Primary retroperitoneal Ewings sarcoma.

The Ewings sarcoma (EWS) family tumors are small, round, cell tumors with different degrees of neuroectodermal differentiation with a peak incidence in children and young adults. About 10-20% of cases are extraskeletal EWS.

An undifferentiated sarcoma with BCOR-CCNB3 fusion transcript - pathological and clinical retrospective study.

The BCOR-CCNB3 positive sarcoma is a recently identified sarcoma morphologically and clinically similar to Ewing sarcoma in adolescents and young adults. The BCOR-CCNB3 fusion transcript originates from a paracentric inversion on the X chromosome with an in-frame fusion between the last codon of BCOR and the exon 5 of CCNB3 gene. We report morphological and molecular genetic analysis of 8 undifferentiated sarcomas positive for the BCOR-CCNB3 fusion. Six of the eight BCOR-CCNB3 positive sarcoma patients were male. Five of the eight patients were in their second decade of life (median of all patients 14 years at diagnosis). The bone marrow involvement was demonstrated in 2 of 4 patients tested. Detection of the fusion transcripts BCOR-CCNB3 in the bone marrow suggests that patients with positive findings are at high risk of the tumor progression.

[Non-Hodgkin´s B-lymphoma of the ovaries with an unfavourable prognosis - incidental finding during caesarean section]. / Non-Hodgkinuv B-lymfom ovarií s nepríznivou prognózou - náhodný nález pri císarském rezu.

OBJECTIVE: Presentation of a rare finding non-Hodgkin´s B-lymphoma of the ovary in a patient during caesarean section. DESIGN: Case report. SETTINGS: Department of Obstetrics and Gynaecology, Regional Hospital Liberec, a.s.; First Internal Clinic - Clinic of Hematology, First Faculty of Medicine and General University Hospital, Charles University in Prague; Department of Pathology and Molecular Medicine, Second Faculty of Medicine, Charles University in Praque and Motol University Hospital. RESULTS: Pregnant woman, 31-years old, primiparous, with a history of caesarean section was examinated in our department due to nonspecific abdominal pain during her pregnancy. During the caesarean section of fetal indication we found bilateral ovarian tumours. We performed unilateral adnexectomy. Preliminary diagnosis from frozen section was thecoma, but final diagnosis (after definitive histology, imunohistochemistry and molecular investigation) was high-grade B-cell non-Hodgkin´s lymphoma with c-myc and bcl-6 gene rearrangement (double-hit lymphoma) resulting in an unfavourable prognosis. The patient consequently completed 6 cycles of chemotherapy with a biological treatment, and achieved a complete remission. However, after 6 months, an early generalisation to the CNS appeared, leading to intracranial hypertension refractory to anithypertensive and anti-oedematous therapy, consequently leading to death. CONCLUSION: Non-Hodgkin´s lymphoma of the ovary in pregnancy is a rare adnexal tumour whose treatment requires interdisciplinary cooperation.

[Clinical and Functional Importance of Selected CASP8 and CASP9 Polymorphisms in Breast Carcinoma]. / Klinický a funkcní význam vybraných polymorfizmu CASP8 a CASP9 u karcinomu prsu.

