Nanopore DNA Sequencing Detected Chromothripsis-Induced PAFAH1B1::USP6 Rearrangement in Periosteal Solid Aneurysmal Bone Cyst Initially Diagnosed as Osteosarcoma.

An aneurysmal bone cyst (ABC) is a benign bone neoplasm that typically occurs during the first and second decades of life. ABC usually presents as a rapidly growing intramedullary expansile mass with multiple blood-filled cysts in the metaphysis of the long tubular bones. Here, we report a case of a periosteal solid ABC that was initially diagnosed as a high-grade surface osteosarcoma. A 10-year-old male was referred to our hospital for swelling and tenderness of the left upper arm. Radiography revealed periosteal mass without fluid-fluid levels. On performing open biopsy, the tumor showed hypercellular proliferation of uniform spindle to epithelioid cells with brisk mitotic activity (up to 12/2 mm2) and lace-like osteoid formation, which was diagnosed as a high-grade surface osteosarcoma. After one course of chemotherapy using adriamycin and cisplatin, peripheral sclerosis was conspicuous, which led to pathological review and revision of diagnosis as "possibly osteoblastoma." The patient was disease-free for 4 years after marginal resection and curettage. Retrospective nanopore DNA sequencing unexpectedly detected a PAFAH1B1::USP6 rearrangement. The fusion gene was further validated using reverse transcription-polymerase chain reaction and the diagnosis was revised to ABC. Chromothripsis involving chromosome 17 has also been identified. Methylation analysis classified the present tumor as an ABC or non-ossifying fibroma using t-distributed stochastic neighbor embedding and unsupervised hierarchical clustering. This case report highlights the utility of nanopore DNA sequencing for soft tissue and bone tumor diagnosis.

Fluorescence in situ hybridization-negative intra-articular myxoid liposarcoma with complex rearrangements involving EWSR1::DDIT3 detected using nanopore sequencing.

Myxoid liposarcoma (MLPS) is a rare sarcoma, typically arising in deep soft tissues during the fourth to fifth decades of life. Histologically, MLPS is composed of uniform oval cells within a background of myxoid stroma and chicken-wire capillaries. Genetically, MLPS is characterized by the FUS/EWSR1::DDIT3 fusion gene, which generally results from balanced interchromosomal translocation and is detectable via DDIT3 break-apart fluorescence in situ hybridization (FISH). Here, we report an unusual intra-articular MLPS case, negative for DDIT3 break-apart FISH but positive for EWSR1::DDIT3. An 18-year-old female was referred to our hospital complaining of an intra-articular mass in the right knee joint. Histologically, the tumor was mainly composed of mature adipocytes, brown fat-like cells, and lipoblasts. Nanopore sequencing detected DNA rearrangements between EWSR1 and DDIT3 and clustered complex rearrangements involving multiple chromosomes, suggesting chromoplexy. Methylation classification using random forest, t-distributed stochastic neighbor embedding, and unsupervised hierarchical clustering correctly classified the tumor as MLPS. The copy number was almost flat. The TERT promoter C-124T was also detected. This report highlights, for the first time, the potential value of a fast and low-cost nanopore sequencer for diagnosing sarcomas.

Allelic loss of HLA class I facilitates evasion from immune surveillance in cervical intraepithelial neoplasia.

Loss of heterozygosity (LOH) has been reported to occur in HLA regions in cervical intraepithelial neoplasia (CIN) and cervical cancer. However, the details of how this is related to the progression of CIN have been unclear. In this study, we examined the human papillomavirus (HPV) antigen-presenting capacity of people with CIN and the significance of LOH of HLA class I in the progression of CIN. It was shown that differences in antigen-presenting capacity among each case depended on HLA types, not HPV genotypes. Focusing on the HLA type, there was a positive correlation between antigen-presenting capacity against HPV and the frequency of allelic loss. Furthermore, the lost HLA-B alleles had a higher HPV antigen-presenting capacity than intact alleles. In addition, frequency of LOH of HLA class I was significantly higher in advanced CIN (CIN2-3) than in cervicitis or early-stage CIN (CIN1): around half of CIN2-3 had LOH of any HLA class I. Moreover, the antigen-presenting capacity against E5, which is the HPV proteins that facilitate viral escape from this immune surveillance by suppressing HLA class I expression, had the most significant impact on the LOH in HLA-B. This study suggests that HPV evades immune surveillance mechanisms when host cells lose the capacity for antigen presentation by HLA class I molecules, resulting in long-term infection and progression to advanced lesions.

