Cytogenetics in the management of chronic lymphocytic leukemia: Guidelines from the Groupe Francophone de Cytogénétique Hématologique (GFCH).

Chromosomal abnormalities are frequent in chronic lymphocytic leukemia (CLL), and most have prognostic value. In addition to the four well-known abnormalities (13q, 11q and 17p deletions, and trisomy 12), other recurrent aberrations have been linked to the disease outcome and/or drug resistance. Moreover, the complex karyotype has recently emerged as a prognostic marker for patients undergoing immunochemotherapy or targeted therapies. Here, we describe the main chromosomal abnormalities identified in CLL and related disorders (small lymphocytic lymphoma and monoclonal B-cell lymphocytosis) by reviewing the most recent literature and discussing their detection and clinical impact. Lastly, we provide technical guidelines and a strategy for the cytogenetic assessment of CLL.

Real-World Diagnostic Accuracy of the On-Site Cytopathology Advance Report (OSCAR) Procedure Performed in a Multidisciplinary One-Stop Breast Clinic.

Fine-needle aspiration (FNA) cytology has been widely used for the diagnosis of breast cancer lesions with the objective of differentiating benign from malignant masses. However, the occurrence of unsatisfactory samples and false-negative rates remains a matter of concern. Major improvements have been made thanks to the implementation of rapid on-site evaluation (ROSE) in multidisciplinary and integrated medical settings such as one-stop clinics (OSCs). In these settings, clinical and radiological examinations are combined with a morphological study performed by interventional pathologists. The aim of our study was to assess the diagnostic accuracy of the on-site cytopathology advance report (OSCAR) procedure on breast FNA cytologic samples in our breast OSC during the first three years (April 2004 till March 2007) of its implementation. To this goal, we retrospectively analyzed a series of 1820 breast masses (1740 patients) radiologically classified according to the American College of Radiology (ACR) BI-RADS lexicon (67.6% being either BI-RADS 4 or 5), sampled by FNA and immediately diagnosed by cytomorphology. The clinicoradiological, cytomorphological, and histological characteristics of all consecutive patients were retrieved from the hospital computerized medical records prospectively registered in the central information system. Histopathological analysis and ultrasound (US) follow-up (FU) were the reference diagnostic tests of the study design. In brief, we carried out either a histopathological verification or an 18-month US evaluation when a benign cytology was concordant with the components of the triple test. Overall, histology was available for 1138 masses, whereas 491 masses were analyzed at the 18-month US-FU. FNA specimens were morphologically nondiagnostic in 3.1%, false negatives were observed in 1.5%, and there was only one false positive (0.06%). The breast cancer prevalence was 62%. Diagnostic accuracy measures of the OSCAR procedure with their 95% confidence intervals (95% CI) were the following: sensitivity (Se) = 97.4% (96.19-98.31); specificity (Sp) = 94.98% (92.94-96.56); positive predictive value (PPV) = 96.80% (95.48-97.81); negative predictive value (NPV) = 95.91% (94.02-97.33); positive likelihood ratio (LR+) = 19.39 (13.75-27.32); negative predictive ratio (LR-) = 0.03 (0.02-0.04), and; accuracy = 96.45% (95.42-97.31). The respective positive likelihood ratio (LR+) for each of the four categories of cytopathological diagnoses (with their 95% CI) which are malignant, suspicious, benign, and nondiagnostic were 540 (76-3827); 2.69 (1.8-3.96); 0.03 (0.02-0.04); and 0.37 (0.2-0.66), respectively. In conclusion, our study demonstrates that the OSCAR procedure is a highly reliable diagnostic approach and a perfect test to select patients requiring core-needle biopsy (CNB) when performed by interventional cytopathologists in a multidisciplinary and integrated OSC setting. Besides drastically limiting the rate of nondiagnostic specimens and diagnostic turn-around time, OSCAR is an efficient and powerful first-line diagnostic approach for patient-centered care.

DNA FISH Diagnostic Assay on Cytological Samples of Thyroid Follicular Neoplasms.

