Recurrent FOSL1 rearrangements in desmoplastic fibroblastoma.

The FOS gene family has been implicated in tumourigenesis across several tumour types, particularly mesenchymal tumours. The rare fibrous tumour desmoplastic fibroblastoma is characterised by overexpression of FOSL1. However, previous studies using cytogenetic and molecular techniques did not identify an underlying somatic change involving the FOSL1 gene to explain this finding. Prompted by an unusual index case, we report the discovery of a novel FOSL1 rearrangement in desmoplastic fibroblastoma using whole-genome and targeted RNA sequencing. We investigated 15 desmoplastic fibroblastomas and 15 fibromas of tendon sheath using immunohistochemistry, in situ hybridisation and targeted RNA sequencing. Rearrangements in FOSL1 and FOS were identified in 10/15 and 2/15 desmoplastic fibroblastomas respectively, which mirrors the pattern of FOS rearrangements observed in benign bone and vascular tumours. Fibroma of tendon sheath, which shares histological features with desmoplastic fibroblastoma, harboured USP6 rearrangements in 9/15 cases and did not demonstrate rearrangements in any of the four FOS genes. The overall concordance between FOSL1 immunohistochemistry and RNA sequencing results was 90%. These findings illustrate that FOSL1 and FOS rearrangements are a recurrent event in desmoplastic fibroblastoma, establishing this finding as a useful diagnostic adjunct and expanding the spectrum of tumours driven by FOS gene family alterations. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Fast Trajectory Tracking Control Algorithm for Autonomous Vehicles Based on the Alternating Direction Multiplier Method (ADMM) to the Receding Optimization of Model Predictive Control (MPC).

In order to improve the real-time performance of the trajectory tracking of autonomous vehicles, this paper applies the alternating direction multiplier method (ADMM) to the receding optimization of model predictive control (MPC), which improves the computational speed of the algorithm. Based on the vehicle dynamics model, the output equation of the autonomous vehicle trajectory tracking control system is constructed, and the auxiliary variable and the dual variable are introduced. The quadratic programming problem transformed from the MPC and the vehicle dynamics constraints are rewritten into the solution of the ADMM form, and a decreasing penalty factor is used during the solution process. The simulation verification is carried out through the joint simulation platform of Simulink and Carsim. The results show that, compared with the active set method (ASM) and the interior point method (IPM), the algorithm proposed in this paper can not only improve the accuracy of trajectory tracking, but also exhibits good real-time performance in different prediction time domains and control time domains. When the prediction time domain increases, the calculation time shows no significant difference. This verifies the effectiveness of the ADMM in improving the real-time performance of MPC.

H3K27me3 expression and methylation status in histological variants of malignant peripheral nerve sheath tumours.

Diagnosing MPNST can be challenging, but genetic alterations recently identified in polycomb repressive complex 2 (PRC2) core component genes, EED and SUZ12, resulting in global loss of the histone 3 lysine 27 trimethylation (H3K27me3) epigenetic mark, represent drivers of malignancy and a valuable diagnostic tool. However, the reported loss of H3K27me3 expression ranges from 35% to 84%. We show that advances in molecular pathology now allow many MPNST mimics to be classified confidently. We confirm that MPNSTs harbouring mutations in PRC2 core components are associated with loss of H3K27me3 expression; whole-genome doubling was detected in 68%, and SSTR2 was amplified in 32% of MPNSTs. We demonstrate that loss of H3K27me3 expression occurs overall in 38% of MPNSTs, but is lost in 76% of histologically classical cases, whereas loss was detected in only 23% cases with heterologous elements and 14% where the diagnosis could not be provided on morphology alone. H3K27me3 loss is rarely seen in other high-grade sarcomas and was not found to be associated with an inferior outcome in MPNST. We show that DNA methylation profiling distinguishes MPNST from its histological mimics, was unrelated to anatomical site, and formed two main clusters, MeGroups 4 and 5. MeGroup 4 represents classical MPNSTs lacking H3K27me3 expression in the majority of cases, whereas MeGroup 5 comprises MPNSTs exhibiting non-classical histology and expressing H3K27me3 and cluster with undifferentiated sarcomas. The two MeGroups are distinguished by differentially methylated PRC2-associated genes, the majority of which are hypermethylated in the promoter regions in MeGroup 4, indicating that the PRC2 target genes are not expressed in these tumours. The methylation profiles of MPNSTs with retention of H3K27me3 in MeGroups 4 and 5 are independent of mutations in PRC2 core components and the driver(s) in these groups remain to be identified. Our results open new avenues of investigation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Synovial chondromatosis and soft tissue chondroma: extraosseous cartilaginous tumor defined by FN1 gene rearrangement.

