Comprehensive clinicopathological, molecular, and methylation analysis of mesenchymal tumors with NTRK and other kinase gene aberrations.

Alterations in kinase genes such as NTRK1/2/3, RET, and BRAF underlie infantile fibrosarcoma (IFS), the emerging entity 'NTRK-rearranged spindle cell neoplasms' included in the latest WHO classification, and a growing set of tumors with overlapping clinical and pathological features. In this study, we conducted a comprehensive clinicopathological and molecular analysis of 22 cases of IFS and other kinase gene-altered spindle cell neoplasms affecting both pediatric and adult patients. Follow-up periods for 16 patients ranged in length from 10 to 130 months (mean 38 months). Six patients were treated with targeted therapy, achieving a partial or complete response in five cases. Overall, three cases recurred and one metastasized. Eight patients were free of disease, five were alive with disease, and two patients died. All cases showed previously reported morphological patterns. Based on the cellularity and level of atypia, cases were divided into three morphological grade groups. S100 protein and CD34 were at least focally positive in 12/22 and 14/22 cases, respectively. Novel PWWP2A::RET, NUMA1::RET, ITSN1::RAF1, and CAPZA2::MET fusions, which we report herein in mesenchymal tumors for the first time, were detected by RNA sequencing. Additionally, the first uterine case with BRAF and EGFR mutations and CD34 and S100 co-expression is described. DNA sequencing performed in 13 cases uncovered very rare additional genetic aberrations. The CNV profiles showed that high-grade tumors demonstrate a significantly higher percentage of copy number gains and losses across the genome compared with low- and intermediate-grade tumors. Unsupervised clustering of the tumors' methylation profiles revealed that in 8/9 cases, the methylation profiles clustered with the IFS methylation class, irrespective of their clinicopathological or molecular features. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements: Biopsy analysis of 70 cases from the Slovak Lymphoma Registry.

We performed a twelve-year retrospective analysis of diffuse large B-cell lymphoma (DLBCL) patients' biopsies with rearrangements of genes MYC, BCL2, and/or BCL6, commonly referred to as double-hit and triple-hit high-grade B-cell lymphomas (DH/TH HGBL). Our aim was to present complex characteristics of the DH/TH HGBL group of patients diagnosed in the Slovak National Lymphoma Register together with the evaluation of the relationship between immunohistochemical (IHC) protein expressions of c-myc, bcl2, bcl6, and cyclin D1 in tissue specimens and the presence of rearrangements of their protein-coding genes by FISH analysis in order to find a clinically relevant diagnostic algorithm that would be the most time- and cost-efficient. For this study, a standard panel of histomorphological, IHC, and FISH methods was used to analyze the characteristics of 70 DH/TH HGBL patients' biopsies. Our study showed a predominance of the immunohistochemical GCB subtype over the non-GCB subtype (59:10 cases) in DH/TH lymphomas. The centroblastic morphology was the most commonly observed (30/70 cases; 43%). Furthermore, our study showed a high predominance of DH lymphoma cases with simultaneous MYC and BCL2 genes rearrangements (40/70; 57%), followed by an almost equal incidence of DH lymphomas with rearrangements of MYC and BCL6 genes (16/70; 23%) and of TH lymphomas (14/70; 20%). 15 of 16 FISH-examined DLBCL cases were negative for CCND1 rearrangement. A great majority of DH/TH cases showed also immunohistochemical overexpression of corresponding proteins (62/70; 89%), mostly in a form of triple expressor of c-myc/bcl2/bcl6 proteins (36/70; 51%), followed by c-myc/bcl2 and c-myc/bcl6 double expressor proteins positivity (20/70 and 6/70, respectively). Comparing preferential FISH testing of DE/TE and GCB DLBCL cases for genetic rearrangements we would be able to detect 89% and 84% of our HGBL-DH, TH group of patients, respectively. None of the examined methods for economically rational FISH testing showed enough concordance with IHC analysis results. We might, therefore, advocate the complex testing of all DLBCL patients' biopsies including FISH analyses.

Clinicopathological analysis of programmed death-ligand 1 testing in tumor cells of 325 patients with non-small cell lung cancer: Its predictive and potential prognostic value.