BACKGROUND: Caspase-8 and caspase-9 (encoded by CASP8 and CASP9) are executive caspases of programmed cell death (apoptosis). Dysregulation of apoptosis plays an important role in cancer development, progression, and resistance to anticancer therapy. The goal of this work was to evaluate potential associations between polymorphisms in CASP8 and CASP9, previously linked to breast cancer risk, and the transcript levels of these genes (including their alternative anti-apoptotic variants) in tumor tissues and the clinical characteristics of the patients. MATERIAL AND METHODS: Sanger sequencing, high resolution melting (HRM) analysis, and allelic discrimination were used to identify polymorphisms in DNA samples isolated from tumor tissues and peripheral blood lymphocytes of 60 breast carcinoma patients. Total transcript levels of CASP8 and CASP9, and levels of alternative splicing variants CASP8L and CASP9B, were quantified by real-time PCR in tumor tissues. Clinically interesting associations were validated in DNA from lymphocytes of 615 breast carcinoma patients. RESULTS: A haplotype in CASP9 composed of three polymorphisms rs4645978-rs2020903-rs4646034 was significantly associated with CASP9 expression in tumors, with the expression of the progesterone receptor and ERBB2, and with the TNBC subtype of breast carcinoma in the validation study. The associations between the rs3834129 polymorphism in CASP8 and stage of disease, rs6435074 with grade, expression of estrogen receptor and ERBB2, and rs6723097 with ERBB2 expression have not yet been validated. However, rs6723097 was associated with disease-free survival in patients treated with hormonal therapy. CONCLUSION: This study reveals a previously unknown and presumably functional (in silico) association between a haplotype in CASP9 and molecular and clinical phenotypes of breast carcinoma. The potential clinical utility of this association for prognostication of breast carcinoma should be evaluated by independent studies.Key words: breast carcinoma - caspases - polymorphisms - functional - clinical - importanceThis work was supported by grant of the CU Grant Agency No. 1444313, and grant of the Internal Grant Agency of the Czech Ministry of Health No. 15-25618A.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 3. 3. 2016Accepted: 26. 10. 2016.

[Histiocytic necrotizing lymphadenitis / Kikuchi-Fujimoto disease (HNL/K-F) and its differential diagnosis: analysis of 19 patients]. / Histiocytární nekrotizující lymfadenitida /Kikuchiho-Fujimotova choroba (HNL/K-F) a její diferenciální diagnostika: analýza 19 prípadu

Histiocytic necrotizing lymphadenitis / Kikuchi-Fujimoto disease (HNL/K-F) is being recognized with an increasing frequency not only in the East Asia but also on the American continents and in the Europe. Still the diagnostics of HNL/K-F is not easy and difficulties with its proper classification persist. In a group of 19 patients diagnosed primarily or as consults at our department there were 12 woman and 7 men. An average age at diagnosis was 28 years, median 25 years. Cervical lymph nodes were involved in 18 patients. Bilateral lymphadenopathy was present in one patient, the remaining 17 were unilateral. Inguinal lymph node was affected in one patient. In one other patient there were enlarged retroperitoneal lymph nodes simultaneously with a cervical lymphadenopathy. The size of the lymph nodes varied between 5 mm to 32 mm. The subclassification showed the necrotizing type in 14 patients, in one there was a predominant xanthomatous tissue reaction around the necrotic areas (xanthomatous type), and in 4 patients the disease was recognized as the proliferative type without necrosis (in two with a variously intense apoptosis of the proliferating lymphocytes). Of 10 consult cases the tumor was primarily evaluated as B cell lymphoma not otherwise specified (1x), peripheral T cell lymphoma (1x), classical Hodgkin lymphoma of mixed cellularity (1x); two patients were submitted with a differential diagnosis between peripheral T cell lymphoma and HNL/K-F; in one diagnosis of probable EBV lymphadenitis and in one diagnosis HNL/K-F was made. There were no data submitted in the remaining three cases. The authors stress diagnostic features which should lead to the diagnosis of the disease and should prevent unnecessary oncological staging investigations and potential chemotherapy for a lymphoma. Among diagnostic features of HNL/K-F identification of the proliferating cells - CD8 activated lymphocytes with apoptotic decay prevail, there are frequent plasmacytoid monocytes and a striking reaction of macrophages which are CD68/myeloperoxidase positive. There are virtually no neutrophil granulocytes and there is a miminal participation of plasma cells. In case of necrotizing and xanthomatous type infectious causes are to be ruled out as well. In case we still need to distinguish HNL/K-F from a lymphoma PCR analysis of a rearrangement of the immunoreceptor gene in T cell population should be investigated.

Burkitt lymphoma (BL): reclassification of 39 lymphomas diagnosed as BL or Burkitt-like lymphoma in the past based on immunohistochemistry and fluorescence in situ hybridization.

