Approach to risk stratification for papillary thyroid carcinoma based on molecular profiling: institutional analysis.

BACKGROUND: Currently, treatment recommendations for papillary thyroid carcinoma are not based on the genetic background causing tumourigenesis. The aim of the present study was to correlate the mutational profile of papillary thyroid carcinoma with clinical parameters of tumour aggressiveness, to establish recommendations for risk-stratified surgical treatment. METHOD: Papillary thyroid carcinoma tumour tissue of patients undergoing thyroid surgery at the University Medical Centre Mainz underwent analysis of BRAF, TERT promoter and RAS mutational status as well as potential RET and NTRK rearrangements. Mutation status was correlated with clinical course of disease. RESULTS: One hundred and seventy-one patients operated for papillary thyroid carcinoma were included. The median age was 48 years (range 8-85) and 69 per cent (118/171) of patients were females. One hundred and nine papillary thyroid carcinomas were BRAF-V600E mutant, 16 TERT promotor mutant and 12 RAS mutant; 12 papillary thyroid carcinomas harboured RET rearrangements and two papillary thyroid carcinomas showed NTRK rearrangements. TERT promoter mutant papillary thyroid carcinomas had a higher risk of distant metastasis (OR 51.3, 7.0 to 1048.2, P < 0.001) and radioiodine-refractory disease (OR 37.8, 9.9 to 169.5, P < 0.001). Concomitant BRAF and TERT promoter mutations increased the risk of radioiodine-refractory disease in papillary thyroid carcinoma (OR 21.7, 5.6 to 88.9, P < 0.001). RET rearrangements were associated with a higher count of tumour-affected lymph nodes (OR 7950.9, 233.7 to 270495.7, P < 0.001) but did not influence distant metastasis or radioiodine-refractory disease. CONCLUSIONS: Papillary thyroid carcinoma with concomitant BRAF-V600E and TERT promoter mutations demonstrated an aggressive course of disease, suggesting the need for a more extensive surgical strategy. RET rearrangement-positive papillary thyroid carcinoma did not affect the clinical outcome, potentially obviating the need for prophylactic lymphadenectomy.

High-Grade Neuroendocrine Carcinoma of the Head and Neck: Human Papillomavirus Status and PD-L1 Expression.

BACKGROUND: Human papillomavirus (HPV)-related head and neck squamous cell carcinoma represents an important subgroup of head and neck cancer, but HPV occurs also in the less common neuroendocrine carcinomas (NEC). The PD-1/PD-L1 pathway appears to be activated in pulmonary NEC and correlates with a higher mutation burden, but the potential of NEC to respond to checkpoint inhibitors is unknown to a large extent. OBJECTIVES: To determine the HPV status of NEC of the head and neck region and to investigate the expression of PD-1 and its ligands PD-L1 and PD-L2. METHODS: Surgical tumor samples from 2006 to 2017 were analyzed. HPV status was determined by p16 immunohistochemistry (IHC) and multiplex PCR. IHC using the Cologne Score was performed for PD-1, PD-L1, and PD-L2. RESULTS: Seven NEC tumor samples were analyzed, three of them showed HPV type 18. Expression of PD-1 and PD-L1 differed widely and showed no correlation to HPV status. IHC showed an overexpression of PD-L2 in most of the patients. CONCLUSIONS AND SIGNIFICANCE: A multicentric analysis of NEC is needed to further evaluate the role of HPV as well as immunocheckpoints with regard to inflammatory immune response in genesis and clinical course of this rare tumor entity. Biomarkers for selection of novel treatment regimens, including immunotherapeutic approaches, are warranted.

ANKRD26-RET - A novel gene fusion involving RET in papillary thyroid carcinoma.

