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.

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.

[Detection, quantification and characterization of disseminated tumor cells]. / Erkennung, Quantifizierung und Charakterisierung von disseminierten Tumorzellen.

There are different reasons why the detection of disseminated tumor cells (DTCs) in the hematopoetic system is important. On the one hand the detection of disseminated tumor cells can provide vital information about a tumor's tendency to develop metastases. In some localized epithelial but also in embryonic tumors, for example a correlation between disseminated tumor cells and unfavorable outcome was observed (6, 14). These studies are based on the assumption that those tumor cells which appear in the hematopoetic system at a very early stage are responsible for the development of metastases. Another important aspect is the monitoring of the disease response to cytotoxic drugs by quantifying DTCs. During and after therapy there is no other possibility (except for an operation) to either directly analyze the effects the therapy has on the tumor cells or to determine their biological characteristics. The dissemination in the hematopoetic system, however, reveals the response to therapy and the biological features of the tumor cells. The prerequisites for such bone-marrow diagnosis, however, is the unequivocal identification of disseminated tumor cells. So in order to avoid false positive results (which are a risk in bone-marrow diagnostics), a system was developed to distinguish tumor cells from non-neoplastic cells and to facilitate insights into the biological make-up of tumor cells (2, 11).

[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.

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.

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.

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.

Automatic detection and genetic profiling of disseminated neuroblastoma cells.

BACKGROUND: Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE: To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS: In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

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.

[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).

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.

Combined metaphase, interphase cytogenetic, and flow cytometric analysis of DNA content of pediatric acute lymphoblastic leukemia.

Eleven pediatric acute lymphoid leukemia patients 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 cytometric derived DNA aneuploidy data. Experiments performed on normal human cells using chromosome specific (peri)centromeric probes indicated that disomy could be recognized in a range of 89.1+/-2.7% (12.9)-96.8+/-0.2% (0.9) for the somatic chromosomes and in 98.1+/-0.4% (1.3) for the sex chromosomes. Using the cutoff level of the mean false monosomy and trisomy in the control cells +2 S.D., chromosome loss or gain for the somatic chromosomes could be revealed beyond a clonal ratio of 3.6-13.2% and 1.1-6.8%, respectively. The same value for the sex chromosomes was 3.5% and 0%, respectively. In 5 of 11 patients the leukemic 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 of the remaining six patients, however, the metaphase analysis indicated numerical aberration in only two 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 a different chromosome. Flow cytometric data showed aneuploidy in three of the six aneuploid leukemia patients. The results suggest that interphase cytogenetics might be more accurate compared with flow cytometry and metaphase analysis to reveal aneuploidy.

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.

[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.

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.

Cell kinetic analysis in recurrent neuro-epithelial tumours.

The biological behaviour of brain tumours is variable. In the majority of cases, recurrence of the tumour is the decisive factor determining the prognosis and individual survival of patients suffering from a neuro-epithelial neoplasm. The time course of recurrences varies significantly according to differences in tumour cell proliferation. In this study, predictive factors concerning the expected prognosis following the resection of neuro-epithelial tumours were investigated with the aim of improving the histological diagnosis. A retrospective analysis of 22 recurrent neuro-epithelial tumours (recurrent tumour group) and 12 neuro-epithelial tumours with a minimum survival rate of 5 years following radical excision (cured tumour group) was performed by means of flow cytometry and immunohistochemistry using the MIB 1 antibody. Histological samples of the subgroups of the recurrent tumour group, i. e., the primary tumours and their recurrences were compared with each other, and the subgroups were compared with the cured tumour group. A multivariate analysis of the data was performed with the BMPD Hotteling T square test. A statistically significant difference was found between the recurrent tumour group (primary tumours + recurrences) and the cured group from every investigated aspect. On the other hand, no difference could be found between the sub-groups primary tumours and their recurrences. All tumours in the recurrent group had an accelerated, active cell cycle, which was expressed in a high proliferation activity. The following conclusion was drawn: an increased risk of recurrence is to be expected in neuro-epithelial tumours characterized by: an S-phase fraction higher than 6-9%, an MIB 1-labelled cell number higher than 2-3/high-power fields, and a number of mitoses higher than 1/10 high-power fields.