A fusion between fibronectin 1 (FN1) and activin receptor 2A (ACVR2A) has been reported previously in isolated cases of the synovial chondromatosis. To analyze further and validate the findings, we performed FISH and demonstrated recurrent FN1-ACVR2A rearrangements in synovial chondromatosis (57%), and chondrosarcoma secondary to synovial chondromatosis (75%), showing that FN1 and/or AVCR2A gene rearrangements do not distinguish between benign and malignant synovial chondromatosis. RNA sequencing revealed the presence of the FN1-ACVR2A fusion in several cases that were negative by FISH suggesting that the true prevalence of this fusion is potentially higher than 57%. In soft tissue chondromas, FN1 alterations were detected by FISH in 50% of cases but no ACVR2A alterations were identified. RNA sequencing identified a fusion involving FN1 and fibroblast growth factor receptor 2 (FGFR2) in the case of soft tissue chondroma and FISH confirmed recurrent involvement of both FGFR1 and FGFR2. These fusions were present in a subset of soft tissue chondromas characterized by grungy calcification, a feature reminiscent of phosphaturic mesenchymal tumor. However, unlike the latter, fibroblast growth factor 23 (FGF23) mRNA expression was not elevated in soft tissue chondromas harboring the FN1-FGFR1 fusion. The mutual exclusivity of ACVR2A rearrangements observed in synovial chondromatosis and FGFR1/2 in soft tissue chondromas suggests these represent separate entities. There have been no reports of malignant soft tissue chondromas, therefore differentiating these lesions will potentially alter clinical management by allowing soft tissue chondromas to be managed more conservatively.

Post-translational regulation contributes to the loss of LKB1 expression through SIRT1 deacetylase in osteosarcomas.

BACKGROUND: The most prevalent form of bone cancer is osteosarcoma (OS), which is associated with poor prognosis in case of metastases formation. Mice harbouring liver kinase B1 (LKB1+/-) develop osteoblastoma-like tumours. Therefore, we asked whether loss of LKB1 gene has a role in the pathogenesis of human OS. METHODS: Osteosarcomas (n=259) were screened for LKB1 and sirtuin 1 (SIRT1) protein expression using immunohistochemistry and western blot. Those cases were also screened for LKB1 genetic alterations by next-generation sequencing, Sanger sequencing, restriction fragment length polymorphism and fluorescence in situ hybridisation approaches. We studied LKB1 protein degradation through SIRT1 expression. MicroRNA expression investigations were also conducted to identify the microRNAs involved in the SIRT1/LKB1 pathway. RESULTS: Forty-one per cent (106 out of 259) OS had lost LKB1 protein expression with no evident genetic anomalies. We obtained evidence that SIRT1 impairs LKB1 protein stability, and that SIRT1 depletion leads to accumulation of LKB1 in OS cell lines resulting in growth arrest. Further investigations revealed the role of miR-204 in the regulation of SIRT1 expression, which impairs LKB1 stability. CONCLUSIONS: We demonstrated the involvement of sequential regulation of miR-204/SIRT1/LKB1 in OS cases and showed a mechanism for the loss of expression of LKB1 tumour suppressor in this malignancy.

EGFR inhibitors identified as a potential treatment for chordoma in a focused compound screen.

Chordoma is a rare malignant bone tumour with a poor prognosis and limited therapeutic options. We undertook a focused compound screen (FCS) against 1097 compounds on three well-characterized chordoma cell lines; 154 compounds were selected from the single concentration screen (1 µm), based on their growth-inhibitory effect. Their half-maximal effective concentration (EC50 ) values were determined in chordoma cells and normal fibroblasts. Twenty-seven of these compounds displayed chordoma selective cell kill and 21/27 (78%) were found to be EGFR/ERBB family inhibitors. EGFR inhibitors in clinical development were then studied on an extended cell line panel of seven chordoma cell lines, four of which were sensitive to EGFR inhibition. Sapitinib (AstraZeneca) emerged as the lead compound, followed by gefitinib (AstraZeneca) and erlotinib (Roche/Genentech). The compounds were shown to induce apoptosis in the sensitive cell lines and suppressed phospho-EGFR and its downstream pathways in a dose-dependent manner. Analysis of substituent patterns suggested that EGFR-inhibitors with small aniline substituents in the 4-position of the quinazoline ring were more effective than inhibitors with large substituents in that position. Sapitinib showed significantly reduced tumour growth in two xenograft mouse models (U-CH1 xenograft and a patient-derived xenograft, SF8894). One of the resistant cell lines (U-CH2) was shown to express high levels of phospho-MET, a known bypass signalling pathway to EGFR. Neither amplifications (EGFR, ERBB2, MET) nor mutations in EGFR, ERBB2, ERBB4, PIK3CA, BRAF, NRAS, KRAS, PTEN, MET or other cancer gene hotspots were detected in the cell lines. Our findings are consistent with the reported (p-)EGFR expression in the majority of clinical samples, and provide evidence for exploring the efficacy of EGFR inhibitors in the treatment of patients with chordoma and studying possible resistance mechanisms to these compounds in vitro and in vivo. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

