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

BACKGROUND: 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. OBJECTIVE: To perform an immunohistochemical study on primary CMM comparing the MT expression of tumours without metastases (n = 23) to that of samples with haematogenous metastases (n = 23) and to examine the correlation between MT staining and immunological markers relevant in CMM progression. METHODS: The immunohistochemical labelling of different tumour sections was analysed using tissue microarrays for the evaluation of the suitability of this method in future studies. RESULTS: Our results suggest that MT overexpression is significantly more frequent in primary CMM with haematogenous metastases (P = 0.018) and that the overexpression is independent of the Breslow tumour thickness (R = 0.102, P = 0.501). Interestingly, MT overexpression of the tumour cells was correlated with the presence of tumour-infiltrating CD68(+) macrophages (P = 0.003), a known predictive factor for melanoma progression, thereby suggesting a role for MT in the development of a defective host immune response. Furthermore, the presence of CD163(+) macrophages infiltrating the tumours correlated with metastasis formation (P < 0.001), whereas the presence CD1a(+) dendritic cells surrounding the tumours was associated with a lower risk of haematogenous spread (P = 0.003). CONCLUSION: Our results demonstrate that MT may represent a suitable prognostic factor that can characterize the metastasising ability of CMM and the tumour-promoting host immune response.

Fecal expression of Escherichia coli lysine decarboxylase (LdcC) is downregulated in E-cadherin negative lobular breast carcinoma.

Breast cancer is characterized by oncobiosis, the abnormal composition of the microbiome in neoplastic diseases. The biosynthetic capacity of the oncobiotic flora in breast cancer is suppressed, as suggested by metagenomic studies. The microbiome synthesizes a set of cytostatic and antimetastatic metabolites that are downregulated in breast cancer, including cadaverine, a microbiome metabolite with cytostatic properties. We set out to assess how the protein expression of constitutive lysine decarboxylase (LdcC), a key enzyme for cadaverine production, changes in the feces of human breast cancer patients (n = 35). We found that the fecal expression of Escherichia coli LdcC is downregulated in lobular cases as compared to invasive carcinoma of no special type (NST) cases. Lobular breast carcinoma is characterized by low or absent expression of E-cadherin. Fecal E. coli LdcC protein expression is downregulated in E-cadherin negative breast cancer cases as compared to positive ones. Receiver operating characteristic (ROC) analysis of LdcC expression in lobular and NST cases revealed that fecal E. coli LdcC protein expression might have predictive values. These data suggest that the oncobiotic transformation of the microbiome indeed leads to the downregulation of the production of cytostatic and antimetastatic metabolites. In E-cadherin negative lobular carcinoma that has a higher potential for metastasis formation, the protein levels of enzymes producing antimetastatic metabolites are downregulated. This finding represents a new route that renders lobular cases permissive for metastasis formation. Furthermore, our findings underline the role of oncobiosis in regulating metastasis formation in breast cancer.

Combined automatic immunological and molecular cytogenetic analysis allows exact identification and quantification of tumor cells in the bone marrow.

PURPOSE: To improve the detection of disseminated tumor cells in bone marrow (BM) and peripheral blood samples of solid tumor patients, a novel computer-assisted scanning system for automatic search, image analysis, and repositioning of these cells was developed. This system allows precise identification and quantification of tumor cells by sequential immunological and molecular cytogenetic analysis. In this study, we attempt to demonstrate the practical use of this approach by analyzing BM samples from neuroblastoma patients. EXPERIMENTAL DESIGN: The disialo-ganglioside (GD2) molecule was used as the immunological target. The GD2 molecule was described as being specific for neuroblastoma cells, although false positive reactions had been suspected. To verify or disprove the neoplastic nature of the immunologically positive cells, sequential fluorescence in situ hybridization was performed on these cells to search for those genetic aberrations found in the corresponding primary tumors. A total of 115 samples from 40 newly diagnosed patients were evaluated for the presence of GD2(+) cells in the BM. RESULTS: GD2 positivity was detected in 95.2% of stage 4 patients, in 100% of stage 4s patients, and in 38.5% of patients with localized/regional disease. In stage 4 and 4s BM samples, the GD2(+) cells were unequivocally identified as tumor cells based on the molecular cytogenetic aberrations found by fluorescence in situ hybridization. However, in BM samples from patients with localized/regional disease, all GD2(+) cells were concluded to represent false positivity due to the absence of genetic aberrations. CONCLUSIONS: Automatic search and sequential molecular cytogenetic analysis of the immunologically positive cells provide precise information on both the number and cytogenetic profile of disseminated tumor cells.

Nucleolin and fibrillarin expression in stimulated lymphocytes and differentiating HL-60 cells. A flow cytometric assay.

