Kristof Egervari

Correlation among metallothionein expression, intratumoural macrophage infiltration and the risk of metastasis in human cutaneous malignant melanoma

Backgroundu2002 The formation of metastases and the efficacy of systemic therapies in cutaneous malignant melanoma (CMM) depend on the characteristics of the tumour cells and the host immune response. Aberrant expression of metallothionein (MT) has been observed in several types of cancers with poor prognoses.

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. n nWe 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. n nNone 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 u200b(Table11). n n n nTable 1 n nResults of IHC of c-Met and FISH of LSI D7S486/CEP7 applied to lobular and ductal carcinomas n n n nOut 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 u200b(Table22). n n n nTable 2 n nIHC and FISH results of MET according to the status of PR receptor in DIC carcinomas n n n nWe 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]. n nOur study suggests that it would be interesting to investigate new therapeutic options for ER+/PR- DIC, which may include c-Met inhibitors.

Intraoperative evaluation of sentinel lymph nodes in breast cancer: comparison of frozen sections, imprint cytology and immunocytochemistry.

M. Francz, K. Egervari and Z. Szollosiu2028Intraoperative evaluation of sentinel lymph nodes in breast cancer: comparison of frozen sections, imprint cytology and immunocytochemistry

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.

Aurora kinase B expression in breast carcinoma: cell kinetic and genetic aspects.

Background: Mitotic deregulations may contribute significantly to cell division errors and the development of aggressive tumor cells. The mitotic kinase Aurora B is essential for chromosome segregation. Its gene is located at 17p13 in close proximity to the TP53 gene. Although the frequent alteration of this locus is well known, the information about the AURKB status and protein expression is limited. Methods: 50 breast carcinoma cases were evaluated for 17p13 status and chromosome 17 ploidy by FISH and for Aurora B protein by immunohistochemistry. Results: Aurora B protein expression showed a strong correlation with cell proliferation (regression coefficient = 0.77). Therefore, the Aurora B/MIB-1 index was used as a measure of expression, which showed a wide range (1–35%, mean 0.32, SD ± 0.28). A gain in the 17p13 chromosome locus could not be shown while a deletion was stated in 10/50 cases including a subset with TP53 and AURKB codeletion in 6/10 cases. The loss of TP53/AURKB loci strongly correlated with aneusomy (p < 0.0001). Conclusion: Elevated Aurora B expression frequently occurs due to an increased cell proliferation rate in breast carcinoma. Codeletion of TP53 and AURKB at 17p13 indicates a concerted mechanism leading to the survival of cell clones with deficient mitotic kinase function which could contribute to the formation of aneuploid cells and an aggressive tumor phenotype.

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.

An alternative and reliable real-time quantitative PCR method to determine HER2/neu amplification in breast cancer.

The overexpression of HER-2/neu is an independent prognostic factor of clinical outcome of breast cancer, therefore determination of HER-2/neu status is now an integral part of the clinicopathologic workup. The ways of measuring the copy number of the HER-2/neu gene in tumor cells comprise in situ hybridization techniques and real-time polymerase chain reaction (PCR). Quantitative real-time PCR is a relatively new technique for assessing HER-2/neu gene amplification with high sensitivity. However, the HER-2/neu Quantification Kit developed by Roche designed for a LightCycler 1.5 platform had been withdrawn from the commercial market; therefore, we were encouraged to design an alternative LightCycler-based method that offers the desired level of reliability. One hundred breast cancer cases with known HER-2/neu status have been examined with the original Roche developed HER-2/neu Quantification kit and the custom real-time PCR assay. The newly developed, custom PCR showed sensitivity of 91.43%, specificity of 90.63%, and accuracy of 90.91% taking fluorescence in situ hybridization results as the end point. We have described a novel real-time PCR technique for the relative quantification of the HER2/neu gene on a LightCycler 1.5 platform. We have determined that our method is eligible and ideal for the supplement of regular fluorescence in situ hybridization reactions, concerning its high sensitivity and reliability.

Comparison of Pathvysion and Poseidon HER2 FISH assays in measuring HER2 amplification in breast cancer: a validation study

Aims The current study was done as a validation study prior to setting up a clinical HER2 testing service using the new commercial Poseidon HER2 fluorescence in situ hybridisation (FISH) assay. However, it was felt that the experience of the authors of this study may be of interest to other laboratories when considering setting up a HER2 diagnostic facility. Methods 122 patients who had been diagnosed with invasive breast cancer were selected. Immunolabelling with HercepTest, PathVysion and Poseidon FISH assays were carried out using tissue microarray blocks. Results Concordance correlation coefficients showed near perfect agreement in average HER2 and centromere specific signal counts per cell and in the HER2/CEP17 ratios between the PathVysion and the Poseidon FISH assays. In addition, the κ measure showed perfect agreement (κ 0.9441, p<0.0001), and if only 2+ cases were considered there was substantial agreement (κ 0.7671, p=0.0006), between the two assays. The sensitivity and the specificity of the Poseidon FISH kit were calculated to be 95.2% and 100%, respectively, whereas the positive predictive value (PPV) and negative predictive value (NPV) were 100% and 99%, respectively. With regard to the ability to presume HER2 polysomy, the Poseidon FISH kit had a sensitivity of 93.3% and a specificity of 99.1%, with PPV and NPV of 93.3% and 99.1%, respectively, as assessed with PathVysion classification as the reference. Conclusions Statistical analysis confirmed that the two FISH assays are comparable in terms of detection of HER2 gene amplification. Proceeding from these findings, the genetic diagnoses obtained with the Poseidon kit can be considered to be as valuable as the results from the Food and Drug Administration approved PathVysion assay, and its utilisation in routine HER2 diagnostics is proposed.