GNAS mutations are not detected in parosteal and low-grade central osteosarcomas.

Parosteal osteosarcoma, low-grade central osteosarcoma, and fibrous dysplasia share similar histological features that may pose a diagnostic challenge. The detection of GNAS mutations in primary bone tumors has been useful in clinical practice for diagnosing fibrous dysplasia. However, the recent report of GNAS mutations being detected in a significant proportion of parosteal osteosarcoma challenges the specificity of this mutation. As the number of cases reported in this study was small we set out to determine if these results could be reproduced. We studied 97 formalin-fixed paraffin-embedded low-grade osteosarcomas from 90 patients including 62 parosteal osteosarcomas, of which MDM2 amplification was detected in 79%, 11 periosteal osteosarcomas and 24 low-grade central osteosarcoma samples. The mutational status of GNAS was analyzed in codons p.R201, p.Q227, and other less common GNAS alterations by bidirectional Sanger sequencing and/or next generation sequencing using the Life Technologies Ion Torrent platform. GNAS mutations were not detected in any of the low-grade osteosarcomas from which informative DNA was extracted. Our findings therefore support prior observations that GNAS mutations are highly specific for fibrous dysplasia and occur rarely, if ever, in parosteal and other low-grade osteosarcomas.

Can Genomic Amplification of Human Telomerase Gene and C-MYC in Liquid-Based Cytological Specimens Be Used as a Method for Opportunistic Cervical Cancer Screening?

OBJECTIVE: To evaluate the effectiveness of five methods including the ThinPrep cytological test (TCT), liquid-based cytology, the human papillomavirus (HPV) test, detection of the TERC and C-MYC genes and visual inspection with acetic acid/Lugol's iodine (VIA/VILI) for opportunistic cervical cancer screening, and to explore whether genomic amplification of the human telomerase gene and C-MYC in liquid-based cytological specimens can be used as a method for opportunistic cervical cancer screening. METHODS: Data were collected prospectively from 1,010 consecutive patients who visited the gynecology clinic and agreed to participate in opportunistic cervical cancer screening at our institution from November 2010 to July 2011. The five methods mentioned above were used for the screening in all cases. The histopathological diagnosis served as the gold standard for the evaluation. A comparison between the five screening methods for the diagnosis of high-grade cervical intraepithelial neoplasia (CIN II and III) was performed for their sensitivity, specificity, false-positive rate, false-negative rate, accuracy rate, positive likelihood ratio and negative likelihood ratio. A comprehensive comparison of the different combination programs for screening was performed according to the analysis of the receiver operating characteristic (ROC) curve area. The accuracy of the five screening methods for the diagnosis of high-grade CIN (CIN II and III) was compared in the different age groups. A joint model for the diagnosis using different combinations of the five methods was developed according to the analysis by the SAS 8.0 software. The model was used to evaluate the accuracy of the different combination programs for the diagnosis of high-grade CIN, and the results were confirmed by the histopathological examination. RESULTS: The sensitivity and specificity of the single screen method (TCT, HPV test, detection of the TERC and C-MYC genes, and VIA/VILI method) for CIN II was 80.9, 70.2, 72.3, 76.6, and 72.3%, as well as 98.0, 95.1, 96.3, 96.3, and 90.4%, respectively. The sensitivity of the single screening method in four different age groups (25-34, 35-44, 45-54 and 55-66 years) was as follows: TCT, 64.3, 90.9 76.5, and 85.7%; HPV test, 78.6, 72.7, 60.0, and 71.4%; the TERC gene, 50.0, 90.9, 80.0, and 71.4%; the C-MYC gene, 50.0, 90.9, 80.0, and 100%; VIA/VILI, 85.7, 81.8, 66.7, and 42.9%. The specificity was: TCT, 98.9, 98.1, 98.8, and 95.2%; HPV test, 96.7, 95.1, 92.2, and 100%; the TERC gene, 95.0, 98.9, 94.0, and 95.2%; the C-MYC gene, 97.2, 97.3, 93.4, and 97.6%; VIA/VILI, 91.2, 90.5, 89.8, and 88.1%, respectively. In the joint model for the diagnosis using different combinations, we found Logit (P) = 5.757 - 4.055 × TCT - 3.724 × HPV. The sensitivity and specificity in the combination program with TCT (primary screening) and HPV testing (adjunct screening) were 78.7 and 99.5%, while in the combination with HPV (primary) and TCT (adjunct), they were 53.2 and 99.7%, respectively. However, in the cytology-HPV parallel test, they were 97.9 and 93.4%. The ROC analysis revealed that the cytology-HPV parallel test is superior to the combinations of either TCT (primary) and HPV (adjunct) or HPV (primary) and TCT (adjunct; AUCTCT-HPV parallel test = 0.956; AUCTCT/primaryHPV/adjunct = 0.764). CONCLUSIONS: Opportunistic cervical cancer screening is a practical approach to improve the efficiency of cervical cancer screening. Although the accuracy of TCT is the highest of the five screening methods for the diagnosis of high-grade CIN, it is still subject to sample acquisition and the practitioner's skill and experience. Since the efficacy of VIA/VILI may vary in all ages, it is not recommended for menopausal and perimenopausal women.