Burkitt lymphoma (BL) is a well characterized entity. For atypical findings a term Burkitt-like lymphoma (B-LL) was applied in the past, but the interpretation of the morphological appearances was subjective and poorly reproducible. We used a combined approach (morphology using classical histological staining; immunohistochemistry-IHC; fluorescence in situ hybridization-FISH on interphase nuclei; cytogenetics) to perform a retrospective study on 39 patients diagnosed as BL and B-LL at our department in the years 1982 to 2002. By FISH we demonstrated t(8;14)(q24;q32) in 31 patients; in further two we found a break at 8q24, suggestive of a variant translocation. In three patients with the cytogenetic investigation available we confirmed the findings of FISH--two lymphomas had the t(8;14)(q24;q32), one had t(2;8)(p12;q24). IHC showed CD20, CD10, BCL-6, p53 expression, and Ki-67 antigen in > 95% of the tumor cell population in a majority of the patients. There was a group of 4 patients in whom the t(8;14)(q24;q32) or a break at 8q24 were not found (FISH). These cases were reclassified within the WHO defined grey zone subgroup of B-cell lymphoma unclassifiable with features intermediate between diffuse large cell lymphoma (DLBCL) and Burkitt lymphoma--I-DLBCL/BL. Two further cases were reclassified as DLBCL based on a combined IHC and FISH findings. A lymphoma of one of these patients had breaks at 3q27 (BCL6) and at 14q32 (IGH) suggestive of t(3;14)(q27;q32). The overall survival estimate of 33 patients with the diagnosis of BL was 54%. Most of deaths occurred within 6 months after the tumor diagnosis. The unfavorable clinical outcome appears to be associated with a strong expression of the p53 protein in the tumor cell population. Individually utilized methods in the diagnosis of BL may lead to false diagnostic conclusions. A combined approach helps to establish a more reliable diagnosis of BL and to separate grey zone lymphomas I-DLBCL/BL and DLBCL with morphological mimics of BL to start adequate treatment. I-DLBCL/BL is a non-homogenous group of lymphomas necessitating further analysis in a prospective study.

Quantitative molecular analysis in mantle cell lymphoma.

A molecular analysis has three major roles in modern oncopathology--as an aid in the differential diagnosis, in molecular monitoring of diseases, and in estimation of the potential prognosis. In this report we review the application of the molecular analysis in a group of patients with mantle cell lymphoma (MCL). We demonstrate that detection of the cyclin D1 mRNA level is a molecular marker in 98% of patients with MCL. Cyclin D1 quantitative monitoring is specific and sensitive for the differential diagnosis and for the molecular monitoring of the disease in the bone marrow. Moreover, the dynamics of cyclin D1 in bone marrow reflects the disease development and it predicts the clinical course. We employed the molecular analysis for a precise quantitative detection of proliferation markers, Ki-67, topoisomerase IIalpha, and TPX2, that are described as effective prognostic factors. Using the molecular approach it is possible to measure the proliferation rate in a reproducible, standard way which is an essential prerequisite for using the proliferation activity as a routine clinical tool. Comparing with immunophenotyping we may conclude that the quantitative PCR-based analysis is a useful, reliable, rapid, reproducible, sensitive and specific method broadening our diagnostic tools in hematopathology. In comparison to interphase FISH in paraffin sections quantitative PCR is less technically demanding and less time-consuming and furthermore it is more sensitive in detecting small changes in the mRNA level. Moreover, quantitative PCR is the only technology which provides precise and reproducible quantitative information about the expression level. Therefore it may be used to demonstrate the decrease or increase of a tumor-specific marker in bone marrow in comparison with a previously aspirated specimen. Thus, it has a powerful potential to monitor the course of the disease in correlation with clinical data.

A whole population study of gastrointestinal stromal tumors in the Czech Republic and Slovakia.