Nucleolin and fibrillarin are two histone-like major proteins in the nucleolus that were found to be overexpressed in proliferating cells. Using specific antibodies to either nucleolin or fibrillarin flow cytometric, measurements were carried out to demonstrate quantitative changes of these proteins during lymphocyte mitogenic activation and differentiation of HL-60 promyelocytic leukaemia cells. The expression of nucleolin increased during lymphocyte stimulation and decreased slowly but constantly in the course of differentiation of HL-60 cells. Expression of fibrillarin reached a maximum in the first cell cycle and then dropped to a basic level in stimulated lymphocytes. Compared to nucleolin, the level of fibrillarin decreased more rapidly and more extensively in differentiating HL-60 cells. The data support other observations that nucleolin is a stabile structural protein at the ribosomal genes while fibrillarin may have a more specific functional role in nucleologenesis and ribosome production.

In situ fluorescent visualization of nucleolar organizer region-associated proteins with a thiol reagent.

Nucleolar organizer regions (NORs) are nucleolus-forming rDNA loops associated with argyrophil proteins, the amount of which varies according to the proliferative state of the cell. It has been presumed that the nucleolar protein-related thiol groups may have a role in selective silver staining. We investigated the nuclear thiol distribution with a fluorescent thiol reagent, coumarinyl-phenyl-maleimide (CPM) in human K-562 myeloblast cultures and found that SH group-related fluorescence was brightest in the area of nucleoli, which became highly selective after RNAse digestion. A remarkable co-localization of AgNOR silver reaction and CPM fluorescence was observed, although occupation of the SH groups by CPM did not prevent the silver staining. We applied the stain to dual-parameter flow cytometry in combination with DNA content measurements, which provide further information on nucleolar function and changes in experimental and pathological specimens.

Phenotypic and genotypic analyses of blastic cell population suggest that pure B-lymphoblastic leukemia may arise from myelodysplastic syndrome.

The case history of a 70-year-old man with myelodysplastic syndrome terminated into acute leukemia in 22 months is presented. The leukemic cells exhibited multifocal acid phosphatase positivity and expressed TdT, CD45, CD34 and HLA-DR but not myeloid, monocytic or megakaryocytic differentiation antigenes. The genotypic analysis revealed clonal immunoglobulin heavy chain gene rearrangement. These phenotypic and genotypic analyses of the blastic cell population suggest that myelodysplastic syndrome may transform to pure acute lymphoblastic leukemia of B-cell origin.

Solid and papillary epithelial neoplasm arising in heterotopic pancreatic tissue of the mesocolon.

AIM: Solid and papillary epithelial neoplasm (SPEN) is an uncommon pancreatic tumour. Very rarely it has also been described outside the pancreas, usually arising from heterotopic pancreatic tissue. This report summarises all the published extrapancreatic SPENs and documents the sixth such case arising from heterotopic pancreatic tissue of the transverse mesocolon in a 15 year old girl. METHODS/RESULTS: Histological and immunohistochemical examination revealed typical papillary and solid areas composed of columnar, cuboidal, and round cells, which were focally positive for vimentin, cytokeratin, neurone specific enolase, carcinoembryonic antigen, alpha1-antitrypsin, alpha1-antichymotrypsin, and negative for neuroendocrine markers (neurofilament, PGP 9.5, chromogranin A, synaptophysin, and S100), p53, and oestrogen and progesterone receptors. Electron microscopy showed scant zymogen but no neurosecretory granules. In agreement with the flow cytometric result s of diploidy, comparative genomic hybridisation (CGH) did not reveal loss or gain of genetic material, and the in situ hybridisation analysis of the RB1 and p53 genes revealed no abnormality in the 13q and 17p arms. CONCLUSIONS: Immunohistochemical and electron microscopic data support exocrine differentiation. The CGH and the flow cytometric results suggest a subtle, yet unknown genetic change, rather than a large genetic alteration. RB1 and p53 in situ hybridisation ruled out the role of deletion at these sites in the pathogenesis of SPEN. Interestingly, review of the published and the present heterotopic pancreatic SPENs identified the mesocolon as the most common anatomical site (four of six), despite the very rare occurrence of ectopic pancreatic tissue at this site.

Renal angiomyolipoma: report of three cases with regional lymph node involvement and/or with renal cell carcinoma.