BACKGROUND: Rearrangements of RET are drivers of oncogenesis, traceable in different cancer types as papillary thyroid carcinoma (PTC), non-small cell lung cancer, colorectal or breast cancer. Anchored multiplex PCR based next-generation sequencing (NGS) can detect RET rearrangements involving previously unknown partner genes. METHODS: A sample of PTC underwent NGS, following detection of RET rearrangement by fluorescence in situ hybridization. Expression analysis of ANKRD26 and RET was performed for the tumor harboring ANKRD26-RET, for corresponding normal thyroid tissue and PTC tumors with representative genetic alterations (BRAFV600E, CCDC6-RET), complemented by a comparative search in the "UniProt" database. RESULTS: NGS analysis resulted in the discovery of the fusion ANKRD26-RET. ANKRD26 mRNA was expressed in all PTC tumors (ANKRD26-RET, BRAFV600E, CCDC6-RET) and in normal thyroid tissue, whereas RET mRNA was detected only in the tumors with RET rearrangement. On protein level, ANKRD26-RET combines the RET tyrosine kinase to ankyrin repeat and coiled-coil domains. CONCLUSIONS: ANKRD26-RET is a novel rearrangement of the RET gene, associated with RET expression in thyroid tissue. The result is a fusion of the RET tyrosine kinase to prominent protein-protein interaction motifs. Further studies are required to investigate the influence of different RET rearrangements on metastasis and disease-free survival in PTC.

PD-L1 Expression and Immune Cell Infiltration in Gastroenteropancreatic (GEP) and Non-GEP Neuroendocrine Neoplasms With High Proliferative Activity.

The potential of neuroendocrine neoplasms (NEN) to respond to checkpoint inhibitors is largely unknown and full of great expectations. Immunohistochemical (IHC) studies of programmed cell death ligand 1 (PD-L1) expression in the tumor microenvironment and its implications in predicting the response to checkpoint inhibition is a very active subject. Currently, the combined analysis of PD-L1 expression and tumor-associated immune cell (TAIC) infiltration is considered the best predictive marker of therapeutic response. Here we investigated the expression of PD-L1 on tumor cells (TC) and tumor-infiltrating immune cells (IC) by IHC in 68 NEN samples with a high proliferation rate (Ki-67 >20%) from 57 patients and in 22 samples we correlated it with TAIC density by assessing intratumoral infiltration of CD3+, CD8+, and CD68+ cells. Furthermore, the tumor microenvironment was evaluated according to the classification of Teng et al. We detected PD-L1 expression in 31.6% of NEN G3. Its expression usually was weak and more IC than TC expressed PD-L1. The proportion of tumors positive for PD-L1 was comparable in NEN from different sites of origin but varied depending on tumor differentiation and disease extension. No positive IHC staining was found in 3 well-differentiated neuroendocrine tumors (NETs) with a proliferation rate above 20% (NET G3). When analyzing TAIC, we rarely (18.2%) detected intratumoral CD8+ cells, whereas infiltration by CD3+ and CD68+ cells was more common (45.5 and 59.1%, respectively). By combining CD3+ cells and PD-L1 status, we identified the immune ignorant phenotype of tumor microenvironment as being the most common phenotype, supporting the concept of a preferably combined immunotherapeutic approach in neuroendocrine carcinoma (NEC).

[Hepatocellular carcinoma in an inflammatory adenoma with ß-catenin mutation in a patient with hepatocellular adenomatosis due to germline mutation in HNF1α]. / Entstehung eines hepatozellulären Karzinoms in einem inflammatorischen Adenom mit ß-Catenin-Mutation bei einem Patienten mit hepatozellulärer Adenomatose und HNF1α-Keimbahnmutation.

Hepatocyte nuclear factor 1 α (HNF1α) mutations cause maturity-onset diabetes of the young (MODY) type 3 and are associated with hepatocellular adenomatosis. Malignant transformation of HNF1α-mutated hepatocellular adenomas is rare. We report a case of a 28-year-old man with HNF1α-inactivated adenomatosis who developed an inflammatory adenoma with ß catenin mutation with malignant transformation into a well-differentiated hepatocellular carcinoma (HCC).

Novel rearrangements involving the RET gene in papillary thyroid carcinoma.