Wild-type K-ras has a tumour suppressor effect on carcinogen-induced murine colorectal adenoma formation.

K-ras mutations are found in ~40% of human colorectal adenomas and carcinomas and contribute to colorectal tumour formation at an early stage. Wild-type K-ras has been reported to be deleted in some tumours, but the consequences of changes in wild-type K-ras copy number for experimental colorectal carcinogenesis have not been investigated. To characterize the effects of K-ras copy number changes on formation of carcinogen-induced colorectal neoplasms in mice, wild-type (K-ras(+/+) ) and heterozygous K-ras exon 1 knockout (K-ras(+/-) ) mice were given 10 weekly treatments of 1, 2-dimethylhydrazine (DMH) to induce colorectal tumours. Colorectal expression levels of K-ras 4A and 4B transcripts in K-ras(+/-) mice were ~50% decreased compared with K-ras(+/+) mice. One year after DMH treatment, survival of K-ras(+/-) mice decreased from 88 to 82% compared with wild-type mice. Colorectal adenomas significantly increased from 0.52 ± 0.15 in K-ras(+/+) mice to 0.87 ± 0.14 in K-ras(+/-) mice (mean ± SEM per mouse, P < 0.01); total tumour volume increased 2.13-fold (P < 0.05). Comparing K-ras(+/+) with K-ras(+/-) murine adenomas, Ki-67-positive proliferating tumour cells significantly increased from 7.77 ± 0.64% to 9.15 ± 0.92% and cleaved caspase-3-positive apoptotic tumour cells decreased from 1.40 ± 0.37% to 0.80 ± 0.22% (mean ± SEM, P < 0.05 for both). No K-ras or B-raf mutations were detected in the adenomas. Immunohistochemical studies showed no significant changes in extracellular signal regulating kinase/mitogen-activated protein kinase (Erk/MapK) or PI3K/Akt pathway activation in the adenomas. In conclusion, the data collectively show that a 50% reduction in K-ras gene dosage and RNA expression promoted experimental colorectal tumourigenesis, consistent with wild-type K-ras having a tumour suppressor effect on carcinogen-induced murine colorectal adenoma formation.

[Delay in formalin fixation and HER2 testing in gastric cancer].

OBJECTIVE: To evaluated HER2 status using immunohistochemistry (IHC) assay and fluorescence in situ hybridization (FISH) at two different time points of tissue fixation after surgical resection of gastric cancer, emphasizing the importance of standard operation and quality control in HER2 testing. METHODS: Forty-one resection specimens of advanced gastric cancer were collected with tissue fixation periods of < 30 min or > 30 min after surgical resection. HER2 status was evaluated by immunohistochemistry (IHC) assay and fluorescence in situ hybridization (FISH). RESULTS: The frequency of HER2 expression by IHC in the samples with fixation time of < 30 min was higher than that in those of > 30 min (P < 0.05). However, no significant difference was observed by FISH (P > 0.05) between the two groups. Samples of < 30 min fixation time had high concordant results between IHC and FISH (100.0% for both positive and negative cases, Rho = 0.724, P < 0.05). In addition, HER2 expression by IHC was significantly correlated with Lauren classification, histologic differentiation, TNM stage and gender (P < 0.05). CONCLUSION: The time to tissue fixation after surgical resection of more than 30 min has deleterious effect on the detection of HER2 by IHC although FISH testing is not affected.