AIMS AND BACKGROUND: Angiomyolipomas (AMLs) are benign hamartoid tumors which frequently occur in tuberous sclerosis (TS). They may be manifest at different organ sites such as kidneys, lymph nodes, liver and lung and may be associated with renal cell carcinoma (RCC). The nature of multiple organ involvement in AML (metastasis versus multicentric synchronous tumors), the malignant transformation and the relation of AML to RCC have not been sufficiently clarified. STUDY DESIGN: Three cases of renal AMLs in patients with tuberous sclerosis associated with lymphangioleiomyomatosis of the paraaortic lymph nodes and/or with RCC are reported. The concise clinical history of the patients as well as the findings of histology, immunohistochemistry and quantitative DNA analysis are presented. RESULTS: The multicentric form of AML and coincidence of renal AML and RCC were observed in 2 patients. AML and RCC were found within the same focus in one of the cases. RCCs were either aneuploid or "near diploid", whereas one of the multicentric AMLs showed a discordant DNA ploidy pattern, namely aneuploidy in the kidney and diploidy in the lymph nodes. CONCLUSIONS: The presented cases (all of them underwent periaortic lymphadenectomy) suggest that lymph node involvement in renal AML may be more frequent than expected (1-2% of all AMLs) on the basis of the few reported cases. The discordant DNA ploidy (renal versus lymph node lesions) observed in one of the cases with multicentric AML implies synchronous tumor growth at different sites rather than metastatic disease. The intimate coexistance of RCC and AML (RCC revealed by immunohistochemistry within a larger mass of renal AML) may indicate that malignant transformation of an AML should only be accepted, if such a coincidence is unequivocally excluded.

[Protothecosis. A new, or rarely recognized disease?]. / Protothecosis. Uj, vagy ritkán felismert megbetegedés?

Two cases of infection caused by Prototheca, belonging to the colourless algae, are discussed. In one of the patients colpitis, in the other one disease of the cubital bursa developed due to Prototheca. In the first case recovery was attained with tetracyclin, in the second case with surgical treatment. Microbiology, epidemiology, pathology and clinical aspects of the insufficiently known in Hungary protothecosis are outlined.

[Incidence and distribution of t(12;21) in prognostic groups of pediatric acute lymphoblastic leukemia]. / A t(12;21) incidenciája és megoszlása a gyermekkori akut lymphoblastos leukaemia prognosztikai csoportjaiban.

The authors investigated by reverse transcription-polymerase chain reaction the incidence of the t(12;21)(p13;q22) translocation among 130 pediatric acute lymphoblastic leukemia registered by the Hungarian Pediatric Oncology Workgroup. The distribution of this translocation was analysed in the ploidy categories as defined by the flow cytometric DNA analysis and interphase cytogenetics. The incidence of the translocation proved to 19%, the positive patients' age ranged between 2 and 14 with an average of 5.8 years. Ninety percent of the leukemic patients harbouring the t(12;21) exhibited the precursor B-cell phenotype, 10% coexpressed myeloid markers. Coexistence of the t(12;21) with the m-bcr type of Philadelphia translocation was not observed. Ninety five percent of the t(12;21) positive children was diploid by flow cytometry whereas the same figure proved to be 58% using interphase cytogenetics. This difference was due to the hypo- and pseudodiploidy undetectable by flow cytometry but revealed by interphase cytogenetics. The authors conclude that the t(12;21) positive patients which seemed to be homogeneous at gross DNA level were markedly heterogeneous with interphase cytogenetics and this might explain the inconsistent data in the literature in connection with prognostic significance of the t(12;21).

[A case of myelodysplastic syndrome transforming into acute B-cell lymphoid leukemia]. / Tiszta B-sejtes akut lymphoid leukaemiába transzformálódó myelodysplasticus szindróma esete.

Case history of a seventy year old man with myelodysplastic syndrome is presented. The disease terminated into acute leukaemia in 22 months. The pure, B lymphoid stem cell nature of the leukaemic cells has been proved, beside morphology and cytochemistry, by detailed flow cytometric phenotyping and PCR amplification as well as sequencing of the immunoglobulin heavy chain gene CDR3 region.

[Application of interphase cytogenetics for the determination of changes in the DNA content in acute childhood lymphoid leukemia]. / Interfázis citogenetika alkalmazása a DNS-tartalom változásának megítélésére gyermekkori akut lymphoid leukaemiában (ALL).

The authors investigated the usefulness of the interphase cytogenetic approach to reveal numerical chromosomal abnormalities. Experiments performed on normal human cells using chromosome specific (peri)centromeric probes indicated that disomy was recognized in a range of 89.1 +/- 5.4%-96.8 +/- 0.4% for the somatic chromosomes and in 98.1 +/- 0.8% for the sex chromosomes. Using positivity threshold of mean percentage of the fals monosomy and trisomy + 2 SD, chromosome loss or gain for the somatic chromosomes could be revealed beyond clonal ratio of 3.6-13.2% and 1.1-6.8%, respectively. The same value for the sex chromosomes was 3.2% and 0%, respectively. Eleven pediatric acute lymphoid leukaemia were investigated for chromosomal aneuploidy by interphase cytogenetics using chromosome specific (peri)centromeric probes for all the somatic and sex chromosomes. Results were compared with metaphase cytogenetic and flow cytometry derived gross DNA aneuploidy data. In 5 cases the leukaemic cells proved to be diploid with all three methods at both gross DNA and chromosome levels. Interphase cytogenetics revealed chromosome loss or gain in all the remaining 6 cases, however, metaphase analysis indicated numerical aberration in only 2 patients. In one of them only the increased chromosome number could have been detected without identifying the chromosomes involved and in the other one the two methods indicated trisomy for not the same chromosome. Flow cytometry data showed aneuploidy in 3 out of the 6 aneuploid leukaemia. The results imply that interphase cytogenetics might be more accurate as compared with flow cytometry and metaphase analysis to reveale aneuploidy.

Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis.

Accurate quantification of disseminated tumor cells in hematological samples is of fundamental importance in clinical oncology. However, even highly standardized protocols allow only a rough estimation of the total analyzed cell number, as sample processing may have adverse effects on the number of cells available for analysis. The fluorescence-based microscopic scanning system (MetaCyte) detects, counts, captures, and relocates immunolabeled tumor cells in hematopoietic samples. We report on a cell-counting approach that has been implemented into the scanning system to precisely quantify the number of cells per slide. The cell-counting function, which was designed to determine the number of all nucleated (DAPI-stained) cells on the slide, allows an accurate counting of the tumor cells and the total number of cells analyzed in the given microscopic sample. The reliability of the cell-counting approach was demonstrated by the analysis of DAPI-stained images with 18-1,363 nucleated cells. A good correlation (r(2) = 0.965) between the manually and automatically gained results was observed. The counting accuracy could even be optimized after implementing a correction factor. To prove or disprove an interslide variation, routine bone marrow cytospin preparations from neuroblastoma patients were immunostained for GD2/FITC and counterstained with DAPI. Automatic cell counting of cytospin preparations from the same patients showed significant differences in the total cell number (up to 67% cell loss during preparation, with a maximum interslide difference of 4.7 x 10(5) mononuclear cells). We conclude that determination of the tumor cell content in hematopoietic samples is only reliable when it is performed together with accurate cell counting.

Objective analysis of centromere separation.

Centromere separation sequence and premature centromere division have gained increasing interest; however, inaccuracy and subjectivity in their investigation have often been criticized. We describe a simple computerized image analysis system that makes an objective and exact staging of centromere division possible.

Detecting disseminated solid tumor cells in hematopoietic samples: methodological aspects.

Detection of tumor cell dissemination in solid tumor patients recently became essential to determine the prognosis of the disease and to monitor response to the therapy. Accurate detection of disseminated tumor cells in hematological samples requires tumor-specific target molecules, which allow sensitive and specific assays and, further, enable the quantification of tumor cells. Currently, numerous applications are in use, including immunological and molecular biological approaches. Theoretically, both ways are sensitive enough to detect less than one tumor cell in 1 million hematopoietic cells. With the improved sensitivity, however, the likelihood that unspecific events will be amplified is also increased. Moreover, biological and analytical variables may fundamentally influence the findings in a particular case. Basic methods, significant pitfalls and the most recent developments in this field are discussed in this overview.

[Cell proliferation and cell death in disseminated tumor cells]. / Zellproliferation und Zelltod in disseminierten Tumorzellen.

The occurrence of occult metastases of solid tumors at initial diagnosis or during follow-up is of crucial therapeutical importance. The sensitive detection of such cells in hematological samples depends on tissue specific cellular markers. The demonstration of minimally disseminated tumor cells at a given timepoint is, however, only a snapshot, which does not give any information about the potential and dynamics of the cells in question. Functional differences may fundamentally influence the impact of a positive finding. The analysis of cell proliferation and cell death (apoptosis) in disseminated tumor cells, for instance, defines, whether the dissemination process is progressive or regressive. With a newly developed automatic image analysis station the investigation of functional parameters in isolated cells from clinical samples became possible. The studies presented here demonstrate, that such techniques allow an improved identification of isolated tumor cells with clinical importance.

Circulating breast cancer cells are frequently apoptotic.

Automatic search for cytokeratin/mucin-1 double immunofluorescence was performed to detect and characterize circulating epithelial tumor cells in patients with advanced breast cancer. The peripheral blood samples in 8 of 19 patients (42.1%) presented with cytokeratin-positive and epithelial-type mucin-positive (CK(+)/MUC1(+)) tumor cells. Detailed microscopic analysis, however, suggested that the majority of the double immunopositive cells was apoptotic according to an "inclusion type" cytokeratin staining pattern and nuclear condensation. Furthermore, apoptosis-related DNA strand breaks could be demonstrated by applying the TdT-uridine nick end labeling assay in these cells. In 3 of 8 positive samples all of the CK(+)/MUC1(+) cells displayed apoptotic features. We conclude that apoptotic cells significantly contribute to the circulating tumor cell fraction in breast cancer patients. As the predictive value of such cells for the outcome of the disease is unclear, they should be considered separately when analyzing tumor cell dissemination.