BACKGROUND: In the field of gene fusions driving tumorigenesis in papillary thyroid carcinoma (PTC), rearrangement of the proto-oncogene RET is the most frequent alteration. Apart from the most common rearrangement of RET to CCDC6, more than 15 partner genes are yet reported. The landscape of RET rearrangements in PTC ("RET-PTC") can notably be enlarged by modern targeted next-generation sequencing, indicating similarities between oncogenic pathways in other cancer types with identical genetic alterations. METHODS: Targeted next-generation sequencing was performed for two cases of BRAF-wild type PTC with confirmation of the results by Sanger sequencing. A "UniProt" database research was performed to assess protein alterations resulting from RET rearrangements. RESULTS: RUFY2-RET and KIAA1468-RET were detected. The fusion genes were not present in normal tissue of the index patients. The rearrangement RUFY2-RET lead to a fusion of the RET tyrosine kinase domain to a RUN domain and a coiled-coil domain. For KIAA1468-RET, a fusion to a LisH domain and two coiled-coil domains resulted. CONCLUSIONS: RUFY2-RET and KIAA1468-RET are novel RET/PTC rearrangements. The fusions were previously described in non-small cell lung cancer. The rearrangement results in a fusion of the RET tyrosine kinase to regulatory domains of RUFY2 and KIAA1468.

Detection of RET rearrangements in papillary thyroid carcinoma using RT-PCR and FISH techniques - A molecular and clinical analysis.

INTRODUCTION: Oncogenic BRAF and RAS mutations as well as multiple known (and yet unknown) RET fusion oncogenes comprise the majority of causative molecular alterations in papillary thyroid carcinoma (PTC). Apparently "mutation-negative" PTCs encompass a heterogenous group impeding analysis of prognostic significance of underlying genetics. MATERIAL AND METHODS: BRAF wild type PTC tissue of 56 patients was analyzed using two established methods: hybrid-specific RT-PCR for the predominant rearrangement RET/PTC1 and fluorescent in situ hybridization (FISH). Clinical features of the cases with and without RET rearrangement were compared (patient age, gender, tumor size, focality, lymph node affection, and iodine avidity). RESULTS: RT-PCR revealed RET/PTC1 rearrangements in five of 56 tumors (9%). FISH confirmed these, and identified four additional RET rearrangements (9/56; 16%). Loss of the iodine avidity only occurred in cases of RET/PTC hybrids (7/9 tumors), but not in RET/PTC-negative PTCs (0/41 tumors with available uptake information; p = 0.029). The risk to develop lymph node metastases was eight times higher in presence of RET rearrangements (p = 0.010). CONCLUSIONS: FISH analysis, in contrast to hybrid-specific RT-PCR, revealed infrequent and unknown RET fusion genes. The presence of RET rearrangements was associated with a significantly elevated risk to develop iodine refractory disease and lymph node metastases. Of note, significant clinical discrimination was only achievable when taking the FISH results into account; differences would have been missed when using the RT-PCR method only. Increasing evidence of the clinical impact of RET/PTC-positivity may influence the decision on the extent of surgical resection, especially on lymph node dissection, in PTCs.

Targeted next-generation sequencing of cancer genes in poorly differentiated thyroid cancer.

Poorly differentiated thyroid carcinoma (PDTC) is a rare malignancy with higher mortality than well-differentiated thyroid carcinoma. The histological diagnosis can be difficult as well as the therapy. Improved diagnosis and new targeted therapies require knowledge of DNA sequence changes in cancer-relevant genes. The TruSeq Amplicon Cancer Panel was used to screen cancer genomes from 25 PDTC patients for somatic single-nucleotide variants in 48 genes known to represent mutational hotspots. A total of 4490 variants were found in 23 tissue samples of PDTC. Ninety-eight percent (4392) of these variants did not meet the inclusion criteria, while 98 potentially pathogenic or pathogenic variants remained after filtering. These variants were distributed over 33 genes and were all present in a heterozygous state. Five tissue samples harboured not a single variant. Predominantly, variants in P53 (43% of tissue samples) were identified, while less frequently, variants in APC, ERBB4, FLT3, KIT, SMAD4 and BRAF (each in 17% of tissue samples) as well as ATM, EGFR and FBXW7 (each in 13% of tissue samples) were observed. This study identified new potential genetic targets for further research in PDTC. Of particular interest are four observed ERBB4 (alias HER4) variants, which have not been connected to this type of thyroid carcinoma so far. In addition, APC and SMAD4 mutations have not been reported in this subtype of cancer either. In contrast to other reports, we did not find CTNNB1 variants.