[Chromosomal translocation involving USP6 gene in nodular fasciitis].

OBJECTIVE: To investigate the frequency of USP6 gene rearrangement in nodular fasciitis (NF) and to evaluate its clinical application. METHODS: Twenty nine cases of previously diagnosed NF were screened for the presence of the USP6 gene rearrangement by interphase fluorescence-in-situ hybridization (FISH) on formalin-fixed paraffin-embedded tissue. Fifteen of these cases, which had available tissue, were also analysed for MYH9-USP6 fusion transcripts by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Twenty four of the 29 cases (83%) were positive for the USP6 gene rearrangement by interphase FISH. The 15 cases with RT-PCR showed the following results: 11 positive, one deletion and three negative for USP6 gene rearrangement. Of these 15 cases, eight (8/15) showed MYH9-USP6 fusion transcript by RT-PCR. Of these eight cases, seven were positive for USP6 gene rearrangement and one showed USP6 deletion by FISH. CONCLUSIONS: USP6 gene rearrangement is a recurrent genetic event in NF. It is a valuable ancillary tool for the pathological diagnosis of these lesions.

Report and literature review of four cases of EWSR1::NFATC2 round cell sarcoma.

BACKGROUND: EWSR1::NFATC2 rearranged sarcomas are a group of rare round, undifferentiated sarcomas with clinicopathological features different from those of Ewing's sarcoma (ES) family and other non-ES sarcomas. We report 4 cases of this rare sarcoma and review their features. MATERIALS AND METHODS: Four cases of EWSR1::NFATC2 rearranged round cell sarcoma of the bone from the Pathology Department of Peking University People's Hospital were retrospectively studied. Clinical and pathological data were summarized, and immunohistochemical staining, fluorescence in situ hybridization (FISH), and Next-generation sequencing (NGS) were performed. Relevant literature reports were also reviewed. RESULTS: Among the four cases of EWSR1::NFATC2 rearranged round cell sarcoma, three were male, and one was female, with the age ranged from 14 to 34 years old at diagnosis (mean age: 27.5 years). All tumors were located in the femur and ranged in size from 4 to 8cm (mean 6cm), involving the surrounding soft tissues. All four patients underwent surgical treatment, and three received chemotherapy and radiotherapy postoperatively. Follow-up results showed that all four patients were alive. Histologically, the tumors exhibited small round cell sarcoma phenotype, with the stroma rich in mucin or exhibiting a glassy appearance. The tumor cells diffusely expressed CD99, NKX2.2, NKX3.1 and focal expression of CK and EMA was observed. FISH analysis showed that EWSR1 gene rearrangement was detected in all 4 cases, accompanied by 5' locus amplification. EWSR1::NFATC2 fusion probe demonstrated multi yellow fusion signals. NGS identified EWSR1::NFATC2 breakpoints in exon 9 and exon 3 in all 4 cases. The average follow-up duration of the study group was 88 months (range from 26-180 months). One case experienced both local recurrence and metastasis to the lung and chest wall. One case presented with local recurrence. The remaining two cases did not have the recurrence or metastasis. CONCLUSION: Although the disease can locally recur and metastasize to the lungs, its mortality rate is significantly lower than that of Ewing sarcoma and other high-grade small round cell undifferentiated sarcomas. Therefore, it supports to classify this tumor as a separate subtype of small round cell sarcoma.

Sensitivity of MDM2 amplification and unexpected multiple faint alphoid 12 (alpha 12 satellite sequences) signals in atypical lipomatous tumor.