INTRODUCTION: Recent studies on check-point inhibitor therapy, which seems to improve the prognosis of patients with advanced non-small cell lung carcinoma increase the importance of immunohistochemical analyses of the programmed-death receptor and of its ligand, PD-L1 protein. MATERIAL AND METHODS: In our study we present results of PD-L1 immunohistochemical tumor cell expression in a series of 325 lung carcinoma patients biopsies, using the clone 22C3 (and DAKO Link 48 immunostainer). Evaluation of the expression using tissue proportion scoring system allowed to distinguish negative cases (either 0 % or < 1 % of positive tumor cells) versus positive cases in the categories 1-9 %, 10-49 % and ≥ 50 % of positive tumor cells. RESULTS: In association to histopathologic parameters we observed similar rates of positive expression in patients with adenocarcinoma types (47,8 % of all the cases) as well as with squamous cell carcinomas (44,4 %). Within these histological categories, the rates of positivity were similar also in patients with small versus large (resectional) biopsies. In the biopsies of patients with adenocarcinoma we identified differences in the PD-L1 protein expression associated with its histological subtype. In the cases with predominant lepidic pattern the PD-L1 positivity was present in 18,8 %, with predominant acinar or papillary pattern in 40,8 % and in cases with predominant solid or micropapillary component in 74,1 % of the cases resp. Keratinizing squamous cell carcinomas were positive in 38,5 % and non-keratinizing in 53,8 % of all the cases. The hiqhest incidence of an extensive posivity was observed in sarcomatoid carcinoma type. DISCUSSION AND CONCLUSION: Immunohistochemically verified PD-L1 protein expression represents a broadly accepted predictive biomarker for immunotherapy of NSCLC patients. The indicated differences of the expression among various NSCLC types and subtypes require to be verified in larger cohorts of patients in relation with clinical parameters to demonstrate whether it could be plausible to use the PD-L1 expression in a role of a negative prognostic parameter.

DNA methylation and detection of cervical cancer and precancerous lesions using molecular methods.

Cervical cancer is the third most common cancer disease affecting the female population, and a key factor in the development of the disease is the human papillomavirus infection (HPV). The disease is also impacted by epigenetic changes such as DNA methylation, which causes activation or exclusion of certain genes. The aim of our review is to summarize and compare the most common molecular methods for detection of methylated promoter regions in biomarkers occurring in cervical carcinoma and also show the importance of connections of HR-HPV testing with methylation analysis in patients with cervical intraepithelial neoplasia. Insight into genetic and epigenetic alterations associated with cervical cancer development can offer opportunities for the molecular biomarkers that can be useful for screening, diagnosis, and also as new ways of treatment of cervical cancer precursor lesions.

Syringocystadenocarcinoma Papilliferum In Situ-Like Changes in Extramammary Paget Disease: A Report of 11 Cases.

The authors report 11 cases of extramammary Paget disease (EMPD), all of which also demonstrated a combination of histological changes highly reminiscent of syringocystadenocarcinoma papilliferum in situ. In addition to the classical features of EMPD, characterized by the intraepidermal spread of individually dispersed neoplastic cells with ample cytoplasm, many of which contained mucin, there were areas of acanthosis with the substitution of spinous layer keratinocytes by neoplastic cells, whereas the native basal cell layer was intact. In addition to acanthosis (and sometimes papillomatosis), the dermal papillae showed a prominent infiltrate of plasma cells, completing the resemblance to syringocystadenocarcinoma papilliferum in situ; this similarity was further enhanced in 2 cases, which showed conspicuous gland formation. One additional case showed multifocal dermal proliferations compatible with eccrine syringofibroadenoma (syringofibroadenomatous hyperplasia). The changes described herein seem to be relatively rare in EMPD, and they can represent a diagnostic pitfall, as evidenced by 2 cases that were originally misinterpreted as syringocystadenocarcinoma papilliferum in situ. Clinically, these microscopic changes sometimes corresponded to nodular lesions, which were specifically noted to have a papillated erosive surface.

[Follicular and mantle cell lymphoma diagnosed in biopsies of gastroenterocolic region]. / Folikulový lymfóm a lymfóm z plástových buniek v biopsiách orgánov zalúdocno-crevnej oblasti.