Detection of cellular senescence within human invasive breast carcinomas distinguishes different breast tumor subtypes.

Oncogene-induced senescence is thought to act as a barrier to tumorigenesis by arresting cells at risk of malignant transformation. Nevertheless, numerous findings suggest that senescent cells may conversely promote tumor progression through the development of the senescence-associated secretome they produce. It is likely that the composition and the physiological consequences mediated by the senescence secretome are dependent on the oncogenes that trigger the senescence program. Breast cancer represents a heterogenous disease that can be divided into breast cancer subtypes due to different subsets of genetic and epigenetic abnormalities. As tumor initiation and progression of these breast cancer subtypes is triggered by diverse oncogenic stimuli, differences in the senescence secretomes within breast tumors might be responsible for tumor initiation, progression, metastasis and therapeutic response. Many studies have addressed the role of senescence as a barrier to tumor progression using murine xenograft models. However, few investigations have been performed to elucidate the degree to which senescent tumor cells are present within untreated human tumors, and if present, whether these senescent tumor cells may play a role in disease progression. In the present study we analysed the appearance of senescent cells within invasive breast cancers. Detection of cellular senescence by the use of SAß-galactosidase (SAß-gal) staining within invasive breast carcinoms from 129 untreated patients revealed differences in the amount of SAß-gal+ tumor cells between breast cancer subtypes. The highest percentages of SAß-gal+ tumor cells were found in HER2-positive and luminal A breast carcinomas whereas triple negative tumors showed either little or no positivity.

Hepatitis e virus detection in liver tissue from patients with suspected drug-induced liver injury.

Hepatitis E virus (HEV) infection is increasingly recognized as a cause of acute hepatitis in the industrialized world. We aimed to determine the frequency of acute HEV infection in cases of suspected drug-induced liver injury (DILI), mainly a diagnosis of exclusion. To this aim, formalin-fixed paraffin-embedded (FFPE) liver tissues of all cases routinely processed in our institute during a 2 1/2 years period in which DILI was among the differential diagnoses (157 liver biopsies, 1 liver explant) were subjected to semi-nested RT-PCR for the detection of HEV RNA. Histopathology was re-evaluated on all cases tested positive. HEV RNA was detectable in 3 of 158 cases (2%) tested, comprising autochthonic as well as travel-related infections with genotypes 1, 3, and 4 each found once, respectively. Histopathologic findings comprised one case with subtotal hepatic necrosis and two cases of acute (cholestatic) hepatitis not distinguishable from acute hepatitis of other etiology. Thus, the overall frequency of acute HEV infection as determined by detection of HEV RNA in liver tissue is substantially increased in patients with suspected DILI compared to the healthy population, emphasizing the need to actively look for HEV infection in cases of suspected DILI. Molecular testing for HEV RNA in routinely processed FFPE liver tissues can be applied to cases with undetermined HEV status.

Phenotypic variability and risk of malignancy in SDHC-linked paragangliomas: lessons from three unrelated cases with an identical germline mutation (p.Arg133*).