This study assessed whether analysis of MDM2 copy number by fluorescence in situ hybridization (FISH) would help distinguish lipomas from atypical lipomatous tumors, otherwise referred to as well-differentiated liposarcomas, using a commercially available MDM2 FISH kit. 227 lipomatous and 201 non-lipomatous tumors were analyzed to assess its sensitivity and specificity. Of 178 mature lipomatous tumors, 86 were classified histologically as lipoma and 92 as atypical lipomatous tumor. Two of the lipomas harboring MDM2 amplification were reclassified as atypical lipomatous tumors. Overall, 13 atypical lipomatous tumors did not reveal MDM2 or CDK4 amplification, although this was reduced to 12 following analysis of multiple slides. Three of these cases revealed very occasional tumor cells harboring high-level MDM2 amplification, two had a dedifferentiated component, and MDM2 amplification was detected when one tumor recurred. The remaining six cases exhibited reactive/inflammatory features and were reclassified as lipomas. The findings indicate that MDM2 amplification is 93.5% sensitive for diagnosing atypical lipomatous tumor. A total of 2 of the 20 dedifferentiated liposarcomas failed to reveal MDM2 amplification. All atypical lipomatous tumors measured >10 cm, two dedifferentiated liposarcoma presented de novo at <10 cm, and ~50% of lipomas measured >10 cm. Spindle cell lipomas, lipoblastomas, hibernomas and pleomorphic liposarcomas did not reveal MDM2 amplification. Of 201 non-lipomatous tumors, eight revealed MDM2 amplification or multiple faint alphoid 12 signals and were reclassified as dedifferentiated liposarcoma. Multiple faint alphoid 12 signals were observed in nine tumors from seven patients, an observation not previously reported on paraffin sections: these included four atypical lipomatous tumors, and three dedifferentiated liposarcomas, one previously diagnosed as a myxofibrosarcoma, all of which also revealed amplification of CDK4, although two lacked MDM2 amplification. MDM2 FISH test is a useful adjunct to histology for distinguishing lipoma from atypical lipomatous tumor. The limitations of molecular genetic tests must be known before introducing them into a clinical service.

The t(14;18)(q32;q21)/IGH-MALT1 translocation in MALT lymphomas contains templated nucleotide insertions and a major breakpoint region similar to follicular and mantle cell lymphoma.

The t(14;18)(q32;q21) involving the immunoglobulin heavy chain locus (IGH) and the MALT1 gene is a recurrent abnormality in mucosa-associated lymphoid tissue (MALT) lymphomas. However, the nucleotide sequence of only one t(14;18)-positive MALT lymphoma has been reported so far. We here report the molecular characterization of the IGH-MALT1 fusion products in 5 new cases of t(14;18)-positive MALT lymphomas. Similar to the IGH-associated translocations in follicular and mantle cell lymphomas, the IGH-MALT1 junctions in MALT lymphoma showed all features of a recombination signal sequence-guided V(D)J-mediated translocation at the IGH locus. Furthermore, analogous to follicular and mantle cell lymphoma, templated nucleotides (T-nucleotides) were identified at the t(14;18)/IGH-MALT1 breakpoint junctions. On chromosome 18, we identified a novel major breakpoint region in MALT1 upstream of its coding region. Moreover, the presence of duplications of MALT1 nucleotides in one case suggests an underlying staggered DNA-break process not consistent with V(D)J-mediated recombination. The molecular characteristics of the t(14;18)/IGH-MALT1 resemble those found in the t(14;18)/IGH-BCL2 in follicular lymphoma and t(11;14)/CCND1-IGH in mantle cell lymphoma, suggesting that these translocations could be generated by common pathomechanisms involving illegitimate V(D)J-mediated recombination on IGH as well as new synthesis of T-nucleotides and nonhomologous end joining (NHEJ) or alternative NHEJ repair pathways on the IGH-translocation partner.

SOX11 is useful in differentiating cyclin D1-positive diffuse large B-cell lymphoma from mantle cell lymphoma.

AIMS: To characterize the frequency and clinicopathological features of cyclin D1-positive diffuse large B-cell lymphoma (DLBCL) and the usefulness of SOX11 in the differential diagnosis from mantle cell lymphoma (MCL). METHODS AND RESULTS: We retrospectively stained 206 consecutive DLBCLs for cyclin D1, and identified three (1.5%) positive cases, comprising two in the elderly with necrosis, and a third with a starry-sky pattern. All three cases shared the same non-germinal centre B-cell (non-GCB) phenotype [CD5-/CD10-/bcl-6+/MUM1+/SOX11-], Epstein-Barr virus (EBV) negativity, and absence of CCND1 aberrations by fluorescence in-situ hybridization. The third case showed both BCL6 and MYC rearrangements: a double-hit lymphoma. In the same period there were 22 MCLs, all expressing cyclin D1, with 89% cases expressing SOX11, a frequency that is statistically different from cyclin D1-positive DLBCL. Notably, we identified a pleomorphic MCL initially misdiagnosed as DLBCL. A separate cohort of 98 DLBCL cases was negative for SOX11, with only one case expressing cyclin D1 with a GCB phenotype (CD10+/bcl-6+/MUM1-). The two patients with tumour necrosis rapidly died of disease. The other two were in complete remission after immunochemotherapy. CONCLUSION: Cyclin D1-positive DLBCLs are rare, and they are negative for SOX11 or CCND1 aberration. SOX11 is useful in differentiating cyclin D1-positive DLBCL from MCL.

