the epstein-barr virus and hodgkin's lymphoma in the north-eastern part of Hungary : Effects on therapy and survival

This retrospective study included 109 patients with Hodgkin’s lymphoma (HL; 45 females, 64 males). In 47 of the 109 HL patients (43%), immunohistochemical analysis of their formalin-fixed, paraffin-embedded histologic samples revealed Epstein-Barr virus (EBV) by latent membrane protein (LMP) 1. The highest virus association (50%) was found with the mixed cellularity histologic subtype, especially in patients aged 11–20 and >50 years. Virus positivity in nodular sclerosis was 35% (negative cases accumulated in patients aged 15–30 years). Regarding clinical stages, histologic subtypes, general symptoms, treatments employed and response to treatment, the EBV-positive group was not significantly different from the virus-negative group. During the mean follow-up time of 83 months (9–300 months), the overall or event-free survival of EBV-negative patients was more favorable than that of EBV-positive patients, although the difference was not significant (p = 0.16 and p = 0.24, respectively). EBV infection may be involved in the pathogenesis of HL in our Hungarian study cohort, but it does not significantly affect clinical symptoms, therapeutic results or complete and event-free survival of HL patients.

Comparison of dual-color dual-hapten brightfield in situ hybridization (DDISH) and fluorescence in situ hybridization in breast cancer HER2 assessment

The most optimal method for assessing HER2 status is still subject to controversy as far as the type of assay used, the optimal method to perform, and the costs of each assay are concerned. The current study was done as a validation study prior to setting up a clinical HER2 testing service using the new commercial dual-color dual-hapten brightfield in situ hybridization (DDISH), but it was felt that our experience may be of interest to other laboratories considering setting up HER2 diagnostic facilities. One hundred and five patients diagnosed with invasive breast cancer were selected. PathVysion FISH and DDISH assays were carried out. Concordance correlation coefficients showed near perfect agreement in average HER2 and centromere-specific signal counts per cell and in HER2/CEN17 ratios between the PathVysion and the DDISH assays, and also the Kappa measure showed near perfect agreement between the two assays (Kappa=0.8712, P<0.0001). Statistical analysis confirmed that the two assays are comparable in terms of detection of HER2 gene amplification and suggests its utilization in routine HER2 diagnostics.

Sarcoidosis in patients with mixed connective tissue disease: Clinical, genetic, serological and histological observations

The objective of this study was to investigate how the development of sarcoidosis influences the disease course of mixed connective tissue disease (MCTD). The cellular composition of MCTD-associated sarcoidosis granulomas was evaluated and also the disease-accompanying T-cell activation and alterations of the serum cytokine levels were measured before and after the therapy. The HLA-DR specific alleles were also assessed. We present two cases with MCTD coexisting sarcoidosis. Serum concentrations of Th1 and Th2 cytokines were assessed by ELISA. Peripheral blood CD3+ total T-cell numbers, CD4+ and CD8+ T-cell subset were determined by flow cytometry. Furthermore, hematoxylin-eosin staining and immunhistochemistry were performed for histological assessment. HLA-DR specific alleles were determined by using PCR-SSP. Elevated number of activated T-cells and high Th1 cytokine levels were detected, mainly IFN-gamma and TNF-alpha. Histologically, CD4+ and CD8+ T-cells were present in the sarcoidosis infiltrations. The haplotypes were to some extent dissimilar from the HLA-DR genotype from patients with MCTD, or sarcoidosis alone. Sarcoidosis enhances the activation of MCTD, based on the laboratory and clinical findings. Our results show that the inflammation is mainly in the effector phase, while granuloma formation is characteristic of the resolution phase of the disease. The assessment of the cytokine network in sarcoidosis-associated MCTD enables us to select the most effective, individualized therapy protocol for these patients.

Impact of chromosome 17 centromere region assessment on HER2 status reported in breast cancer.

Recent studies have indicated that polysomy 17 is a rare event in breast cancer, and polysomy is usually mimicked in FISH analysis by gain or amplification of the centromere covered by the chromosome 17 centromere probe. To estimate the impact of chromosome 17 centromere assessment on routine practice, we conducted a retrospective re-classification study. Four hundred and five consecutive cases were selected. The original molecular pathology reports were available. Centromere 17 copy counts were ignored in the reassessment. Altogether, nineteen (4.69%) discrepant cases were found, from which five (1.23%) were considered originally non-amplified but had an HER2 copy number >6. Therefore, we reclassified them as HER2-amplified, while fourteen (3.46%) cases were originally considered amplified with 6 or fewer HER2 signals/cell. The discrepant cases found in our reassessment study would require further high-resolution genetic analysis to resolve the disagreement. On the other hand, our result also highlights that for the vast majority of breast cancer cases traditional FISH examination is still adequate to reach the correct diagnosis. This diagnostic gap must be filled by more sophisticated genetic examinations. Moreover, upcoming HER2 guidelines should consider the aid that high-resolution karyotyping can give to the diagnostic algorithm.