Expanding the spectrum of tyrosine kinase fusions in calcified chondroid mesenchymal neoplasms: Identification of a novel PDGFRA::USP8 gene fusion.

Calcified chondroid mesenchymal neoplasms represent a distinct, and recently recognized, spectrum of tumors. To date most cases have been reported to be characterized by FN1 gene fusions involving multiple potential tyrosine kinase partners. Following incidental identification of a tumor morphologically corresponding to calcified chondroid mesenchymal neoplasm, but with a PDGFRA::USP8 gene fusion, we undertook a retrospective review to identify and characterize additional such cases. A total of four tumors were identified. Each was multilobulated and composed of polygonal-epithelioid-stellate cells with a background of chondroid matrix containing distinctive patterns of calcification. Targeted RNA sequencing revealed an identical PDGFRA (exon 22)::USP8 (exon 5) gene fusion in each case. Subsequent immunohistochemical staining confirmed the presence of PDGFRα overexpression. In summary, we report a series of four tumors within the morphologic spectrum of calcified chondroid mesenchymal neoplasms. In contrast to prior reports, these tumors harbored a novel PDGFRA::USP8 gene fusion, rather than FN1 rearrangement. Our findings expand the molecular diversity of these neoplasms, and suggest they are united through activation of protein kinases.

The E2F1-HOXB9/PBX2-CDK6 axis drives gastric tumorigenesis and serves as a therapeutic target in gastric cancer.

Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.

The novel finding of an FGFR1::TACC1 fusion in an undifferentiated spindle cell sarcoma of soft tissue with aggressive clinical course.

We report an aggressive soft tissue sarcoma with FGFR1-TACC1 fusion occurring in the thigh of an 83-year-old man. Microscopically, the tumor was composed of monomorphic spindle cells arranged in intersecting fascicles. The tumor cells showed ovoid nuclei, fine chromatin, indistinct nucleoli, and elongated eosinophilic cytoplasm. Focal increase in nuclear atypia was noted. Immunohistochemistry showed only focal rare positivity to S100, but negative to SOX10, CD34, STAT6, TLE-1, SMA, and other myogenic markers. An extensive panel of immunostains did not reveal a definite lineage of cellular differentiation. The fusion junction of the chimeric transcript involved FGFR1 exon 16 and TACC1 exon 7, which was similar to those reported in other types of neoplasms such as glioblastomas and epithelial cancers. The transcript was predicted to be in-frame and confirmed by Sanger sequencing after reverse transcriptase-polymerase chain reaction. The patient was treated by marginal excision of tumor without receiving adjuvant therapy. He experienced rapid tumor recurrence with distant metastases and succumbed at 3.5 months after presentation. The finding of FGFR1-TACC1 fusion in a high-grade, undifferentiated spindle cell sarcoma of soft tissue is novel and may offer a potential therapeutic target in the near future.

Expanding the spectrum of mesenchymal neoplasms with NR1D1-rearrangement.

Undifferentiated mesenchymal neoplasms can be morphologically subclassified based on cell shape; epithelioid tumors may be diagnostically challenging, particularly since they can show morphologic and immunohistochemical overlap with epithelial neoplasms. Following the recent report of an NR1D1::MAML1 gene fusion in an undifferentiated pediatric neoplasm, we performed a retrospective archival review and identified four additional cases of undifferentiated mesenchymal neoplasms with NR1D1-rearrangement. All four tumors occurred in adult women. The tumors involved superficial and/or deep soft tissues of the extremities or abdomen. Morphologically, they showed a spectrum of overlapping features. In addition to epithelioid cells, two cases also had a prominent spindle cell component. Two cases also had admixed polygonal cells containing prominent cytoplasmic vacuoles with amorphous debris. The immunophenotype was nonspecific but all cases had at least focal keratin expression; this was extensive in two tumors. Targeted RNA-sequencing revealed two cases each with NR1D1::MAML1 and NR1D1::MAML2 gene fusions. One patient developed lung and liver metastases, and one patient required amputation due to multifocal disease and underlying bone involvement. This study confirms undifferentiated NR1D1-rearranged sarcoma represents a distinct mesenchymal neoplasm with an epithelioid morphology and potential for aggressive behavior. Further, we offer new insight into the spectrum of clinical, morphologic, immunohistochemical, and molecular findings possible in these rare neoplasms. An awareness of this entity is especially important given the potential for misclassification as a carcinoma.