The authors present a retrospective analysis of follicular lymphomas (FL) and mantle cell lymphomas (MCL) diagnosed according to the WHO classification (2008) in consecutive biopsies of GI organs in a period of 11 years. The series includes 18 patients with FL verified in 22 biopsies and 44 patients with MCL diagnosed in 54 biopsies. FL represented always a solitary tumor, most often - up to ¾ of all the cases - of a small intestine, more often in its jejunoileal than duodenal parts. The biopsies were obtained almost equally by endoscopical approach, they were usually mucosal and rarely polypectomic, as well as by surgical resections (54,5 % and 45,5 % of the cases respectively). FL of grade 3 was identified in approximately 11 % of the cases, while majority of the patients showed FL of grade 1 or 2. Only 2 patients with duodenal FL relapsed and bioptically verified recidives did not show signs of a high grade transformation. Although it was difficult to identify a nodular growth pattern in more common small biopsies, a typical histomorphology and phenotype mostly allowed the FL diagnosis in the majority of the cases. The FL diagnosis had to be supported by detection of BCL2 translocation only in one case. MCL appeared most often in the stomach and large intestine, the small intestinal cases represented less than 23 %. In » of the patients the lymphoma was multifocal and manifested as lymphomatoid polyposis affecting most often both large and small intestine. In a majority of the MCL patients, the diagnosis was done in mucosal and polypectomic endoscopic biopsies, surgical intervention and resection was recorded in less than 10 % of the cases. Most of the patients showed conventional "centrocytic" MCL morphology and approximately » of the cases showed blastoid MCL. The rebiopsies of 9 patients revealed a relaps of the disease which was locoidentical in 5 of them; in other 4 patients the biopsies documented a dissemination to other GI organs. The blastic transformation was recognized only in one of the relapsing patients. In contrast to FL, in 18 % of the biopsies the MCL diagnosis required a CCND1 gene rearrangement detection by FISH. In summary both of the studied GI lymphomas showed different and heterogenous incidence, clinical manifestation and necessity to support the biopsy diagnosis also by FISH examination. The correct FL and MCL diagnosis represents a conditio sine qua non for an indication of different therapeutical modalities as well as for the prognostic stratification of the patients.

[Clinically relevant possibilities and limits of differential diagnosis of megaloblastic anemia and myelodysplastic syndrome - refractory anemia type in bone marrow biopsies]. / Klinicky relevantné moznosti a limity diferenciálnej diagnostiky megaloblastovej anémie a myelodysplastického syndrómu typu refraktérnej anémie v trepanobioptických vzorkách kostnej drene.

INTRODUCTION: Megaloblastic anemia (MA) represents a subtype of macrocytic anemia caused by impaired DNA synthesis, mostly due to folate and vitamin B12 deficiency. Its mildest forms lead to macrocytosis without concomitant anemia, but more severe forms to thrombocytopenia and/or leucopenia as well. In majority of the cases, the diagnosis of MA dose not represent a serious clinical problem, however, other causes of macrocytosis including myelodysplastic syndrome (MDS) must be excluded. MATERIAL AND METHODS: In the period 2004-2015 we identified in our registry 126 consecutive bone marrow (BM) biopsies of patients with cytopenia/s in peripheral blood and suspicion either on MA or MDS of refractory anemia (RA) type. We performed a retrospective analysis of BM biopsies focused on evaluation of parameters useful for the differential diagnosis, as represented by (a) cellularity and proportions of BM precursors, (b) and their topography, (c) presence of maturation defects and dysplastic changes, (d) grade and extent of myelofibrosis, (e) iron deposits and (f) presence of "inflammatory" response in BM. Histological analyses were supported by immunohistochemical examinations. RESULTS: The series consisted of biopsies of 126 patients (61 men and 65 women) with average age 63 (14-88 years) - almost all patients (121/126) presented with anemia. Based on the findings we distinguished three diagnostic groups - MA (31 patients), MDS-RA (39) and bioptically unclasifiable case ("DIF DG" - 56 patients). Abnormalities of the BM cellularity were observed in 81 % and of topography in 73 % of all cases respectively. Megalobastic differentiation of erythropoesis was detected in 79 % and diagnostic dysplastic changes in 25 % of all biopsy cases. In 29 % of all biopsies ring sideroblasts were present, megakaryocytic nuclear lobulisation defects density changes were found in 61 % of all patients. In 14 % of all biopsies the BM myelofibrosis was absent, in contrast 5 % of the biopsies showed severe diffuse fibrosis. "Inflammatory" response was developed in 44 % of all biopsies. Iron deposits were absent in 26 %, decreased in 35 % and increased in 33 % of all the cases. CONCLUSIONS: From the point of view of histopathologist it seems to be difficult to distinguish BM hematopoietic changes in patients with MA and MDS-RA respectivelly, as histological examinations allowed determination of a definitive and correct diagnosis in about 55% of the cases. The crucial problem represents a decision whether the observed changes really result from the development of a clonal disease.Key words: megaloblastic anemia - megaloblastic differentiation - refractory anemia.