CONTEXT: Mutations in the four subunits of succinate dehydrogenase (SDH) are the cause for the hereditary paraganglioma (PGL) syndrome types 1-4 and are associated with multiple and recurrent pheochromocytomas and PGLs. SDHC mutations most frequently result in benign, nonfunctional head-and neck PGLs (HNPGLs). The malignant potential of SDHC mutations remains unclear to date. OBJECTIVES: We report a patient with malignant PGL carrying a SDHC mutation and compare her case with two others of the same genotype but presenting with classic benign HNPGLs. Loss of heterozygosity (LOH) was demonstrated in the malignant PGL tissue. DESIGN: In three unrelated patients referred for routine genetic testing, SDHB, SDHC, and SDHD genes were sequenced, and gross deletions were excluded by multiplex ligation-dependent probe amplification (MLPA). LOH was determined by pyrosequencing-based allele quantification and SDHB immunohistochemistry. RESULTS: In a patient with a nonfunctioning thoracic PGL metastatic to the bone, the lungs, and mediastinal lymph nodes, we detected the SDHC mutation c.397C>T predicting a truncated protein due to a premature stop codon (p.Arg133*). We demonstrated LOH and loss of SDHB protein expression in the malignant tumor tissue. The two other patients also carried c.397C>T, p.Arg133*; they differed from each other with respect to their tumor characteristics, but both showed benign HNPGLs. CONCLUSIONS: We describe the first case of a malignant PGL with distant metastases caused by a SDHC germline mutation. The present case shows that SDHC germline mutations can have highly variable phenotypes and may cause malignant PGL, although malignancy is probably rare.

JAK2-V617F-mutated myeloproliferative neoplasms reveal different allele burden within hematopoietic cell lineages: a microdissection study of bone marrow trephine biopsies.

The JAK2-V617F mutation is prevalent in almost all patients with polycythemia vera (PV) and about half of the cases of essential thrombocythaemia (ET) and primary myelofibrosis (PMF). A different allele burden in these entities has long been noticed, but little is known about its distribution among the neoplastic hematopoietic cell lineages within the bone marrow. We conducted a microdissection study of JAK2-V617F-mutated myeloproliferative neoplasms (MPN); 10 cases each of ET, PV, and PMF, with separate analysis of the JAK2 mutation status in three hematopoietic cell lines (i.e., megakaryo-, granulo-, and erythropoiesis). Different numbers of cell lineages harboring the JAK2-V617F mutation were found, being the lowest in ET (17/30), higher in PV (24/30) and in PMF (22/30). The megakaryopoiesis was the most commonly mutated cell lineage (24/30 cases). By analyzing microdissectates we were able to demonstrate a different allele burden of the JAK2-V617F mutation in the megakaryo-, erythro-, and granulopoiesis within the bone marrow of a given case of MPN. We demonstrated differences in the number of mutated cell lineages. The different mutation status may contribute to the different phenotypes of ET, PV, and PMF.

Silver-enhanced in situ hybridization for detection of polyomavirus DNA in patients with BK virus nephropathy.

BK virus nephropathy is not an infrequent complication of renal transplantation associated with high rates of graft loss. Although antibodies against SV40 antigen detect different viruses of the polyomavirus family, immunohistochemistry is widely used to confirm the diagnosis of BK virus nephropathy in renal biopsies. Here we aimed to validate the novel silver-enhanced in situ hybridization (SISH) technique for the automated detection of BK virus in renal transplant biopsies. Two different patient cohorts were included. Twenty-nine consecutive patients suspicious for BK virus infection were investigated by SISH and chromogenic in situ hybridization. An additional 26 renal biopsies positive by SV40 immunohistochemistry from 19 patients were analyzed by SISH. Polyomavirus DNA serum levels, as determined by nested PCR analysis, were available for all of these patients. The presence of BK virus DNA in renal tubular cells was identified in 5 of the suspicious cases by both, SISH and chromogenic in situ hybridization . One additional patient was only positive in the SISH. In the second cohort, SISH was positive in all SV40 positive biopsies, but SISH signals were less extensive than SV40 immunohistochemistry. Our results show that the BK virus SISH is an ancillary tool for the detection of polyomavirus DNA in renal biopsies using bright-field microscopy. However, its diagnostic value in comparison with standard immunohistochemistry seems to be limited.

Oropharyngeal lesions and cervical lymphadenopathy: syphilis is a differential diagnosis that is still relevant.