Epidemiology of Acute Myocarditis/Pericarditis in Hong Kong Adolescents Following Comirnaty Vaccination.

BACKGROUND: Age-specific incidence of acute myocarditis/pericarditis in adolescents following Comirnaty vaccination in Asia is lacking. This study aimed to study the clinical characteristics and incidence of acute myocarditis/pericarditis among Hong Kong adolescents following Comirnaty vaccination. METHODS: This is a population cohort study in Hong Kong that monitored adverse events following immunization through a pharmacovigilance system for coronavirus disease 2019 (COVID-19) vaccines. All adolescents aged between 12 and 17 years following Comirnaty vaccination were monitored under the COVID-19 vaccine adverse event response and evaluation program. The clinical characteristics and overall incidence of acute myocarditis/pericarditis in adolescents following Comirnaty vaccination were analyzed. RESULTS: Between 14 June 2021 and 4 September 2021, 33 Chinese adolescents who developed acute myocarditis/pericarditis following Comirnaty vaccination were identified. In total, 29 (87.88%) were male and 4 (12.12%) were female, with a median age of 15.25 years. And 27 (81.82%) and 6 (18.18%) cases developed acute myocarditis/pericarditis after receiving the second and first dose, respectively. All cases are mild and required only conservative management. The overall incidence of acute myocarditis/pericarditis was 18.52 (95% confidence interval [CI], 11.67-29.01) per 100 000 persons vaccinated. The incidence after the first and second doses were 3.37 (95% CI, 1.12-9.51) and 21.22 (95% CI, 13.78-32.28 per 100 000 persons vaccinated, respectively. Among male adolescents, the incidence after the first and second doses were 5.57 (95% CI, 2.38-12.53) and 37.32 (95% CI, 26.98-51.25) per 100 000 persons vaccinated. CONCLUSIONS: There is a significant increase in the risk of acute myocarditis/pericarditis following Comirnaty vaccination among Chinese male adolescents, especially after the second dose.

SETD2 alterations and histone H3K36 trimethylation in phyllodes tumor of breast.

PURPOSE: SETD2 is one of the key epigenetic regulatory genes involved in histone modifications. Its alterations were potentially oncogenic and commonly found in cancers. Interestingly, SETD2 is one of the most frequent mutated genes found exclusively in phyllodes tumor of the breast (PT). However, little has been done to further characterize SETD2 alterations in PT. METHODS: In this study, we examined the alterations of SETD2 gene and protein expression in a large cohort of PTs. Their correlations with SETD2 downstream target, H3K36me3 expression, and clinicopathologic features in PT were also assessed. RESULTS: SETD2 mutation was found in 15.9% of our cases and was mostly predicted to be damaging mutations. Interestingly, SETD2 mutations were associated with lower H3K36me3 expression, particularly those with damaging mutations (p = .041). Neither SETD2 mutations nor H3K36me3 expression was associated with PT grading and other clinicopathological features. By contrast, the SETD2 protein expression cannot reflect its mutation status and showed a different trend of clinicopathological correlations from H3K36me3. CONCLUSIONS: Our findings may suggest a potential involvement of epigenetic regulation via SETD2 alterations and downstream H3K36me3 on PT development. SETD2 mutations may occur early in the pathogenic process of PTs and its loss per se may not be sufficient for progression to malignancy. Exclusive alterations of SETD2 in PT can be used as markers for the diagnosis of fibroepithelial lesions. The association of H3K36me3 with SETD2 mutations may also indicate the value of evaluation of H3K36me3 expression in the diagnosis of fibroepithelial lesions.

Receptor tyrosine kinase fusions act as a significant alternative driver of the serrated pathway in colorectal cancer development.