Survival and risk factors associated with uterine sarcomas and carcinosarcomas in stage I and II.

OBJECTIVE: Uterine sarcomas are rare mesodermal malignant tumors with an incidence between 0.5 and 3.3 cases per 100,000 females per year. Most sarcomas are aggressive tumors leading to poor overall survival rates and only limited therapeutic options. The aim of this study was to evaluate the risk factors for uterine sarcomas and carcinosarcomas, and to identify the factors influencing the survival rate. SUBJECTS AND METHODS: We conducted a retrospective study with twenty-nine patients who were diagnosed with uterine sarcoma and thirty-four patients with carcinosarcoma between the years 1990 and 2006 at the Oncogynecologic center at the University Hospital in Martin, Slovakia. We focused on the analysis of the risk factors and survival rate of early stages I and II. RESULTS: We confirmed highly statistically significant values for the inverse correlation between survival and tumor size, positive lymph nodes, high mitotic activity, vascular invasion, positive peritoneal cytology, elevated CA-125, smoking and BMI in sarcoma and carcinosarcoma group (p<0.001 for all factors). The use of lymphadenectomy had no effect on survival of all patients. DISCUSSION: Sarcomas and carcinosarcomas are aggressive tumors leading to poor overall survival rates and only limited therapeutic options. As there is no consensus on specific treatment, an individual approach based on evaluation of known risk factors is essential.

New molecular aspects of endometrial carcinoma.

Endometrial cancer is the most commonly diagnosed gynecological cancer and its incidence is increasing worldwide. The number of patients with this disease is likely to continue to grow, including younger patients. It is a complex disease driven by abnormal genetic and epigenetic alterations, as well as environmental factors. Many endometrial cancers show estrogen-dependent proliferation. The carcinogenic mechanisms are unknown or not completely explained beyond mutations of single oncogenes and tumor suppressor genes. Possible carcinogenic mechanisms include imbalance between endometrial proliferation by unopposed estrogen and the mismatch repair (MMR) system; rmethylation changes and mutation of genes. Epigenetic changes resulting in aberrant gene expression are dynamic and modifiable features of many cancer types. A significant epigenetic change is aberrant DNA methylation. In this review, we review evidence on the role of different changes in relation to endometrial carcinogenesis. Carcinogenic mechanisms of endometrial cancer involve both genetic and epigenetic changes. Determination of the detailed carcinogenic mechanisms will be useful for prevention and diagnosis of endometrial cancer, risk assessment, and development of new treatment strategies targeting genes.

Tissue distribution of ethanol after intraprostatic injection using a porous needle.

Purpose: To develop a safe and precise method for intraprostatic injection, and to establish correlation between the volume of ethanol injectate and the volume of subsequent infiltrated prostate tissue. Materials and methods: We performed intraprostatic injection of 96% ethanol using a needle which has a segment of its wall made of capillary membrane with hundreds of pores in an acute and chronic canine experiment, in heart-beating cadaveric organ donors, and in a xenograft model of human prostate cancer. Whole mount tissue sections were used for three-dimensional reconstruction of the necrotic lesions and calculation of their volumes. Results: The ethanol injection resulted in oval shaped lesions of well-delineated coagulative necrosis. In both healthy human and canine prostates, the prostatic pseudocapsule and neurovascular bundle remained intact without evidence of disruption. There was a linear correlation between administered volume of ethanol and the volume of necrotic lesion. Regression analysis showed strong correlation in the acute canine experiments and in experiments performed on xenografts of human prostate cancer. A formula was calculated for each experiment to estimate the relationship between the injected volume and the volume of infiltrated prostate tissue area. Conclusions: Intraprostatic injection using a porous needle allows for effective and predictable tissue distribution of the injectate in the prostate. Through varying the volume of the agent injected and use of needles with a different length of the porous segment, the volume of infiltrated tissue could be adjusted allowing for targeted focal treatment.