Targeting PAK1 is effective against cutaneous squamous cell carcinoma in a syngenic mouse model.

By taking advantage of forward genetic analysis in mice, we have demonstrated that Pak1 plays a crucial role during DMBA/TPA skin carcinogenesis. Although Pak1 has been considered to promote cancer development, its overall function remains poorly understood. To clarify the functional significance of Pak1 in detail, we sought to evaluate the possible effect of an allosteric inhibitor against PAK1 (NVS-PAK1-1) on a syngeneic mouse model. To this end, we established two cell lines, 9AS1 and 19AS1, derived from DMBA/TPA-induced squamous cell carcinoma (SCC) that engrafted in FVB mice. Based on our present results, NVS-PAK1-1 treatment significantly inhibited the growth of tumors derived from 9AS1 and 19AS1 cells in vitro and in vivo. RNA-sequencing analysis on the engrafted tumors indicates that NVS-PAK1-1 markedly potentiates the epidermal cell differentiation and enhances the immune response in the engrafted tumors. Consistent with these observations, we found an expansion of Pan-keratin-positive regions and potentially elevated infiltration of CD8-positive immune cells in NVS-PAK1-1-treated tumors as examined by immunohistochemical analyses. Together, our present findings strongly suggest that PAK1 is tightly linked to the development of SCC, and that its inhibition is a promising therapeutic strategy against SCC.

Metastasizing aneurysmal dermatofibroma initially diagnosed as angiosarcoma confirmed by CD63::PRKCD fusion gene detection with nanopore sequencing.

Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.

Correction: Histone Demethylase JMJD2B Functions as a Co-Factor of Estrogen Receptor in Breast Cancer Proliferation and Mammary Gland Development.

[This corrects the article DOI: 10.1371/journal.pone.0017830.].

CD106 in Tumor-Specific Exhausted CD8+ T Cells Mediates Immunosuppression by Inhibiting TCR Signaling.

T-cell exhaustion is a major contributor to immunosuppression in the tumor microenvironment (TME). Blockade of key regulators of T-cell exhaustion, such as programmed death 1, can reinvigorate tumor-specific T cells and activate antitumor immunity in various types of cancer. In this study, we identified that CD106 was specifically expressed in exhausted CD8+ T cells in the TME using single-cell RNA sequencing. High CD106 expression in the TME in clinical samples corresponded to improved response to cancer immunotherapy. CD106 in tumor-specific T cells suppressed antitumor immunity both in vitro and in vivo, and loss of CD106 in CD8+ T cells suppressed tumor growth and improved response to programmed death 1 blockade. Mechanistically, CD106 inhibited T-cell receptor (TCR) signaling by interacting with the TCR/CD3 complex and reducing its surface expression. Together, these findings provide insights into the immunosuppressive role of CD106 expressed in tumor-specific exhausted CD8+ T cells, identifying it as a potential biomarker and therapeutic target for cancer immunotherapy. Significance: CD106 is specifically expressed in tumor-specific exhausted CD8+ T cells and inhibits the TCR signaling pathway by reducing surface expression of the TCR/CD3 complex to suppress antitumor immunity.

Stem-like progenitor and terminally differentiated TFH-like CD4+ T cell exhaustion in the tumor microenvironment.

Immune checkpoint inhibitors exert clinical efficacy against various types of cancer through reinvigoration of exhausted CD8+ T cells that attack cancer cells directly in the tumor microenvironment (TME). Using single-cell sequencing and mouse models, we show that CXCL13, highly expressed in tumor-infiltrating exhausted CD8+ T cells, induces CD4+ follicular helper T (TFH) cell infiltration, contributing to anti-tumor immunity. Furthermore, a part of the TFH cells in the TME exhibits cytotoxicity and directly attacks major histocompatibility complex-II-expressing tumors. TFH-like cytotoxic CD4+ T cells have high LAG-3/BLIMP1 and low TCF1 expression without self-renewal ability, whereas non-cytotoxic TFH cells express low LAG-3/BLIMP1 and high TCF1 with self-renewal ability, closely resembling the relationship between terminally differentiated and stem-like progenitor exhaustion in CD8+ T cells, respectively. Our findings provide deep insights into TFH-like CD4+ T cell exhaustion with helper progenitor and cytotoxic differentiated functions, mediating anti-tumor immunity orchestrally with CD8+ T cells.