Mutant K-ras promotes carcinogen-induced murine colorectal tumourigenesis, but does not alter tumour chromosome stability.

K-ras (KRAS) mutations are observed in around 40% of human colorectal adenomas and carcinomas. Previously, we developed and characterized a strain of transgenic mice with inducible intestinal epithelial expression of K-ras{Val12} via a Cre/LoxP system. To evaluate the influence of mutant K-ras on carcinogen-induced colorectal tumourigenesis, we induced neoplastic alterations in the large intestines of wild-type and K-ras{Val12} mice using the colon-selective carcinogen 1,2-dimethylhydrazine (DMH), which has been widely used to induce colorectal tumours that are histopathologically similar to those observed in humans. K-ras{Val12} expression significantly promoted DMH-induced colorectal tumourigenesis: the average lifespan of the mice decreased from 38.52 ± 1.97 weeks for 40 control mice to 32.42 ± 2.17 weeks for 26 K-ras{Val12} mice (mean ± SEM, p < 0.05) and the abundance of large intestinal tumours increased from 2.27 ± 0.15 per control mouse to 3.85 ± 0.20 in K-ras{Val12} mice (mean ± SEM, p < 0.01). Adenomas from DMH-treated K-ras{Val12} mice showed significantly higher proportions of Ki-67-positive proliferating cells (10.9 ± 0.69%) compared with those from DMH-treated wild-type mice (7.77 ± 0.47%) (mean ± SEM, p < 0.01) and a mild increase in apoptotic nuclei staining for cleaved caspase-3 (1.94 ± 0.21% compared with 1.15 ± 0.14%, mean ± SEM, p < 0.01). In the adenomas from DMH-treated K-ras{Val12} mice, K-ras{Val12} transgene recombination and expression were confirmed, with immunohistochemical evidence of strong Erk/MapK and mild PI3K/Akt pathway activation compared with adenomas from DMH-treated wild-type mice. Microarray hybridization and clustering analysis demonstrated different expression profiles in adenomas from DMH-treated wild-type and DMH-treated K-ras{Val12} mice, indicating involvement of different molecular mechanisms including Erk/MapK and PI3K/Akt signalling in K-ras{Val12}-expressing adenomas. Array-comparative genomic hybridization analysis showed chromosome stability in both cohorts, with only a very few tiny alterations observed in one adenoma from a DMH-treated K-ras{Val12} mouse. Taken together, these data show that mutant K-ras significantly promotes DMH-induced colorectal tumourigenesis, resulting in distinct changes in cell signalling and proliferation, but does not alter chromosome stability in the tumours.

Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study.

A variety of analyses, including fluorescence in situ hybridization (FISH), quantitative PCR (qPCR) and array CGH (aCGH), have been performed on a series of chordomas from 181 patients. Twelve of 181 (7%) tumours displayed amplification of the T locus and an additional two cases showed focal amplification; 70/181 (39%) tumours were polysomic for chromosome 6, and 8/181 (4.5%) primary tumours showed a minor allelic gain of T as assessed by FISH. No germline alteration of the T locus was identified in non-neoplastic tissue from 40 patients. Copy number gain of T was seen in a similar percentage of sacrococcygeal, mobile spine and base of skull tumours. Knockdown of T in the cell line, U-CH1, which showed polysomy of chromosome 6 involving 6q27, resulted in a marked decrease in cell proliferation and morphological features consistent with a senescence-like phenotype. The U-CH1 cell line was validated as representing chordoma by the generation of xenografts, which showed typical chordoma morphology and immunohistochemistry in the NOD/SCID/interleukin 2 receptor [IL2r]gammanull mouse model. In conclusion, chromosomal aberrations resulting in gain of the T locus are common in sporadic chordomas and expression of this gene is critical for proliferation of chordoma cells in vitro.

The role of epidermal growth factor receptor in chordoma pathogenesis: a potential therapeutic target.