Prevalence and significance of M541L single nucleotide polymorphism in the central European cohort of gastrointestinal stromal tumor patients.

BACKGROUND: Single nucleotide polymorphisms can create a genetic microenvironment in some tumors that affects the course of treatment, resistance, etc. Whether single nucleotide polymorphisms have an impact on gastrointestinal stromal tumor (GIST) development and disease progression is not yet accurately verified. KIT SNPM541L in exon 10 correlates with a worse prognosis of many cancers. The impact of KIT SNPM541L in GISTs is relatively unknown and, therefore, its analyses could have potential in patient therapy and could provide more detailed information on tumor character, clinical presentation, or tumor behavior in treatment. AIM: The aim of the study was the analysis of the biological and clinical significance of the KIT SNPM541L polymorphism in exon 10. MATERIALS AND METHODS: Paraffin sample tissues were obtained from the National GIST Register in Martin. Retrospective samples from 177 GIST patients were divided into several groups. Detection of SNPM541L was performed by Sanger sequencing. Statisitical analyses were performed to determine the prevalence of KIT SNPM541L in the Slovak GIST cohort, to search for correlation between c-KIT status and clinicopathological, molecular and biological data. RESULTS: Overall, 29 samples out of 177 showed KIT SNPM541L polymorphism. CONCLUSION: Our results do not support the association between KIT SNPM541L and increased risk of relapse in localized primary GISTs. Additionally, we found a positive correlation between KIT SNPM541L occurrence and earlier onset of relapse in PDGFRa and WT subgroup of GISTs.

BRAF mutations in KIT/PDGFRA positive gastrointestinal stromal tumours (GISTs): Is their frequency underestimated?

BRAF V600E mutations in GISTs are considered to be one of the mutational events in KIT/PDGFRA negative or positive GISTs, respectively. BRAF mutated GISTs usually do not respond to imatinib treatment, even more GISTs with imatinib sensitive KIT mutation. However, they are almost phenotypically and morphologically identical with KIT/PDGFRA positive GISTs. In general, due to the small number of BRAF mutations in GIST and because of the rarity of concomitant BRAF/KIT or BRAF/PDGFRA mutations, their frequency may be depreciated. The aim of this study was BRAF mutation detection in KIT/PDGFRA positive GISTs and their verification by other molecular methods. We applied the sensitive droplet digital PCR on 35 randomly selected KIT/PDGFRA positive GISTs to detect V600E mutations. We have established two criteria for the evaluation of samples: false positive rate (FPR) based on the negative controls; Limit of Detection (LoD) based on the serial dilution of positive control from RKO cell line harboring heterozygous V600E mutation in constant wild-type DNA background. Results from ddPCR were verified by other molecular methods: allele-specific PCR, dideoxysequencing, competitive allele-specific TaqMan PCR (castPCR). FPR was determined as 5 (∼4.4) positive droplets, and LoD was assessed to 3.4293 copies/µL what is the method sensitivity of 0.0162 %. We identified eight KIT/PDGFRA positive patients with concomitant V600E mutation. The five of them were in coexistence with KIT mutation and three with PDGFRA mutation. We also included the liver metastasis, but data from primary tumour were not available. We achieved the very high sensitivity of the ddPCR method for detecting BRAF mutation in GISTs to have importance from the point of view of therapy.

Fibro-osseous pseudotumor of digits and myositis ossificans show consistent COL1A1-USP6 rearrangement: a clinicopathological and genetic study of 27 cases.

Myositis ossificans (MO) and fibro-osseous pseudotumor of digits (FOPD) are localized, self-limiting bone-producing pseudosarcomatous lesions characterized by nodular fasciitis-like proliferation and osteoid and immature woven bone production, which may eventually develop into more mature lamellar bone. Traditionally, MO and FOPD were thought to be of reactive, non-neoplastic nature. USP6 gene rearrangement was recently reported as a consistent finding in MO and FOPD, thus expanding the spectrum of transient, USP6-rearranged neoplasms. COL1A1 was described as the fusion partner of USP6 in a subset of MO cases, but the fusion partners of USP6-rearranged FOPD have not been uncovered so far. Initially, we carefully reviewed all 27 cases of MO/FOPD from our archives, documenting the remarkable morphological overlap between both lesions. Sixteen cases were seen in consultation, and our review was requested to rule in or rule out tentative diagnoses by referring pathologists. Malignant diagnosis (osteosarcoma) was suggested by the submitting pathologists in 3 cases, whereas 7 cases were sent by the referring pathologists to "rule out sarcoma." In the following step, using next-generation sequencing, we confirmed the COL1A1-USP6 rearrangement in 5/7 cases of MO and found the same abnormality in 4/5 of FOPD. Overall, 9 of the 12 analyzable cases (75%) of MO and FOPD harbored this gene fusion. The presence of COL1A1-USP6 gene rearrangement in MO/FOPD links these lesions to other USP6-driven tumors and represents a very useful supportive marker, which may help to avoid overdiagnosis of MO/FOPD as a sarcoma.