BACKGROUND: Syphilis (lues), a chronic infectious disease caused by Treponema pallidum, has been increasing in incidence during the last few years. Therefore, while clinically it is often not suspected, syphilis is increasingly becoming a differential diagnosis in routine pathology. AIM: To report our experience with five cases of cervical lymphadenopathy and/or oropharyngeal lesions, clinically thought to be lymphomas, lymph node metastases or carcinoma, in which we made the mostly clinically unsuspected diagnosis of syphilis. METHODS: Fine needle aspiration of enlarged cervical lymph nodes was evaluated by cytology and flow cytometry (fluorescence-activated cell sorting analysis), and biopsies were examined by using histology. In addition, all materials were also subjected to immunostaining, silver staining and molecular (PCR) testing. RESULTS: Fine needle aspiration cytology revealed follicular hyperplasia in two cases and granulomatous lymphadenitis in one case. In three patients, concomitant biopsy of co-existing oropharyngeal lesions revealed histological findings compatible with syphilis. T pallidum was detected in all cytological and histological samples by immunohistochemistry/immunocytochemistry and PCR. Subsequently, a diagnosis of syphilis was confirmed clinically and by serology. CONCLUSIONS: Syphilitic lymphadenitis is still a relevant differential diagnosis of cervical lymphadenopathy, and it is clinically often not suspected. Co-existing oropharyngeal lesions should alert the physician to this differential diagnosis; and lesions with compatible morphology should be tested with immunohistochemistry and immunocytochemistry and/or molecular analysis to confirm the diagnosis of syphilis.

Impact of pathognomonic genetic alterations on the prognosis of papillary thyroid carcinoma. ESES vienna presentation.

INTRODUCTION: BRAF mutations and RET or NTRK1 rearrangements were identified as causing events that drive the malignant transformation of the thyroid follicular cell. The impact of these alterations on the course of papillary thyroid carcinoma (PTC) is still unsettled. PATIENTS AND METHODS: Tumor tissues of 290 (98 male, 192 female) patients were intra-operatively snap frozen or harvested from archival paraffin-embedded blocks and used for extraction of DNA and RNA. Comprehensive analysis of RET/PTC and NTRK1 rearrangements was carried out by multiplex screening RT-PCR, hybrid-specific RT-PCR and sequencing of detected hybrids. A mutation-specific PCR was used for BRAF analysis. RESULTS: The BRAF V600E mutation was detected in 122/290 (42%), RET rearrangements in 20/137 (14.6%), and NTRK1 rearrangements in 15/93 (16.1%) PTCs. One hundred forty one out of 290 (48.6%) PTCs demonstrated none of the genetic alterations studied. Eight PTCs expressed two different mutations (1 RET/PTC + BRAF, 6 NTRK1 + BRAF, 1 RET/PTC + NTRK1). Tumor-specific survival analysis (mean follow-up, 5.5 years) demonstrated no significant difference, but a tendency toward worse prognosis of BRAF-positive patients compared to BRAF-negative patients or rearrangement-positive patients, respectively. CONCLUSION: Long-term follow-up data on large tumor panels are needed to disclose significant survival differences of prognostic predictors on PTC. This study provides further evidence that patients harboring BRAF-V600E-positive PTCs may experience an unfavorable course of the disease compared to patients with tumors carrying other genetic alterations.

Detection of papillary thyroid carcinoma by analysis of BRAF and RET/PTC1 mutations in fine-needle aspiration biopsies of thyroid nodules.