Serrated polyps are a clinically and molecularly heterogeneous group of lesions that can contribute to the development of colorectal cancers (CRCs). However, the molecular mechanism underlying the development of serrated lesions is still not well understood. Here, we combined multiple approaches to analyze the genetic alterations in 86 colorectal adenomas (including 35 sessile serrated lesions, 15 traditional adenomas, and 36 conventional adenomatous polyps). We also investigated the in vitro and in vivo oncogenic properties of a novel variant of the NCOA4-RET fusion gene. Molecular profiling revealed that sessile serrated lesions and traditional serrated adenomas have distinct clinicopathological and molecular features. Moreover, we identified receptor tyrosine kinase translocations exclusively in sessile serrated lesions (17%), and the observation was validated in a separate cohort of 34 sessile serrated lesions (15%). The kinase fusions as well as the BRAF and KRAS mutations were mutually exclusive to each other. Ectopic expression of a novel variant of the NCOA4-RET fusion gene promoted cell proliferation in vitro and in vivo, and the proliferation was significantly suppressed by RET kinase inhibitors. All of these underscored the importance of mitogen-activated protein kinase (MAPK) pathway activation in the serrated pathway of colorectal tumorigenesis. In addition, we demonstrated that the kinase fusion may occur early in the precursor lesion and subsequent loss of TP53 may drives the transformation to carcinoma during serrated tumorigenesis. In conclusion, we identified kinase fusions as a significant alternative driver of the serrated pathway in colorectal cancer development, and detecting their presence may serve as a biomarker for the diagnosis of sessile serrated lesions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Serologic Responses in Healthy Adult with SARS-CoV-2 Reinfection, Hong Kong, August 2020.

In March 2020, mild signs and symptoms of coronavirus disease developed in a healthy 33-year-old man in Hong Kong. His first infection did not produce virus neutralizing antibodies. In August, he had asymptomatic reinfection, suggesting that persons without a robust neutralizing antibody response might be at risk for reinfection.

Genomic landscape of lymphoepithelioma-like hepatocellular carcinoma.

Lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) is a distinct variant of HCC that is characterized by dense tumor-infiltrating lymphocytes (TILs). Patients with LEL-HCC also show better clinical outcomes compared to conventional HCC (c-HCC), which is commonly presented with low TIL. Emerging evidence has begun to highlight tumor-intrinsic genetic abnormalities in the tumor-host immune interfaces. However, genome-wide characterization of LEL-HCC remains largely unexplored. Here, we defined the genomic landscape of 12 LEL-HCC using whole-exome sequencing, and further underpinned those genetic alterations related to an immune active microenvironment by comparing findings to 15 c-HCC that were sequenced in parallel. Overall, the mutational load between LEL-HCC and c-HCC was similar. Interestingly, SNV incidences of specific genes (CTNNB1, AXIN1, NOTCH1, and NOTCH2) were significantly higher in c-HCC than LEL-HCC, suggesting a plausible link between activated Wnt/ß-catenin and Notch signaling pathways and immune avoidance. Marked focal amplification of chromosome 11q13.3 was prevalent in LEL-HCC. Using The Cancer Genome Atlas dataset, we further established oncogenes expressed from chromosome 11q13.3 (CCND1, FGF19, and FGF4) to be strongly associated with the immune checkpoint signature (CD274, PDCD1, BTLA, CTLA4, HAVCR2, IDO1, and LAG3). Our results have illustrated for the first time the somatic landscape of LEL-HCC, and highlighted molecular alterations that could be exploited in combinatory therapy with checkpoint inhibitors in targeting HCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PRRX-NCOA1/2 rearrangement characterizes a distinctive fibroblastic neoplasm.

Fibroblastic/myofibroblastic neoplasms represent a broad, and occasionally diagnostically challenging, category of soft tissue neoplasms. A subset of these tumors defy conventional classification. However, with the advent of next-generation sequencing, the identification of disease-defining molecular alterations is gradually improving their subclassification. Following identification of two index cases of a distinctive fibroblastic neoplasm with a fusion gene involving PRRX1 and NCOA1, we performed a retrospective review to further characterize this entity. We identified two additional cases, including one with a fusion between PRRX1 and NCOA2. The average patient age was 38 years, and three patients were female. Two tumors occurred on the neck, and the others involved the groin and thigh. Tumors were centered in the subcutis and ranged from 2.3 to 14.0 cm (average 5.8 cm). Morphologically, they were predominantly hypocellular, with focal hypercellularity. They were composed of monomorphic spindle-stellate cells with a vague fascicular pattern. The nuclei were bland with only rare mitotic activity, and occasional multinucleation. The intervening stroma was typically abundant and ranged from myxoid to collagenous, with frequent rope-like collagen bundles. Three of the cases had a prominent vasculature ranging from numerous small curvilinear vessels to ectatic and branching staghorn-like vessels. Immunohistochemistry was negative for desmin, smooth muscle actin, S100, CD34, keratin, and epithelial membrane antigen. Each of the patients was treated by simple excision and none of the tumors were associated with local recurrence or metastasis. Based on their unique morphological and molecular attributes, we believe this represents a novel fibroblastic tumor for which we have tentatively proposed the name "PRRX-NCOAx-rearranged fibroblastic tumor."