Different methylation levels in the KLF4, ATF3 and DLEC1 genes in the myometrium and in corpus uteri mesenchymal tumours as assessed by MS-HRM.

Mesenchymal tumours of the corpus uteri comprise common benign lesions - leiomyomas and very rare malignant variants - sarcomas. It can be difficult to distinguish between the particular types of mesenchymal tumours pre-surgically. Primarily, leiomyomas and the very aggressive leiomyosarcomas can be easily misdiagnosed when using only imaging devices. Therefore, a reliable non-invasive marker for these tumour types would provide greater certitude for patients that the lesion remains benign. Our collection comprises 76 native leiomyomas, an equal number of healthy myometrium samples and 49 FFPE samples of various types of sarcomas. The methylation level was assessed by MS-HRM method and we observed differences in the methylation level between healthy, benign and (semi)malignant tissues in the KLF4 and DLEC1 genes. The mean methylation levels of leiomyomas compared to myometrium and leiomyosarcomas were 70.7% vs. 6.5% vs. 39.6 % (KLF4) and 66.1% vs. 14.08% vs. 37.5% (DLEC1). The ATF3 gene was differentially methylated in leiomyomatous and myometrial tissues with 98.1% compared to 76.6%. The AUC values of the predictive logistic regression model for discrimination between leiomyomas and leiomyosarcomas based on methylation levels were 0.7829 (KLF4) and 0.7719 (DLEC1). Finally, our results suggest that there should be distinct models for the methylation events in benign leiomyomas and sarcomas, and that the KLF4 and DLEC1 genes can be considered potential methylation biomarkers for uterine leiomyomas.

Spiradenocylindroma-like basaloid carcinoma of the anus and rectum: case report, including HPV studies and analysis of the CYLD gene mutations.

The authors report a case of basaloid carcinoma involving the anus and rectum of a 57-year-old woman. Microscopically, the tumor showed unusual morphologic features strongly resembling a spiradenocylindroma because it consisted, in most parts, of basaloid cell nodules arranged in a jigsaw-puzzle fashion containing or surrounded by eosinophilic basal membrane material; in addition, there were intratumoral lymphocytes. The overlying squamous epithelium manifested dysplastic changes compatible with in situ squamous carcinoma that gradually became invasive and blended with basaloid cell islands; additionally, there were koilocytes in the squamous epithelium. A molecular biology study identified HPV-16 in the lesional tissue. Analysis of the CYLD gene did not prove any mutation.

A unique clinical presentation of de novo acute promyelocytic leukemia as a myeloid sarcoma of the breast.

Myeloid sarcoma is a rare presentation of acute leukemia as a solid tumor at various extramedullary sites. It may present concurrently, before or after the onset of systemic bone marrow leukemia. Unusual clinical localization may lead to misdiagnosis, or delayed diagnosis and treatment. We describe the first case, to our knowledge, of de novo myeloid sarcoma of the breast confirmed as acute promyelocytic leukemia. Immunohistochemical analysis, flow cytometry, fluorescent in situ hybridization analysis and molecular analysis using RQ-PCR of tissue samples should be routine in determining the correct diagnosis in this setting.