Although fine-needle aspiration cytology (FNAC) is helpful in determining whether thyroid nodules are benign or malignant, this distinction remains a cytological challenge in follicular neoplasms. Identification of genomic alterations in cytological specimens with direct and routine techniques would therefore have great clinical value. A series of 153 cases consisting of 72 and 81 histopathologically confirmed classic follicular adenomas (cFAs) and classic follicular thyroid carcinomas (cFTCs), respectively, was studied by means of different molecular techniques in three different cohorts of patients (pts). In the first cohort (training set) of 66 pts, three specific alterations characterized by array comparative genomic hybridization (aCGH) were exclusively found in half of cFTCs. These structural abnormalities corresponded to losses of 1p36.33-35.1 and 22q13.2-13.31, and gain of whole chromosome X. The second independent cohort (validation set) of 60 pts confirmed these data on touch preparations of frozen follicular neoplasms by triple DNA fluorescent in situ hybridization using selected commercially available probes. The third cohort, consisting of 27 archived cytological samples from an equal number of pts that had been obtained for preoperative FNAC and morphologically classified as and histologically verified to be follicular neoplasms, confirmed our previous findings and showed the feasibility of the DNA FISH (DNA fluorescent in situ hybridization) assay. All together, these data suggest that our triple DNA FISH diagnostic assay may detect 50% of cFTCs with a specificity higher than 98% and be useful as a low-cost adjunct to cytomorphology to help further classify follicular neoplasms on already routinely stained cytological specimens.

In vitro functional characterization of biosimilar therapeutic antibodies.

The key factor in successful development and marketing of biosimilar antibodies is a deep understanding of their critical quality attributes and the ability to control them. Comprehensive functional characterization is therefore at the heart of the process and is a crucial part of regulatory requirements. Establishment of a scientifically sound molecule-specific functional in vitro assay panel requires diligent planning and high flexibility in order to respond to both regulatory requirements and the ever-changing demands relevant to the different stages of the development and production process. Relevance of the chosen assays to the in vivo mechanism of action is of key importance to the stepwise evidence-based demonstration of biosimilarity. Use of a sound interdisciplinary approach and orthogonal state-of-the-art techniques is also unavoidable for gaining in-depth understanding of the biosimilar candidate. The aim of the present review is to give a snapshot on the methodic landscape as depicted by the available literature discussing the in vitro techniques used for the functional characterization of approved biosimilar therapeutic antibodies. Emerging hot topics of the field and relevant structure-function relationships are also highlighted.

Regarding the rights and duties of Clinical Laboratory Geneticists in genetic healthcare systems; results of a survey in over 50 countries.

Specialists of human genetic diagnostics can be divided into four groups: Medical Geneticists (MDG), Genetic Nurses and/or Counsellors (GN/GC), Clinical Laboratory Geneticists (CLG) and Laboratory Genetics Technicians (LGT). While the first two groups are in direct patient contact, the work of the latter two, of equal importance for patient care, are often hidden as they work behind the scenes. Herein the first study on the rights and duties of CLGs is presented. We present the results of a survey performed in 35 European and 18 non-European countries with 100 participating specialists. A national CLG title is available in 60% of European countries, and in 77% of the surveyed European countries a CLG can be the main responsible head of the laboratory performing human genetic tests. However, in only 20% of European countries is a lab-report valid with only a CLGs' signature - even though the report is almost always formulated by the CLG, and an interpretation of the obtained results in a clinical context by the CLG is expected in nearly 90% of European countries. Interestingly, CLGs see patients in 30% of European countries, and are also regularly involved in student education. Overall, the CLG profession includes numerous duties, which are quite similar in all regions of the world. Strikingly, the CLG's rights and responsibilities of leading a lab, or signing a report are regulated differently according to country specific regulations. Overall, the CLG is a well-recognized profession worldwide and often working within a multidisciplinary team of human genetic diagnostics professionals.

Molecular Screening for Cancer Treatment Optimization (MOSCATO-01) in Pediatric Patients: A Single-Institutional Prospective Molecular Stratification Trial.