BACKGROUND: Activating mutations of the oncogene BRAF or rearrangements of the tyrosine kinase receptor RET are observed in up to 80% of papillary thyroid carcinomas (PTCs). The predominant BRAF V600E mutation has not been detected in benign thyroid tissue so far, so consequently, this assumedly pathognomonic alteration is qualified to improve the preoperative diagnosis of PTC. METHODS: Two hundred ninety preoperatively harvested fine-needle aspiration biopsies (FNABs) underwent routine cytologic assessment. BRAF V600E mutation analysis was performed by mutation-specific PCR using the same cell material; a hybrid-specific RT-PCR assay was used for detection of RET/PTC1 rearrangements. Detected genetic alterations were verified by direct sequencing. Definitive histopathology was obtained in 93/290 lesions following surgery of the respective thyroid nodule. RESULTS: While cytology alone diagnosed 13/30 malignancies (22 PTCs, 4 FTCs, 1 MTC, 1 UTC, 2 metastases), five additional malignancies were identified by supplementary mutation analysis. Cytology classified eight FNABs as benign, while postoperative histology demonstrated a thyroid malignancy (6 PTCs, 1 FTC, 1 metastasis). In four of these eight cases, the genetic analysis detected a BRAF V600E mutation or a RET/PTC1 rearrangement. Classifying both suspicious and malignant FNAB results as positive cytology results, supplementary genetic testing increased the overall sensitivity of FNAB from 70.4 to 85.7%, the positive predictive value (PPV) from 59.4 to 64.9%, and the negative predictive value (NPV) from 84.0 to 91.3%. CONCLUSIONS: Supplementary mutation analysis of RET and especially of the BRAF V600E mutation in FNABs is a fast and probably cost-effective assay in routine diagnostic setting. Mutation analyses of PTC-specific genetic alterations improve the preoperative identification and prognostic assessment of thyroid malignancies and therefore enable an optimized surgical strategy.

The TFIIH subunit p89 (XPB) localizes to the centrosome during mitosis.

BACKGROUND: The general transcription factor II H (TFIIH), comprised of a core complex and an associated CAK-complex, functions in transcription, DNA repair and cell cycle control. Mutations of the two largest subunits, p89 (XPB) and p80 (XPD), cause the hereditary cancer-prone syndrome xeroderma pigmentosum. METHODS: The TFIIH subunit p89 was monitored during interphase and cell division by immunofluorescence staining, GFP-fusion constructs including deletions, live cell imaging and immuno-precipitations. RESULTS: Here we demonstrate that during cell division, from prophase until telophase, the TFIIH core subunit p89, but not other subunits of TFIIH, associates with the centrosomes and the adjacent parts of the mitotic spindle. With overall constant levels throughout mitosis, p89 re-localizes to the newly formed nuclei by the end of mitosis. Furthermore, p89 interacts with the centrosomal protein gamma-tubulin. Truncations of p89 result in an abnormal subcellular distribution during interphase and abolished centrosomal association during mitosis. CONCLUSIONS: Our observations suggest a so far unappreciated role for p89 in cell cycle regulation, and may be the structural basis for a long known, but hitherto unexplained interaction between p89 and tubulin.

Wild-type JAK2 secondary acute erythroleukemia developing after JAK2-V617F-mutated primary myelofibrosis.

A 54-year-old female patient developed acute erythroleukemia after an 8-year course of primary myelofibrosis. The latter harbors the JAK2-V617F mutation and was treated with hydroxyurea and anagrelide. A bone marrow trephine biopsy disclosed 2 morphologically distinct areas of chronic primary myelofibrosis and acute erythroleukemia. Microdissection and a separate molecular pathological analysis was performed. Although the activating JAK2-V617F mutation was not maintained in blasts of acute erythroleukemia, it was detectable in the chronic phase of primary myelofibrosis, indicating that this mutation did not play a role in the leukemic transformation of erythroid cells.

Biomaterial-induced sarcomagenesis is not associated with microsatellite instability.

Sarcomagenesis, in contrast to carcinogenesis, is poorly understood. Microsatellite instability has been implicated in the development of many cancers, in particular those associated with chronic inflammatory conditions. In an experimental animal model, rats developed not only a peri-implantational chronic inflammatory reaction, but also malignant mesenchymal tumors in response to different biomaterials. Therefore, it was the aim of our study to test if the development of biomaterial-induced sarcomas is characterized by a mutator phenotype. A multiplex-PCR approach was designed to screen biomaterial-induced sarcomas for the presence of microsatellite instability. Seven different microsatellite loci were tested in ten tumors for microsatellite instability using a fluorochrome-labelled multiplex-PCR and subsequent fragment analysis. All tumors provided a microsatellite-stable phenotype at all loci tested. Our data suggest that microsatellite instability is rarely or not at all a feature of malignant transformation of biomaterial-induced soft tissue tumors. Thus, there is no evidence that a mutator phenotype is a hallmark of biomaterial-induced sarcomagenesis.