Detection of mutations in the BRAF gene in patients with KIT and PDGFRA wild-type gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are characterized by mutations in exons 9, 11, 13, and 17 of KIT or exons 12, 14, and 18 of PDGFRA gene. However, approximately 10 to 15 % of GISTs lack the mutations in KIT and PDGFRA, and these are referred to as wild-type GISTs which are less sensitive to tyrosine-kinase inhibitors. The aim of this study was to detect BRAF mutations in patients with wild-type GISTs. We applied a sensitive allele-specific PCR, which was optimized using the V600E mutation-harboring cell line RKO, followed by verification of the results by dideoxy sequencing. We selected 149 GIST patients without detectable mutations in KIT and PDGFRA genes from the Slovak national GIST register and analyzed biopsy specimens for the presence of BRAF mutations in exon 15. We identified nine patients with the V600E mutation. The BRAF-driven GISTs were primary gastric (n = 3), small intestinal (n = 3), colon (n = 1), and of uncertain origin (n = 1). We also included a liver metastasis of a patient with a simultaneous KIT exon 11-mutated intra-abdominal metastasis. We conclude that genome analysis of wild-type GISTs for mutations should include the BRAF gene, as its mutation status contributes to understanding of pathogenesis and might be important for decisions on therapy.

Mantle cell lymphoma: improved diagnostics using a combined approach of immunohistochemistry and identification of t(11;14)(q13;q32) by polymerase chain reaction and fluorescence in situ hybridization.

INTRODUCTION: Mantle cell lymphoma (MCL) is a clinicopathological entity characterized by an aggressive clinical course, morphological features, and overexpression of cyclin D1 due to juxtaposition of the bcl-1 locus (and CCND1 gene coding for the cyclin D1) to the IgH gene. This phenomenon is caused by t(11;14)(q13;q32). The morphological diagnosis of MCL may pose difficulties. Ancillary methods are available to support the diagnosis. PATIENTS AND METHODS: We studied a group of 32 patients with MCL; 24 men and 8 women. The median age at the diagnosis was 64 years. We characterized the investigated group by histology, and to analyze the immunohistochemical (IHC) profile we used a panel of antibodies including anti-cyclin D1. Polymerase chain reaction (PCR) was used to detect the rearrangement of bcl-1/IgH in 26 cases (in 11 patients, the DNA was isolated from frozen tissues or from nucleated cells of bone-marrow aspirate or peripheral blood, in 15 patients we utilized paraffin-embedded material). Dual color fluorescence in situ hybridization (FISH) on interphase nuclei detecting the t(11;14)(q13;q32) was applied in all 32 cases. RESULTS: Cyclin D1 IHC was positive in 29 of 30 cases tested (97%). In six, the result was weak and difficult to rely on to support the diagnosis. PCR revealed the fusion gene in 14 of the 26 cases (54%). The best yield was obtained from fresh and frozen samples (8 of 11 positive). Using FISH, we identified the translocation in all 32 patients, the findings being easily interpretable in 29 patients. In three cases, the intensity of red and green signals was weaker and difficult to read though the co-hybridized signals were identified. The classical pattern of the translocation was observed in 26 patients, while in 3 we found variant patterns suggesting a loss of the V segment of the IgH gene (2x) and a shift in the breakpoint region at chromosome 11 (1x). CONCLUSION: The diagnosis of MCL should be supported by a complex laboratory approach. Interphase FISH seems a useful complementary method to morphology and IHC. It is applicable to various tissues and cells prepared as tissue imprints or histological sections.

Spectrum of mutations in gastrointestinal stromal tumor patients - a population-based study from Slovakia.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of gastrointestinal tract and are characterized by presence of mutations in tyrosine kinases cKIT (KIT) and PDGFRα (PDGFRA). Mutations identified are highly heterogeneous, but some mutations are associated with specific clinical features of the tumor. Samples from 278 GIST patients collected during the period 2004-2011 were screened for mutations in exons 9, 11, 13, and 17 of KIT and 12, 14 and 18 of PDGFRA. Results of mutation screening were summarized and tested for possible association with clinical parameters of tumors. Mutations were identified in 83.81% of patients. Most frequent mutations were found in KIT exon 11 reaching frequency of 62.95%. Other exons contributed to the mutation pool with frequencies 8.27%, 7.55%, 2.52%, 1.44%, 1.08%, and 0.00%, in decreasing order KIT exon 9, PDGRFA exons 18 and 12, KIT exon 13, PDGFRA exon 14, and KIT exon 17. General linear model analysis showed no effect of any individual analyzed mutation on the phenotypic variables, but we confirmed association between mutations KIT exon 9 p. 503-504_dup2, and PDGFRA exon 18 p. D842V and intestinal and gastric localization of tumors.