Due to problems with identification and an incomplete understanding on the gastrointestinal stromal tumors (GIST) before 2001, there has been a lack of comprehensive long-term population-based studies on GIST epidemiology at present date. We used data from the online registry of Czech and Slovak GIST patients (http://gist.registry.cz/), which has been compiled and maintained since 2006 and involves patients diagnosed from the year 2000. 278 patients were included in this study. Most of the tumors fell into the high-risk category (58.7%), followed by the intermediate (21.4%), low (16.6%) and very low (3.3%) categories. Locations other than the small intestine and stomach had significantly higher contribution of high-risk tumors. The median time of overall survival was 93.2 months, 5-year relative survival was 78.3% overall, 71.9% for patients with high-risk tumors, 91.1% for intermediate patients, and 91.9% for patients from the low- and very low-risk category. The annual crude incidence between the years 2001-2005 was 0.52 cases per 100,000 inhabitants. The annual European ASR and World ASR were 0.44 and 0.31 per 100,000 inhabitants, respectively. Presented data generally correspond to the whole-population studies recently published, including actual data on epidemiology, clinical characteristics, and survival of patients. The registry helps in improving GIST diagnostics, knowledge about the properties and behaviour of tumors, communication among physicians, and, last but not least, therapeutical options and results.

Clinical, pathological and molecular characteristics of newly diagnosed breast cancers.

Selection of breast carcinoma therapy is based on standard prognostic markers, such as tumor size, infiltration of regional lymph nodes, tumor grade, and expression of hormonal receptors. Insufficient treatment results stimulate a search for new markers which may lead to a more precise characterization of these tumors and to a more effective treatment. In our study we determined essential clinical and histopathological characteristics of non-metastasizing breast cancer - primary tumor size, involvement of the regional lymph nodes, expression of hormonal receptors and a status of ERBB-2 protein (HER-2), DNA ploidy, and their possible inter-correlation. In this study 77 patients were analyzed. The mean age was 59.3 years. Tumor stage T1 was found in 53%, T2 in 39%, T3 in 5% of patients. 57% of patients did not show any metastases in the axillary lymph nodes. A higher tumor grade 3 was seen mainly in larger tumors, in 62% of T2 and 66% of T3 tumors; 77% of carcinomas expressed hormonal receptors. HER-2 expression was shown in 21 T1 tumors, 13 T2 tumors, and 1 T3 tumor. 47 tumors were diploid. 13 T1 tumors, 14 T2 tumors, and 2 T3 tumors were aneuploid. Any significant correlation among staging T, N and ERBB-2 expression, hormonal receptors expression, tumor grade and DNA ploidy was found.

Rhabdomyosarcoma: molecular diagnostics of patients classified by morphology and immunohistochemistry with emphasis on bone marrow and purged peripheral blood progenitor cells involvement.

Two histologically distinct subtypes of rhabdomyosarcomas (RMS), embryonal and alveolar, are different in many aspects, such as age distribution, primary site, and clinical outcome. We analyzed a group of 30 patients with RMS. The aim was to broaden the spectrum of diagnostic tools in evaluating the primary tumors, their recurrences and/or metastases, and to extend the diagnostic boundary to bone marrow and purged peripheral progenitor blood cell samples. We have performed the RT-PCR assay to analyze RMS for the presence of expression of MyoD1 gene and for the presence of chimeric transcripts PAX3/FKHR or PAX7/FKHR. MyoD1 gene expression was found in all 30 patients in samples from primary tumors. The chimeric transcripts PAX/FKHR were identified in 13 of 15 patients with alveolar RMS. Furthermore, the fusion transcript PAX7/FKHR was identified in 2 of 15 patients with RMS classified as embryonal by histology. Bone marrow samples (12) and peripheral blood progenitor cell specimens (13) in ten patients were examined by RT-PCR. We were able to identify 7 patients with bone marrow involvement and/or with contamination of peripheral blood progenitor cells by the tumor cells. We demonstrate that employing molecular diagnostics has an impact on staging, therapy monitoring and recognition of malignant cells at the tumor resection margins.

[Treatment results of Langerhans cell histiocytosis with LSH II protocol]. / Výsledky lécby histiocytózy z Langerhansových bunek protokolem LCH II.