Purpose: This single-institutional feasibility study prospectively characterized genomic alterations in recurrent or refractory solid tumors of pediatric patients to select a targeted therapy.Experimental Design: Following treatment failure, patients with signed consent and ages above 6 months, underwent tumor biopsy or surgical resection of primary or metastatic tumor site. These newly acquired samples were analyzed by comparative genomic hybridization array, next-generation sequencing for 75 target genes, whole-exome and RNA sequencing. Biological significance of the alterations and suggestion of most relevant targeted therapies available were discussed in a multidisciplinary tumor board.Results: From December 2012 to January 2016, 75 patients were included, 73 patients underwent 79 interventions, 56 of which were research biopsies with a low complication rate. All patients were pretreated, 37.0% had a brain tumor, and 63.0% had an extra-cranial solid tumor. Median tumor cell content was 70% (range, 0%-100%). Successful molecular analysis in 69 patients detected in 60.9% of patients an actionable alteration in various oncogenic pathways (42.4% with copy-number change, 33.3% with mutation, 2.1% with fusion), and change in diagnosis in three patients. Fourteen patients received 17 targeted therapies; two had received a matched treatment before inclusion.Conclusions: Research biopsies are feasible in advanced pediatric malignancies that exhibit a considerable amount of potentially actionable alterations. Genetic events affecting different cancer hallmarks and limited access to targeted agents within pediatric clinical trials remain the main obstacles that are addressed in our two subsequent precision medicine studies MAPPYACTS and AcSé-ESMART. Clin Cancer Res; 23(20); 6101-12. ©2017 AACR.

Integrated molecular portrait of non-small cell lung cancers.

BACKGROUND: Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC. METHODS: Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. RESULTS: At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1 and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944. CONCLUSIONS: Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.

Homogeneous assay for evaluation of aptamer-protein interaction.

We introduce Amplified Luminescent Proximity Homogenous Assay (ALPHA) to assess the K(D) value of aptamer-protein complexes as demonstrated through the study of apple stem pitting virus coat protein-specific aptamers. This method can be used as a simple, cost-effective method for screening aptamer-target protein interactions during aptamer selection.

Impact of the reactive microenvironment on the bone marrow involvement of follicular lymphoma.

AIMS: Follicular lymphoma (FL) is associated with bone marrow (BM) involvement in approximately 40-70% of cases. Previous studies have suggested that the immune-microenvironment of FL plays an important role in the clinical behaviour of the disease. To investigate the role of the microenvironment in BM involvement of FL, we performed immunophenotypical analysis of the reactive cell populations in lymph nodes (LN) and corresponding BM of 35 patients with FL. Microenvironment patterns of BM infiltrates were compared to the corresponding features of the LN in cases with BM manifestation, and the LN microenvironment was compared in FL cases with and without BM involvement. METHODS AND RESULTS: Automated image-segmentation-based quantitation was performed in whole digital slides of tissue microarrays of formalin-fixed paraffin-embedded tissue biopsies. We found significantly more CD8(+) T lymphocytes, forkhead box protein 3 (FoxP3)(+) T lymphocytes and CD68(+) macrophages and fewer PD1(+) T lymphocytes in the BM than in the matching LN samples. Furthermore, we observed significantly fewer CD8(+) T cells and CD68(+) macrophages in cases involving the BM compared to those localized only to the LNs. CONCLUSIONS: Our study showed that different tumour cell growth in the LN and BM may generate different microenvironments, and suggested that the reduced number of cytotoxic T lymphocytes and macrophages in LNs favours BM infiltration of neoplastic cells in FL.

Correlation between DNA ploidy, metaphase high-resolution comparative genomic hybridization results and clinical outcome of synovial sarcoma.

BACKGROUND: Although synovial sarcoma is the 3rd most commonly occurring mesenchymal tumor in young adults, usually with a highly aggressive clinical course; remarkable differences can be seen regarding the clinical outcome. According to comparative genomic hybridization (CGH) data published in the literature, the simple and complex karyotypes show a correlation between the prognosis and clinical outcome. In addition, the connection between DNA ploidy and clinical course is controversial. The aim of this study was using a fine-tuning interpretation of our DNA ploidy results and to compare these with metaphase high-resolution CGH (HR-CGH) results. METHODS: DNA ploidy was determined on Feulgen-stained smears in 56 synovial sarcoma cases by image cytometry; follow up was available in 46 cases (average: 78 months). In 9 cases HR-CGH analysis was also available. RESULTS: 10 cases were found DNA-aneuploid, 46 were DNA-diploid by image cytometry. With fine-tuning of the diploid cases according to the 5c exceeding events (single cell aneuploidy), 33 cases were so called "simple-diploid" (without 5c exceeding events) and 13 cases were "complex-diploid"; containing 5c exceeding events (any number). Aneuploid tumors contained large numbers of genetic alterations with the sum gain of at least 2 chromosomes (A-, B- or C-group) detected by HR-CGH. In the "simple-diploid" cases no or few genetic alterations could be detected, whereas the "complex-diploid" samples numerous aberrations (equal or more than 3) could be found. CONCLUSIONS: Our results show a correlation between the DNA-ploidy, a fine-tuned DNA-ploidy and the HR-CGH results. Furthermore, we found significant correlation between the different ploidy groups and the clinical outcome (p < 0.05).