A comparability study of immunohistochemical assays for PD-L1 expression in hepatocellular carcinoma.

Programmed death ligand 1 (PD-L1) protein expression by immunohistochemistry is a promising biomarker for PD-1/PD-L1 blockade in hepatocellular carcinoma. There are a number of commercially available PD-L1 assays. Our study aimed to compare the analytical performance of different PD-L1 assays and evaluate the reliability of pathologists in PD-L1 scoring. Consecutive sections from tumor samples from 55 patients with surgically resected primary hepatocellular carcinoma were stained with four standardized PD-L1 assays (22C3, 28-8, SP142, and SP263). We also correlated the PD-L1 protein level by immunohistochemistry with the mRNA level of those genes associated with tumor immune microenvironment by the NanoString platform. Five pathologists independently assessed PD-L1 expression on tumor cells [tumor proportion score] together with tumor-infiltrating immune cells (combined positive score). The 22C3, 28-8, and SP263 assays had comparable sensitivity in detecting PD-L1 expression, whereas the SP142 assay was the least sensitive assay. The inter-assay agreement measured by intraclass correlation coefficients for the tumor proportion score and combined positive score were 0.646 and 0.780, respectively. The inter-rater agreement was good to excellent (the overall intraclass correlation coefficient for the tumor proportion score and combined positive score was 0.946 and 0.809, respectively). Pathologists were less reliable in scoring combined positive score than tumor proportion score, particularly when using the SP142 assay. Up to 18% of samples were misclassified by individual pathologists in comparison to the consensus score at the cutoff of combined positive score ≥ 1. The combined positive score by the 22C3 assay demonstrated the strongest correlation with immune-related gene mRNA signatures, closely followed by combined positive scores by the 28-8 and SP263 assays. In conclusion, the 22C3, 28-8, and SP263 assays are highly concordant in PD-L1 scoring and suggest the interchangeability of these three assays. Further improvement of the accuracy in assessing PD-L1 expression at a low cutoff is still necessary.

Specific targeting of point mutations in EGFR L858R-positive lung cancer by CRISPR/Cas9.

Cancer cells are defined genetically by the mutations they harbor, commonly single nucleotide substitutions. Therapeutic approaches which specifically target cancer cells by recognizing these defining genetic aberrations are expected to exhibit minimal side-effects. However, current protein-based targeted therapy is greatly limited by the range of genes that can be targeted, as well as by acquired resistance. We hypothesized that a therapeutic oligonucleotide-based strategy may address this need of specific cancer targeting. We used CRISPR/Cas9 system to target a commonly occurring EGFR point mutation, L858R, with an oligonucleotide guide that recognizes L858R as the suitable protospacer-adjacent motif (PAM) sequence for DNA cleavage. We found that this strategy, which utilized PAM to differentiate cancer mutation from normal, afforded high specificity to the extent of a single nucleotide substitution. The anti-L858R vehicle resulted in selective genome cleavage only in L858R mutant cells, as detected by Sanger sequencing and T7 Endonuclease I assay. Wild-type cells were unaffected by the same treatment. Digital PCR revealed 37.9 ± 8.57% of L858R gene copies were targeted in mutant. Only treated mutant cells, but not wild-type cells, showed reduction in EGFR expression and decreased cell proliferation. Treated mutant cells also formed smaller tumor load in vivo. This targeting approach is expected to be able to target a significant subset of the 15-35% cancer mutations with C > G, A > G, and T > G point mutations. Thus, this strategy may serve as a useful approach to target cancer-defining mutations with specificity, to the extent of differentiating the change of a single nucleotide.

Assessment of programmed cell death ligand-1 expression by 4 diagnostic assays and its clinicopathological correlation in a large cohort of surgical resected non-small cell lung carcinoma.