BACKGROUND: The aim of study was to evaluate outcome of international treatment protocol LCH II for children with Langerhans cell histiocytosis treated in FN Motol. METHODS AND RESULTS: Between November 1995 and December 2003, 46 children were treated, sex ratio M:F 29:17 and median age at diagnosis 6 years 8 months. 28 children (60.9%) suffered from monosystem disease with majority of bone lesions (23 times) with skull predominance (16 times). Surgery was primary treatment modality for monosystem disease. Five children with recurrence were successfully treated by protocol LCH II - LR (3x) and LCH III - LR /G2/, respectively. Eighteen children (39.1%) suffered from multisystem disease. 6 out of 18 patients were treated according to low-risk protocol LCH II - LR and 12 children by high-risk scheme LCH II - HR at the non-randomized branch included etoposide. Recurrence was revealed in 11 patients and 10 of them reached 2nd or 3rd complete remission (CR) by 2 - chlorodeoxyadenosine (CDA) monotherapy, and 1 child reached 2nd CR by LCH II - HR scheme. Two children underwent irradiation after bone lesion excision as well as 1 child as supplemental treatment. Totally, 29 children (63.0%) achieved 1st CR, 14 (30.4%) 2nd CR, 2 (4.4%) 3rd CR, and 1 child died because of LCH progression. There were no severe side effects of chemotherapy. Follow-up median time was 5 years 8 months (range 9 months - 9 years 6 months). CONCLUSIONS: LCH II protocol is safe and effective. Results revealed that treatment of patients with multisystem disease might demand some treatment modification.

Epidermal growth factor receptor--its expression and copy numbers of EGFR gene in patients with head and neck squamous cell carcinomas.

Signaling pathways activated by epidermal growth factor receptor (EGFR) are pathogenetically involved in the development of head and neck squamous cell carcinomas (HNSCC). A monoclonal antibody against the EGFR protein blocking the receptor activity (cetuximab - Erbitux - C225) is now available for therapeutic applications. The mechanisms of EGFR protein overexpression are poorly understood. Regulatory pathways, EGFR gene structural changes or its amplification may be involved. The aim of the study was to evaluate expression of the EGFR protein in patients with HNSCC, to identify EGFR gene copy numbers, and to find out whether the protein overexpression is associated with the EGFR gene amplification. In the case of a pathogenetical link of the EGFR gene amplification and the protein overexpression it would be useful to employ both diagnostic approaches to identify patients eligible for cetuximab therapy. We investigated 33 patients with HNSCC. The expression of EGFR protein was evaluated by immunohistochemistry, copy numbers of EGFR gene and the numbers of chromosome 7 centromeric signals were investigated by fluorescence in situ hybridization on interphasic nuclei (I-FISH). Histological sections from formalin fixed and paraffin embedded tissues were used. We observed three types of EGFR protein expression (homogeneous 3+ membrane positivity in 13 patients; membrane positivity varying from 1+ to 3+ in 12 patients; a strong membrane positivity at the periphery of the tumor cell clusters in 5 patients). In two cases the results were difficult to interpret. In one case single tumor cells only were positive. Numerical changes of chromosome 7 were present in 23 patients. We found the EGFR gene amplification in seven patients. The tumor cells with amplification of the EGFR gene were generally infrequent and were localized in small clusters, or they were randomly dispersed between the tumor cell population without the gene amplification. We did not find any correlation between the EGFR gene amplification and the EGFR protein overexpression. Thus, amplification of the EGFR gene is not pathogenetically involved in the EGFR protein overexpression. From the diagnostic aspect a standardized immunohistochemical assessment of the EGFR protein expression appears sufficient for detection of the EGFR status. Criteria for cetuximab treatment in patients with HNSCC may differ from those already used for patients with colorectal carcinomas and should take different patterns of the EGFR protein overexpression into consideration.

Paget's disease of the nipple: a copy number of the genes ERBB2 and CCND1 versus expression of the proteins ERBB-2 and cyclin D1.