Lymphoid aggregates may contribute to the migration and epithelial commitment of bone marrow-derived cells in colonic mucosa.

AIMS: Colonic inflammation is followed by regeneration supported by bone marrow-derived cells (BMDCs) including multipotent cells. They migrate to the colonic epithelial layer and may transdifferentiate into epithelial-like cells or keep their stem cell characteristics and produce progenies. The aim was to study the role of lymphoid aggregates in the migration and transition of BMDCs in both healthy colons and non-specific colitis (NSC). METHODS: Samples of normal colon (n=5) and NSC (n=5) from female patients who were initially transplanted with male bone marrow were studied. After detecting XY chromosomes using fluorescent in situ hybridisation, tissue sections were digitalised, the coverslips were eliminated and the samples were double stained for CD45 and cytokeratin with immunofluorescence. Then CDX2 expression, as a sign of intestinal epithelial commitment of Musashi-1+ stromal BMDCs, was also tested with both immunoperoxidase and parallel immunofluorescence stainings. The slides were digitalised again and analysed simultaneously. RESULTS: A significant increase in intraepithelial CD45-BMDCs was found in regions adjacent to lymphoid aggregates (median: 1.01) compared with healthy epithelial regions (median: 0.0175) or NSC (median: 0.04) samples. The stromal Musashi-1+ cells were positive for CDX2 as well, as a sign of epithelial differentiation. The CDX2+ cells bearing the Y chromosome proved the epithelial commitment of several stromal BMDCs. CONCLUSION: Elevated number of intraepithelial CD45-BMDCs at lymphoid aggregates suggests that BMDCs play a role in epithelial regeneration and that lymphoid aggregates serve as their migration route.

High rate of neoplastic cells with genetic abnormalities in proliferation centers of chronic lymphocytic leukemia.

In lymph nodes, chronic lymphocytic leukemia (CLL) cells (prolymphocytes and paraimmunoblasts) form proliferation centers (PCs), which are also known as pseudofollicles. To reveal whether PCs play a role in the accumulation of genetic alterations in CLL, we compared deletion at 11q22.3, 13q14.3, and 17p13.1 loci and trisomy 12 by the fluorescence in situ hybridization (FISH) technique in PCs versus surrounding small lymphocytes (SLs) in 12 formalin-fixed paraffin-embedded (FFPE) lymph nodes. The FFPE sections were stained with methylene blue and PCs were marked by laser beam. Subsequent FISH analysis was performed, relocalizing the previously defined regions. Loss of 11q was detected in five cases, loss of 13q in two cases, loss of 17p in two cases, and trisomy 12 in one case. In seven cases PCs contained a significantly higher ratio of cells with genetic alterations compared with the surrounding SLs. Our results show that CLL cells with genetic alterations tend to accumulate in PCs. The clonal expansion of the cell population carrying genetic alterations within PCs may contribute to CLL progression.

The role of the bone marrow derived mesenchymal stem cells in colonic epithelial regeneration.

Bone marrow derived mesenchymal stem cells (BM-MSCs) take part in the colonic mucosal regeneration. They are multipotent cells, which can be identified with both negative (i.e. CD13, CD 14, CD45, c-Kit, major histocompatibility complex /MHC class I and II) and positive (i.e. CD54 (ICAM1), CD133, CD146 (MCAM), CD166, Flk-1, Sca-1, Thy-1, stage-specific antigen I /SSEA-I and Musashi-1, HLA class I) markers. These cells can repopulate the gastrointestinal mucosa as they may differentiate into stromal- (i.e. myofi-broblast) or epithelial-like (Paneth-, epithel-, goblet or enteroendocrin) cells without proliferation. During the mesenchymal to epithelial transition (MET) stem cells enter the epithelial layer and take up epithelial cell-like properties. Rarely BM-MSCs may retain their stem cell characteristics and are capable of producing progeny. The isolated lymphoid aggregates may serve as a platform from where BM-MSCs migrate to the nearby crypts as mediated by several chemoattractant proteins, which are expressed in injured tissue. The number of BM-MSCs is influenced by the degree of inflammation. In this review we summarize the current information about the role of BM-MSCs in the repair progress of injured colonic epithelium and their potential clinical applications.