Immune checkpoint blockade targeting the PD-1/PD-L1 axis has recently demonstrated efficacy and promise in cancer treatment. Appropriate biomarker selection is therefore essential for improving treatment efficacy. However, the establishment of PD-L1 assay in pathology laboratories is complicated by the presence of multiple testing platforms using different scoring systems. Here we assessed the PD-L1 expression in 713 consecutive non-small cell lung carcinomas by four commercially available PD-L1 immunohistochemical assays, namely, 22C3, 28-8, SP142 and SP263. The analytical performances of the four assays and diagnostic performances across clinically relevant cutoffs were evaluated. The prevalence of PD-L1 (22C3) expression was 21% with a ≥50% cutoff and 56% with a ≥1% cutoff. High PD-L1 expression (using a ≥50% cutoff) was significantly associated with male sex (P = 0.001), ever smoking history (P < 0.001), squamous cell carcinoma (P = 0.001), large cell carcinoma (P < 0.001), lymphoepithelioma-like carcinoma (P = 0.006), sarcomatoid carcinoma (P < 0.001), mutant KRAS (P = 0.005) and wild-type EGFR (P = 0.003). Elevated PD-L1 expression was also significantly associated with shorter survival in patients with adenocarcinoma (log-rank P = 0.026) and remained an independent prognostic factor by multivariable analysis. Among the four assays, 22C3, 28-8 and SP263 were highly concordant for tumor cell scoring. With a cutoff of ≥50% (i.e., the threshold for first-line patient selection), inter-rater agreement was high among the three assays with percentage agreement >97%. In conclusion, three PD-L1 assays showed good analytical performance and a high agreement with each other, but not all cases were correctly classified using the same clinical cutoff. Further studies comparing the predictive value of these assays are required to address the interchangeability of these assays for clinical use.

Comprehensive characterization of the patient-derived xenograft and the paralleled primary hepatocellular carcinoma cell line.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive cancer with high mortality and morbidity worldwide. The limited clinically relevant model has impeded the development of effective HCC treatment strategy. Patient-derived xenograft (PDX) models retain most of the characteristics of original tumors and were shown to be highly predictive for clinical outcomes. Notably, primary cell line models allow in-depth molecular characterization and high-throughput analysis. Combined usage of the two models would provide an excellent tool for systematic study of therapeutic strategies. Here, we comprehensively characterized the novel PDX and the paralleled primary HCC cell line model. METHODS: Tumor tissues were collected from HCC surgical specimens. HCC cells were sorted for in vivo PDX and in vitro cell line establishment by the expression of hepatic cancer stem cell marker to enhance cell viability and the rate of success on subsequent culture. The PDX and its matching primary cell line were authenticated and characterized in vitro and in vivo. RESULTS: Among the successful cases for generating PDXs and primary cells, HCC40 is capable for both PDX and primary cell line establishment, which were then further characterized. The novel HCC40-PDX and HCC40-CL exhibited consistent phenotypic characteristics as the original tumor in terms of HBV protein and AFP expressions. In common with HCC40-PDX, HCC40-CL was tumorigenic in immunocompromised mice. The migration ability in vitro and metastatic properties in vivo echoed the clinical feature of venous infiltration. Genetic profiling by short tandem repeat analysis and p53 mutation pattern consolidated that both the HCC40-PDX and HCC40-CL models were derived from the HCC40 clinical specimen. CONCLUSIONS: The paralleled establishment of PDX and primary cell line would serve as useful models in comprehensive studies for HCC pathogenesis and therapeutics development for personalized treatment.

MicroRNA-183 suppresses cancer stem-like cell properties in EBV-associated nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy that exhibits distinct geographical and ethnic prevalence. Although the contemporary therapeutic approach of radio-/chemotherapy provides excellent results for patients with early-stage disease, it is far from satisfactory for those with disease remission and distant metastasis. Promising therapeutic strategies for advanced and relapsed NPC are still lacking. We recently identified and characterized a cancer stem-like cell (CSC) subpopulation in NPC that appeared to play an important role in tumor progression. Microarray analysis revealed downregulation of several stemness-inhibiting miRNAs in these CSC cells. Among these miRNAs, miR-96 and miR-183 showed the highest fold change and were selected to elucidate their role in repressing NPC CSC properties. METHODS: MiR-96 and miR-183 expression in NPC CSCs was detected by qRT-PCR. Transient and stable transfection was performed in EBV-positive NPC C666-1 cells to examine the effects of ectopic expression of miR-96 and miR-183 on repressing cell growth and CSC properties. Anchorage-dependent (colony formation) and anchorage-independent (tumor sphere formation) growths of these miR-96 and miR-183 expressing cells were determined. Expression of multiple CSC markers and related molecules were accessed by flow cytometry and Western blotting. The tumorigenicity of the stable miR-96- and miR-183-transfected NPC cells was examined in an in vivo nude mice model. RESULTS: Downregulation of miR-96 and miR-183 was confirmed in NPC spheroids. Using transient or stable transfection, we showed that ectopic expression of miR-96 and miR-183 suppressed cell growth and tumor sphere formation in NPC. Reduced NICD3 and NICD4 in miR-96- and miR-183-expressing NPC cells suggests the involvement of the NOTCH signaling pathway in their tumor suppressive function. Finally, we showed that the tumorigenicity of cells stably expressing miR-183 was significantly inhibited in the in vivo nude mice model. CONCLUSIONS: miR-183 is a tumor-suppressive miRNA in EBV-associated NPC. Its abilities to suppress CSC properties in vitro and effectively reduce tumor growth in vivo shed light on its role as a potential therapeutic target.