Paget's disease of the nipple (PDN) is characterized by Paget's cells infiltrating from an underlying breast carcinoma into the epidermis of the nipple. The tumor cells were reported to overexpress ERBB-2 protein. There is no evidence, whether the overexpression is caused by amplification of the ERBB2 gene (as is the case of invasive duct carcinomas) or by other causes. Because the Paget's cells proliferate vividly we were interested also in cyclin D1 expression and in its relation to the number of CCND1 gene copies. To address these issues, we performed a study using fluorescence in situ hybridization on interphasic nuclei (I-FISH) on formalin fixed / paraffin embedded tissue sections from twelve women with PDN. We compared immunohistochemical (IHC) expression of the ERBB-2 protein and cyclin D1 with the copy number of the gene ERBB2 and CCND1, respectively (I-FISH). In all cases of our series we found overexpression of the ERBB-2 protein (3+), and in all of them there was a strong amplification of the ERBB2 gene (more than 10 signals per a nucleus, usually about 20). Keratinocytes of the epidermis had two signals of the gene. IHC and I-FISH revealed comparable findings in successive biopsies in two patients. Cyclin D1 was positive in seven cases. We found a moderate amplification of the CCND1 gene (up to 10 signals per a nucleus) in three patients only. Any correlation between the cyclin D1 overexpression anda copy number of the CCND1 gene was observed. Combined numerical changes of chromosome 17 and of chromosome 11 were found in seven women. It is concluded that in PDN, the ERBB-2 protein overexpression is caused by amplification of the ERBB2 gene. A specific immuno-therapy with trastuzumab (Herceptin) used in patients with invasive duct carcinoma may be also effective in patients with PDN.

[Anaplastic large-cell lymphoma: review]. / Anaplastický velkobunecný lymfom: prehled problematiky.

Anaplastic large cell lymphomas (ALCLs) represent a heterogeneous group of malignant lymphoproliferative diseases. Most of the cases are of T-cell line with a loss of cell surface receptors but with a production of cytotoxic cytoplasmatic granules--immunohistochemically (IHC) positive perforin, granzyme B, and TIA-1. The diagnostics of ALCL is based on morphological findings and results of IHC, which further stratify ALCLs to basic immunophenotypes according to ALK (anaplastic lymphoma kinase) protein expression--ALCL CD30+ ALK+ and ALCL CD30+ ALK+. The morphological investigations are supplemented by karyotyping and/or by a demonstration of breakpoint at 2p23 harboring ALK gene (FISH), and by molecular detection of chimeric genes characteristic of ALK+ lymphomas (NPM-ALK, ATIC-ALK, TPM3-ALK, TFG-ALK, and some even rarer rearrangements). Molecular diagnostics is important in monitoring minimal residual disease. As some of the characteristic molecular changes were demonstrated in healthy individuals and in Hodgkin's disease by quantitative PCR, the validation of these findings demands further studies. ALK protein positive ALCLs affect patients in age categories up to the third decade, whereas ALK protein negative cases occur in older patients with an average age of 60 years. Both subgroups of lymphomas are aggressive but ALK+ lymphomas react well to systemic treatment, and have a more favorable prognosis. Primary skin ALCLs belong to a group of T-cell lymphoproliferative diseases of the skin and have, in the majority of cases, a favorable course without generalization.

[Follicular lymphomas: molecular diagnosis using t(14;18)(q32;q21)--fluorescence in situ hybridization, qualitative and quantitative PCR]. / Folikulární lymfomy: molekulární diagnostika t(14;18)(q32;q21)--fluorescencní in situ hybridizace, kvalitativní a kvantitativní PCR.

Diagnosis of follicular lymphoma (FL) is based on histology and immunohistochemical profile (CD20+, CD79alfa+, CD10+, BCL-2+, CD5-). A chromosomal marker--translocation t(14;18)(q32;q21) supporting the tumor diagnosis and useful for monitoring bone marrow or peripheral blood infiltration by the tumor cells is also used. The BCL2 gene (18q21) is controlled by an enhancer of the IGH gene (14q32) resulting in BCL-2 protein overexpression. The translocation is present in the majority of patients with FL. The aim of the study was to introduce the quantitative PCR (RQ-PCR, real-time quantification) method for the assessment of the quantity of cells bearing the translocation t(14;18) in patients with FL. The fluorescence in situ hybridization on interphasic nuclei (I-FISH) in histologic sections was used for screening of patients with the t(14;18). A search for the break of the BCL2 gene at the major breakpoint region (mbr) was performed by means of qualitative PCR. We determined the relative number of the tumor cells bearing t(14;18) translocation (mbr) in patients with FL by the RQ-PCR. The relative quantity of these cells was significantly higher in the lymph nodes than in the bone marrow or peripheral blood. The RQ-PCR is a tool of choice to monitor the activity of the disease in individual patients, and to detect an early disease relapse before its manifestation at the level diagnosed by morphology.