Selection and versatile application of virus-specific aptamers.

Although the significance of molecular diagnostics in routine plant virus detection is rapidly growing, the preferred methods are still antibody-based enzyme immunoassays. In the past decade, aptamers have been demonstrated to be viable alternatives of antibodies in many applications. We set out to select apple stem pitting virus (ASPV)-specific aptamers and to apply them as antibody substitutes in various immunoassay methods. The applied systematic evolution of ligands by exponential enrichment (SELEX) procedure resulted in highly discriminative aptamers selectively binding to the target virus coat protein even in complex protein matrixes. We developed protocols for exploitation of aptamers in diverse plant virus diagnosis methods, such as dot and Western blot analyses and enzyme-linked oligonucleotide assay (ELONA). Our selected aptamers proved to be superior to the available antibody in all aspects. In contrast to the antibody, the aptamers decorate both native and denaturated proteins, and ELONA produces higher signal intensity than traditional enzyme-linked immunosorbent assay (ELISA) with virus-infected plant extract. Summarily, our results present the selection and practical utilization of first plant virus-specific aptamers. Most important, the first application of ELONA for virus detection is demonstrated, which proposes a novel, more flexible, and cost-effective means of virus diagnostics.

Aptamer-based biochips for label-free detection of plant virus coat proteins by SPR imaging.

Specific detection of virus strains by affinity-based bioassays is often limited by the availability of ligands able to differentiate among close homologues of virus coat proteins. As viruses are prone to mutation, the ligand generation should, in addition, be fast enough to allow rapid identification of new varieties. These two criteria are difficult to be fulfilled by antibodies; however, they open up opportunities for aptamer-based detection. Here we report on the feasibility of selectively detecting the apple stem pitting virus (ASPV) coat proteins (PSA-H, MT32) using original DNA aptamers. Surface plasmon resonance (SPR) imaging was used together with aptamer-modified sensor chips to optimize the aptamer immobilization for highest sensitivity and to characterize the aptamer-virus coat protein binding. Different parameters affecting this binding, such as the aptamer flanking, surface coverage, and type of spacer molecules, were identified and their influence was determined. A direct label-free method is proposed for assessing the ASPV based on the detection of the respective virus coat proteins in plant extracts.

[Appearing of bone marrow derived stem cells in healthy and regenerating colonic epithelium]. / A csontvelô eredetû ôssejtek megjelenése az ép vastagbélhámban és a gyulladást követô hámregenerációban.

UNLABELLED: In the process of regeneration following colon inflammation mesenchymal stem cells (MSC) of bone marrow origin may also take part besides their local counterparts. These cells migrate in the colon epithelium where they may contribute to epithelial regeneration or form progeny for keeping up local stem cell pool. MSC cells probably leave circulation around lymphoid aggregates to then migrate into nearby crypts. During migration they change their phenotype upon the influence of local microenvironment. AIMS: In this study epithelial migration and transition of bone marrow stem cells were examined. Samples from normal healthy individuals and from aspecific inflammation were used. The possible role of lymphoid aggregates in the epithelial regeneration was also studied. MATERIALS AND METHODS: Samples of normal colon (2) and those showing mild aspecific colitis (3) from female patients who were initially transplanted with male bone marrow were studied. First we detected gender chromosomes with fluorescent in situ hybridization (FISH) and the samples were archived with digital scanning. Then CD45 and cytokeratin (CK) double immunofluorescent reactions (IF) were made followed by digitalization again. Digitalized samples were estimated simultaneously with virtual microscopy (Mirax Viewer). RESULTS: Significant elevation of CD45 negative/Y-FISH positive potential MSCs were found in crypts locating to the neighborhood of lymphoid aggregates (1,075%) compared with both normal (0,027%, p = 0,002) and mild colitis (0,045%, p = 0,004) samples. CONCLUSION: Local stem cells probably have enough regeneration capacity in case of minor inflammation. However, in aspecific inflammation the number of MSCs contributing to epithelial regeneration was elevated, suggesting their facilitated contribution to the repair process with less probable forming of local stem cell progeny.

Somatic hypermutation of IGVH genes and aberrant somatic hypermutation in follicular lymphoma without BCL-2 gene rearrangement and expression.