Establishment and characterization of a novel primary hepatocellular carcinoma cell line with metastatic ability in vivo.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly aggressive and heterogeneous disease. HCC cell lines established from different patients would be useful in elucidating the molecular pathogenesis. However, success of HCC primary culture establishment remains at low rate. We aim to establish and characterize HCC primary culture and the derived cell line. METHODS: Fresh tumor tissues were collected from 30 HCC patients. Culture conditions were optimized for the attachment and growth of the isolated hepatocytes. Granulin-epithelin precursor (GEP), a growth factor reported to associate with cancer stem cell properties, was examined by flow cytometry to elucidate its role on primary culture establishment. The primary cell line was characterized in detail. RESULTS: Cells isolated from 16 out of 30 HCC cases (53%) had viability more than 70% and were subject to subsequent in vitro culture. 7 out of 16 cases (44%) could give rise to cells that were able to attach and grow in culture. GEP expression levels significantly correlated with the viability of isolated hepatocytes and success rate of subsequent primary culture establishment. Cells from HCC patient 21 grew and expanded rapidly in vitro and was selected to be further characterized. The line, designated HCC21, derived from a Hong Kong Chinese female patient with HCC at Stage II. The cells exhibited typical epithelial morphology and expressed albumin, AFP and HBV antigens. The cell line was authenticated by short tandem repeat analysis. Comparative genome hybridization analysis revealed chromosomal loss at 1p35-p36, 1q44, 2q11.2-q24.3, 2q37, 4q12-q13.3, 4q21.21-q35.2, 8p12-p23, 15q11.2-q14, 15q24-q26, 16p12.1-p13.3, 16q, 17p, 22q and gain at 1q21-q43 in both HCC21 cells and the original clinical tumor specimen. Sequence analysis revealed p53 gene mutation. Subcutaneous injection of HCC21 cells into immunodeficient mice showed that the cells were able to form tumors at the primary injection sites and metastatic tumors in the peritoneal cavity. CONCLUSIONS: The newly established cell line could serve as useful in vitro and in vivo models for studying primary HCC that possess metastasis ability.

Constitutive activation of distinct NF-κB signals in EBV-associated nasopharyngeal carcinoma.

As a distinct type of head and neck cancer, non-keratinizing nasopharyngeal carcinoma (NPC) is closely associated with EBV infection and massive lymphoid infiltration. The unique histological features suggest that local inflammation plays an important role in NPC tumourigenesis. We comprehensively characterized NF-κB signalling, a key inflammatory pathway which might contribute to the tumourigenesis of this EBV-associated cancer. By EMSA, western blotting, and immunohistochemical staining, constitutive activation of distinct NF-κB complexes, either p50/p50/Bcl3 or p50/RelB, was found in almost all EBV-positive NPC tumours. siRNA or chemical inhibition of NF-κB signalling significantly inhibited the growth of EBV-positive NPC cells C666-1. Gene expression profiling identified a number of NF-κB target genes involved in cell proliferation, apoptosis, immune response, and transcription. We further confirmed that p50 signals modulate the expression of multiple oncogenes (MYB, BCL2), chemokines, and chemokine receptors (CXCL9, CXCL10, CX3CL1, and CCL20). The findings support a crucial role of these constitutively activated NF-κB signals in NPC tumourigenesis and local inflammation. In addition to expression of the viral oncoprotein LMP1, genetic alteration of several NF-κB regulators (eg TRAF3, TRAF2, NFKBIA, A20) also contributes to the aberrant NF-κB activation in EBV-associated NPC. Except for LMP1-expressing C15 cells, all NPC tumour lines harbour at least one of these genetic alterations. Importantly, missense mutations of TRAF3, TRAF2, and A20 were also detected in 3/33 (9.1%) primary tumours. Taken together with the reported LTBR amplification in 7.3% of primary NPCs, genetic alterations in NF-κB pathways occurred in at least 16% of cases of this cancer. The findings indicate that distinct NF-κB signals are constitutively activated in EBV-positive NPC cells by either multiple genetic changes or EBV latent genes.