[Large-cell diffuse B-cell lymphoma: heterogenous origin and prognosis from the aspect of modern diagnosis]. / Velkobunecné difuzní B lymfomy: heterogenní puvod a prognóza z hlediska soucasné diagnostiky.

BACKGROUND: Diffuse large B-cell lymphomas represent a heterogeneous group of tumors with a different origin, morphological findings and a variable clinical prognosis. These tumors have been recently classified into two prognostically relevant subgroups differing in the gene expression. The key genes suitable for routine diagnostics of DLBCL have not been yet identified. The aim of this work was to study changes and expression of several genes and proteins participating in the genesis of DLBCL. METHODS AND RESULTS: We analysed a group of 31 patients with diffuse large B-cell lymphomas. Basic clinical data including follow-up of the patients were available. Tumors were examined by a panel of immunohistochemical reactions with antibodies against CD20, CD79a, BCL-2, BCL-6, CD10, Ki-67 and TP53. FISH was used to detect a translocation t(14;18)(q32;q21) and/or a break in BCL6 region (3q27) suggestive of a translocation with a variable translocation partner t(3;?). PCR was utilized to detect the translocation t(14;18) and a clonal rearrangement of heavy and/or kappa chain of the immunoglobin genes. CONCLUSIONS: The expression of BCL-2 protein appeared to correlate with a higher mortality rate. The expression of other proteins examined in the study did not correspond significantly with the clinical development of the disease. Tumors with follicular lymphoma as a component had significantly higher mortality rate than the tumors developing de novo. Moreover, higher mortality was evident in cases with higher values of the International Prognostic Index (IPI).

[Indications for treatment in invasive ductal carcinoma of the breast with Herceptin from the aspect of laboratory diagnosis--study of the ERBB-2 protein and determination of the number of copies of the ERBB2 gene. Review]. / Indikace k lécbe nemocných s invazivními duktálními karcinomy mlécné zlázy Herceptinem z pohledu laboratorní diagnostiky--vysetrení ERBB-2 proteinu a stanovení poctu kopií ERBB2 genu. Prehled problematiky.

Overexpression of the ERBB-2 protein has become a target of the anti-neoplastic treatment in patients with invasive duct carcinomas of the breast with a monoclonal antibody trastuzumab (Herceptin, Genentech). From this reason the immunohistochemical (IHC) evaluation of ERBB-2 protein expression is crucial. However, there are patients in whom the IHC investigation is biased by a subjective evaluation among cases considered negative (in the range of 1+) and positive (2+), and among cases considered positive 2+ and 3+. In such cases it is advisable to complement the IHC investigation by detection of copy numbers of the ERBB2 gene with fluorescence in situ hybridization (FISH). The overexpression of the protein is caused by the gene amplification in a majority of cases. The patients, whose carcinomas show overexpression of the ERBB-2 protein and the overexpression is caused by a significant gene amplification, profit from the Herceptin therapy. In this review we focus also on the methodology of FISH in paraffin sections and tissue imprints with respect to the detection of copy numbers of chromosome 17 and the ERBB2 gene.

[Immunohistochemical detection of HER2 protein and its evaluation in breast carcinoma: comparative study of two methods (HercepTest DAKO versus the routine immunohistochemical approach with the DAKO polyclonal antibody) and the results from two laboratories]. / Imunohistochemický (IHC) prukaz HER2 proteinu a jeho hodnocení u nemocných s karcinomy mlécné zlázy: srovnávací studie dvou pracovních postupu (HercepTest DAKO versus bezný IHC postup s polyklonální protilátkou DAKO) a výsledku dvou pracovist.