BACKGROUND: Follicular lymphoma is characterized by the t(14;18) translocation resulting in constitutive expression of BCL-2 protein; however approximately 10-15% of follicular lymphomas do not express BCL-2 protein, and a small fraction of these cases does not exhibit translocation of the BCL-2 gene either. It is highly debated whether cases of follicular lymphoma without BCL-2 gene rearrangement and expression represent a separate lymphoma entity with distinct biological characteristics, different from the BCL-2-positive cases. DESIGN AND METHODS: To further characterize follicular lymphomas without BCL-2 gene rearrangement and expression, we analyzed and compared the mutational status of IGVH genes as well as other genes (C-MYC, PAX-5 and RHOH) frequently involved in the specific type of genomic instability called aberrant somatic hypermutation in 11 cases of BCL-2-negative and 7 cases of BCL-2-positive follicular lymphomas. We also determined the levels of expression of activation-induced cytidine deaminase in these cases. RESULTS: The analyzed cases were grade 2 and grade 3A follicular lymphomas. Our findings demonstrate that follicular lymphomas without BCL-2 gene rearrangement and expression are associated with ongoing somatic hypermutation of the IGVH genes, low activity of aberrant somatic hypermutation and elevated activation-induced cytidine deaminase expression. These results were in concordance with the results found in the cases of BCL-2-positive follicular lymphoma. CONCLUSIONS: Although, BCL-2 protein overexpression is considered to be a critical pathogenic event in the development of follicular lymphoma, our findings suggest that follicular lymphomas with the same morphology, immunophenotype, mutational pattern and activation-induced cytidine deaminase expression may develop without the involvement of BCL-2 gene. The present data support the hypothesis that BCL-2-positive and BCL-2-negative follicular lymphomas (grades 1-3A) represent a homogenous group with different initial but several common additional molecular pathways.

Malignant myoepithelioma of soft tissue: a case report with cytogenetic findings.

Soft tissue malignant myoepithelioma (STMM) is a particularly rare tumor displaying myoepithelial elements and lacking obvious ductal differentiation. From the one case report with cytogenetic data available in the literature, STMM seems to be a distinct entity with some resemblance to chordoma on the one hand and myoepithelioma on the other. The present case of STMM yielded novel data from high-resolution comparative genomic hybridization (HR-CGH) analysis. An 82-year-old female patient presented with a soft tissue tumor within the deep soft tissues in the right gluteal muscle measuring 16 x 13 x 11 cm. Histologically, the lesion was diagnosed as a myoepithelial carcinoma. Immunohistochemistry was focally positive for pancytokeratin, EMA, S-100 protein, and alpha smooth muscle actin. HR-CGH analysis revealed gains of 1p31 approximately p34, 1q21 approximately q23, 9q12 approximately q33, and 16q22 and losses of 1p11 approximately p22, 1q24 approximately q44, 3p, 10q11.1 approximately q22, 13q, 14q13 approximately q24, and 15q. Subsequent fluorescence in situ hybridization analysis confirmed deletion of 3p, gain of 16q, and monosomy of chromosomes 13 and 15. These results support the hypothesis that STMM is a distinct entity, not sharing the cytogenetic alterations of salivary gland myoepithelial carcinomas and ductal carcinomas of breast with myoepithelial differentiation.

IgVH gene mutation status and genomic imbalances in chronic lymphocytic leukaemia with increased prolymphocytes (CLL/PL).

Chronic lymphocytic leukaemia (CLL) with increased prolymphocytes (CLL/PL) has been defined by the World Health Organization (WHO) classification and considered as a progressive and clinically aggressive variant of CLL. To further characterize the biological features of this disease, we performed IgVH gene mutational status, FISH and high-resolution comparative genomic hybridization (HR-CGH) analysis in 17 cases of CLL/PL. All CLL/PL utilized members of VH1, VH3 and VH4 families, with the highest prevalence of the VH1-69 gene. In all but one cases analyzed, the VH genes were unmutated. The FISH and HR-CGH analyses showed frequent occurrence of trisomy 12, del(11)(q23), del(17)(p13) and genetic imbalances, but recurrent genetic lesion characteristic for CLL/PL was not found. The follow-up HR-CGH analysis of two cases showed that increase of prolymphocytes in the course of CLL or CLL/PL are associated with clonal evolution and selection of the tumour clone. In conclusion, this study suggests that CLL/PL is a relatively homogeneous disease regarding VH gene mutation, but heterogeneous regarding genetic lesions. The heterogeneity and high number of genomic imbalances found in CLL/PL suggest that a genome-wide instability of the neoplastic cells may play a role in the development of the disease.