Chordoma, the molecular hallmark of which is T (brachyury), is a rare malignant bone tumour with a high risk of local recurrence and a tumour from which metastatic disease is a common late event. Currently, there is no effective drug therapy for treating chordomas, although there is evidence that some patients respond to the empirical use of epidermal growth factor receptor (EGFR) antagonists. The aim of this study was to determine the role of EGFR in the pathogenesis of chordoma. Paraffin-embedded material from 173 chordomas from 160 patients [sacro-coccygeal (n = 94), skull-based (n = 50), and mobile spine (n = 16)] was analysed by immunohistochemistry and revealed total EGFR expression in 69% of cases analysed. Of 147 informative chordomas analysed by FISH, 38% revealed high-level EGFR polysomy, 4% high-level polysomy with focal amplification, 18% low-level polysomy, and 39% disomy. Phospho-receptor tyrosine kinase array membranes showed EGFR activation in the chordoma cell line U-CH1 and all of the three chordomas analysed. Direct sequencing of EGFR (exons 18-21), KRAS, NRAS, HRAS (exons 2, 3), and BRAF (exons 11, 15) using DNA from 62 chordomas failed to reveal mutations. PTEN expression was absent by immunohistochemistry in 19 of 147 (13%) analysed chordomas, only one of which revealed high-level polysomy of EGFR. The EGFR inhibitor tyrphostin (AG 1478) markedly inhibited proliferation of the chordoma cell line U-CH1 in vitro and diminished EGFR phosphorylation in a dose-dependant manner, a finding supported by inhibition of phosphorylated Erk1/2. p-Akt was suppressed to a much lesser degree in these experiments. There was no reduction of T as assessed by western blotting. These data implicate aberrant EGFR signalling in the pathogenesis of chordoma. This study provides a strategy for patient stratification for treatment with EGFR antagonists.

A genetic model for central chondrosarcoma evolution correlates with patient outcome.

BACKGROUND: Central conventional chondrosarcoma (CS) is the most common subtype of primary malignant bone tumour in adults. Treatment options are usually limited to surgery, and prognosis is challenging. These tumours are characterised by the presence and absence of IDH1 and IDH2 mutations, and recently, TERT promoter alterations have been reported in around 20% of cases. The effect of these mutations on clinical outcome remains unclear. The purpose of this study was to determine if prognostic accuracy can be improved by the addition of genomic data, and specifically by examination of IDH1, IDH2, and TERT mutations. METHODS: In this study, we combined both archival samples and data sourced from the Genomics England 100,000 Genomes Project (n = 356). Mutations in IDH1, IDH2, and TERT were profiled using digital droplet PCR (n = 346), whole genome sequencing (n=68), or both (n = 64). Complex events and other genetic features were also examined, along with methylation array data (n = 84). We correlated clinical features and patient outcomes with our genetic findings. RESULTS: IDH2-mutant tumours occur in older patients and commonly present with high-grade or dedifferentiated disease. Notably, TERT mutations occur most frequently in IDH2-mutant tumours, although have no effect on survival in this group. In contrast, TERT mutations are rarer in IDH1-mutant tumours, yet they are associated with a less favourable outcome in this group. We also found that methylation profiles distinguish IDH1- from IDH2-mutant tumours. IDH wild-type tumours rarely exhibit TERT mutations and tend to be diagnosed in a younger population than those with tumours harbouring IDH1 and IDH2 mutations. A major genetic feature of this group is haploidisation and subsequent genome doubling. These tumours evolve less frequently to dedifferentiated disease and therefore constitute a lower risk group. CONCLUSIONS: Tumours with IDH1 or IDH2 mutations or those that are IDHwt have significantly different genetic pathways and outcomes in relation to TERT mutation. Diagnostic testing for IDH1, IDH2, and TERT mutations could therefore help to guide clinical monitoring and prognostication.

NPM-ALK inhibits the p53 tumor suppressor pathway in an MDM2 and JNK-dependent manner.

Anaplastic large cell lymphoma (ALCL) is characterized by the presence of the t(2;5)(p23;q35) generating the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, a hyperactive kinase with transforming properties. Among these properties is the ability to regulate activity of the p53 tumor suppressor protein. In many human cancers, p53 is inactivated by mutation or other means, in some cases as a result of up-regulation of the negative regulator MDM2. However, the majority of ALK-expressing ALCL carry wild-type p53 and do not over express MDM2. We demonstrate a novel p53-dependent pathogenetic mechanism in ALK-expressing lymphoma. We confirm previously published reports of NPM-ALK-induced activation of the phosphoinositide (PI) 3-kinase and Jun N-terminal kinase (JNK) stress-activated protein (SAP) kinase proteins, but in this study demonstrate a role for these in the regulation of p53 activity in an intricate signaling system. Specifically, constitutive ALK signaling leads to the functional inactivation and/or degradation of p53 in JNK and MDM2 dependent manners. We also show nuclear exclusion of p53 in a PI 3-kinase-dependent manner. Furthermore, we demonstrate that reactivation of p53 in ALK-expressing cells as a result of pharmacologic inhibition of JNK, PI 3-kinase, and/or MDM2 activities results in the induction of apoptosis suggesting a novel therapeutic modality.