Routine assessing of HER2 status is necessary for successful treatment of carcinoma of breast by Herceptin. Our results prove comparability of well standardized semiquantitative method by Hercep Test DAKO: both negative findings (73.4% in laboratory A, and 77.4% in laboratory B) and positive findings (26.6% in laboratory A, and 22.6% in laboratory B) corresponded both with each other and with the DAKO's reference data. The study also testifies to problems with interpretation of the IHC data (primary antibody c-erbB-2-oncoprotein DAKO). The problems concern particularly the processing standards, the dilution of antibodies, the application of IHC automatic machine, and probably also the detection system. It is suggested that each laboratory develops a standardized IHC protocol, in coordination with other laboratories. Thus, standardization of both processing and evaluation of the results will be achieved. As the indication for treatment by Herceptin is connected with HER2 overexpression, it is suggested to follow the algorhythm of HER2 examination presented by Nenutil at the ROCHE Satellite symposium.

[Invasive ductal carcinoma of the breast: study of the number of copies of the CCND1 gene and chromosome 11 using fluorescence in situ hybridization (FISH) in comparison with expression of cyclin D1 protein and estrogen receptors (ER alpha) with immunohistochemical detection]. / Invazivní duktální karcinomy mlécné zlázy: vysetrení poctu kopií genu CCND1 a poctu chromozómu 11 metodou fluorescencní in situ hybridizace (FISH) v porovnání s expresí proteinu cyklin D1 a receptoru pro estrogen (ER alpha) detegovanou imunohistochemicky (IHC).

BACKGROUND: Overexpression of oncogenic proteins may be caused by gene amplifications. Cyclin D1 participates in regulation of the cell cycle. Relations between cyclin D1 expression and amplification of CCND1 gene encoding this protein in invasive duct breast carcinomas (IDC) are not fully elucidated. An increased interest is also focused on relations to the estrogen receptor (ER alpha). METHODS AND RESULTS: We investigated copy numbers of the CCND1 gene, expression of cyclin D1 and expression of ER alpha in a group of 60 females and 1 male with IDC. The age range varied from 33 to 89 years (median 57 years). The number of CCND1 gene copies and the number of chromosome 11 was evaluated using FISH, the expression of cyclin D1 and ER alpha was investigated by IHC. We detected a strong amplification of CCND1 gene (> 10 copies per tumor cell nuclei) in 9 patients, weak amplification (< or = copies) in 16 patients. Amplification of the CCND1 gene correlated well with the overexpression of cyclin D1. We observed the overexpression of cyclin D1 also in 13 of 36 patients without the gene amplification; therefore, the mechanism of the protein overexpression is different than that caused by the gene amplification in a proportion of patients. Amplification of the CCND1 gene was associated with a high histologic grade of IDC, whereas cases with cyclin D1 overexpression only were not. 24 of 31 patients with overexpression of cyclin D1 coexpressed ER alpha. We did not find correlation between expression/amplification of cyclin D1/CCND1 gene and the size of carcinomas and with metastases to the axillary lymph nodes. CONCLUSIONS: Amplification of the CCND1 gene is associated with overexpression of cyclin D1 in a majority of IDC. Overexpression of cyclin D1 is related to an increased expression of ER alpha. Interaction between cyclin D1 and ER alpha may explain low response to anti-estrogen therapy of some patients.

Kinetics of heme interaction with heme-binding proteins: the effect of heme aggregation state.

The kinetics of the interaction of heme with hemopexin and albumin was monitored by measuring the time dependence of changes in the Soret absorption spectra. Since the protein binding sites can only bind heme monomers, the binding kinetics apparently reflected the slow dissociation of heme dimers, resulting from dimer/monomer equilibria in aqueous heme solutions. The dissociation of heme dimers is characterized by the rate constant of (3-4) x 10(-3) s(-1). The measurements further revealed significant differences in the kinetic profiles (slowing down the binding interaction) that were dependent on the storage time of heme solutions at room temperature. These presumably responded to the gradual formation of higher aggregates of heme, which cannot dissociate into dimers/monomers. Alternatively, partial autooxidation of heme molecules could increase the stability of heme dimers and obstruct specific binding of heme to the proteins.