Identification of a recurrent transforming UBR5-ZNF423 fusion gene in EBV-associated nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer which is prevalent in southern China, south-east Asia and northern Africa. The development and stepwise progression of NPC involves accumulation of multiple gross genetic changes during the clonal expansion of Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cell population. Here, using paired-end whole-transcriptome sequencing, we discovered a number of chimeric fusion transcripts in a panel of EBV-positive tumour lines. Among these transcripts, a novel fusion of ubiquitin protein ligase E3 component n-recognin 5 (UBR5) on 8q22.3 and zinc finger protein 423 (ZNF423) on 16q12.1, identified from the NPC cell line C666-1, was recurrently detected in 12/144 (8.3%) of primary tumours. The fusion gene contains exon 1 of UBR5 and exons 7-9 of ZNF423 and produces a 94 amino acid chimeric protein including the original C-terminal EBF binding domain (ZF29-30) of ZNF423. Notably, the growth of NPC cells with UBR5-ZNF423 rearrangement is dependent on expression of this fusion protein. Knock-down of UBR5-ZNF423 by fusion-specific siRNA significantly inhibited the cell proliferation and colony-forming ability of C666-1 cells. The transforming ability of UBR5-ZNF423 fusion was also confirmed in NIH3T3 fibroblasts. Constitutive expression of UBR5-ZNF423 in NIH3T3 fibroblasts significantly enhanced its anchorage-independent growth in soft agar and induced tumour formation in a nude mouse model. These findings suggest that expression of UBR5-ZNF423 protein might contribute to the transformation of a subset of NPCs, possibly by altering the activity of EBFs (early B cell factors). Identification of the oncogenic UBR5-ZNF423 provides new potential opportunities for therapeutic intervention in NPC.

PRAME expression in genital melanocytic lesions - Potential diagnostic pitfall of intermediate expression in atypical genital nevi.

INTRODUCTION: The Preferentially Expressed Antigen in Melanoma (PRAME) immunostain has seen significant diagnostic use in confirming malignancy for melanocytic lesions. However, the expression of PRAME in genital melanocytic lesions have not been reported. In this study, PRAME staining was performed on a cohort of genital melanocytic lesions, aiming to investigate the diagnostic role of PRAME in genital melanocytic lesions and its expression in atypical genital nevi. METHODOLOGY: A cohort including genital invasive melanoma, melanoma-in-situ, atypical genital nevus (AGN), compound nevus, intradermal nevus, blue nevus, lentigo and melanosis was retrieved with histology reviewed and PRAME immunostaining performed. RESULTS: A total of 66 cases were reviewed. The average proportion expression of PRAME were 56.75 % and 57.43 % for invasive melanoma and melanoma-in-situ, with average H-scores of 153.5/300 and 163.14/300 respectively, which were greater than AGN (3.25 %, 7.75/300, p<0.001), compound/intradermal nevi, lentigo/melanosis, and background junctional melanocytes (<1 %, <1/300, p<0.001). The different cutoffs of PRAME expression, the sensitivity and specificity were 65.22 % and 100 % (>100/300); 69.57 % and 95.83 % (>10/300); and 82.61 % and 93.75 % (≥1/300) respectively. Low level PRAME expression was seen in half of the cases of AGN (n=2/4, 50 %), and at low cutoffs (>10/300 and ≥1/300) unable to differentiate invasive melanoma from AGN (p>0.05). CONCLUSIONS: For genital melanocytic lesions, PRAME immunostain shows high specificity at strong and diffuse staining. AGN not uncommonly display low level expression. Focal and/or weak PRAME expression should not be considered as an absolute indication of malignancy, and comprehensive histological assessment remains the key to accurate diagnosis of melanocytic lesions.