Hematopoietic Wnts Modulate Endochondral Ossification During Fracture Healing.

Wnt signaling plays a critical role in bone formation, homeostasis, and injury repair. Multiple cell types in bone have been proposed to produce the Wnts required for these processes. The specific role of Wnts produced from cells of hematopoietic origin has not been previously characterized. Here, we examined if hematopoietic Wnts play a role in physiological musculoskeletal development and in fracture healing. Wnt secretion from hematopoietic cells was blocked by genetic knockout of the essential Wnt modifying enzyme PORCN, achieved by crossing Vav-Cre transgenic mice with Porcnflox mice. Knockout mice were compared with their wild-type littermates for musculoskeletal development including bone quantity and quality at maturation. Fracture healing including callus quality and quantity was assessed in a diaphyseal fracture model using quantitative micro computer-assisted tomographic scans, histological analysis, as well as biomechanical torsional and 4-point bending stress tests. The hematopoietic Porcn knockout mice had normal musculoskeletal development, with normal bone quantity and quality on micro-CT scans of the vertebrae. They also had normal gross skeletal dimensions and normal bone strength. Hematopoietic Wnt depletion in the healing fracture resulted in fewer osteoclasts in the fracture callus, with a resultant delay in callus remodeling. All calluses eventually progressed to full maturation. Hematopoietic Wnts, while not essential, modulate osteoclast numbers during fracture healing. These osteoclasts participate in callus maturation and remodeling. This demonstrates the importance of diverse Wnt sources in bone repair.

Epigenetic Regulation of the PTEN-AKT-RAC1 Axis by G9a Is Critical for Tumor Growth in Alveolar Rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. As transient and stable modifications to chromatin have emerged as critical mechanisms in oncogenic signaling, efforts to target epigenetic modifiers as a therapeutic strategy have accelerated in recent years. To identify chromatin modifiers that sustain tumor growth, we performed an epigenetic screen and found that inhibition of lysine methyltransferase G9a significantly affected the viability of ARMS cell lines. Targeting expression or activity of G9a reduced cellular proliferation and motility in vitro and tumor growth in vivo. Transcriptome and chromatin immunoprecipitation-sequencing analysis provided mechanistic evidence that the tumor-suppressor PTEN was a direct target gene of G9a. G9a repressed PTEN expression in a methyltransferase activity-dependent manner, resulting in increased AKT and RAC1 activity. Re-expression of constitutively active RAC1 in G9a-deficient tumor cells restored oncogenic phenotypes, demonstrating its critical functions downstream of G9a. Collectively, our study provides evidence for a G9a-dependent epigenetic program that regulates tumor growth and suggests targeting G9a as a therapeutic strategy in ARMS. SIGNIFICANCE: These findings demonstrate that RAC1 is an effector of G9a oncogenic functions and highlight the potential of G9a inhibitors in the treatment of ARMS.

Primary Epithelioid Angiosarcoma of Finger Masquerading as Epithelioid Hemangioma: Report of a Case and Analysis of Mutational Pattern in Epithelioid Hemangiomas and Angiosarcomas by Next-generation Sequencing.

AIMS: We report an unusual case of epithelioid angiosarcoma (AS) mimicking an epithelioid hemangioma (EH) and analyze mutational patterns in EHs and ASs. METHODS AND RESULTS: A 58-year-old woman presented with a finger lump and metastatic lung nodules. Initial needle biopsies showed an EH, with only focal atypical histologic features. The patient underwent finger amputation and resection of lung nodules. The amputation specimen and lung nodules revealed features of AS. Fluorescence in situ hybridization for FOS and FOSB gene rearrangements were negative in the primary tumor as well as in the lung metastasis. Intrigued by the unique morphologic features of an AS masquerading as an EH, we expanded our study by analyzing mutations in EHs versus ASs using a targeted next-generation sequencing of 50 cancer-related genes. Seven EHs and 6 ASs including the present case were subjected to mutation analysis using the Ion AmpliSeq Cancer Hotspot Panel v2 assay of 50 cancer-related genes. The present case lacked mutation. Novel somatic variants were detected in 2 of 7 EHs and 1 of 6 ASs. Sorting intolerant from tolerant and polymorphism phenotyping analysis revealed benign/tolerated and deleterious variants in both tumor types. Deleterious variants TP53 c.707T>C (p.Tyr236Cys), FLT3 c.1995C>T (p.Met665Ile), and SMO c.1919C>T (p.Thr640Ile) were detected in EH, while AS revealed deleterious variant PTPN11 c.226G>A (p.Glu76Lys). CONCLUSIONS: We present an epithelioid AS mimicking EH. We report novel somatic variants in EHs and AS. Benign variants may not be associated with development of these tumors. Whereas, deleterious variants, especially PTPN11 c.226G>A, may be linked to tumorigenesis of AS.

Are we ready for the use of intraoperative salvaged blood in metastatic spine tumour surgery?

PURPOSE: To evaluate the feasibility of using intraoperative cell salvage (IOCS) in combination with leucocyte depletion filter (LDF) in eliminating tumour cells from blood salvaged during metastatic spine tumour surgery (MSTS). This is with the view to pave the path for use of IOCS-LDF in MSTS and musculoskeletal oncological surgery. METHODS: Sixty consecutive patients with known primary epithelial tumour, who were offered surgery for metastatic spine disease at our university hospital, were recruited. Blood samples were collected at three different stages during surgery: from operative field prior to IOCS processing, after IOCS processing and after IOCS-LDF processing. Three separate samples (5 ml each) were taken at each stage. Samples were examined by cell block technique using immunohistochemical monoclonal antibodies to identify tumour cells of epithelial origin in the samples. RESULTS: Of 60 patients, ten were excluded for not fulfilling the inclusion criteria leaving 50 patients. Malignant tumour cells were detected in the samples from operative field prior to IOCS processing in 24 patients and in the samples from the transfusion bag post-IOCS processing in 4 patients. No viable malignant cells were detectable in any of the blood samples after passage through both IOCS and LDF. CONCLUSIONS: The findings support the notion that IOCS-LDF combination works effectively in eliminating tumour cells from salvaged blood so this technique can possibly be applied in MSTS and even musculoskeletal oncological surgery. This concept can then be extended to other oncological surgeries in general with further appropriate clinical studies.

Angiomatoid Fibrous Histiocytoma With Prominent Myxoid Stroma: A Case Report and Review of the Literature.

Angiomatoid fibrous histiocytoma is a rare neoplasm of intermediate malignant potential that usually occurs in the dermis or subcutaneous tissues of the extremities in children or young adults. It is characterized by a nodular growth of spindled, histiocytic, or epithelioid cells and blood-filled spaces, surrounded by a fibrous pseudocapsule that contains a lymphocytic cuff. The histological spectrum of this condition has expanded to include cases that contain prominent myxoid stroma. We herein present another instance of myxoid angiomatoid fibrous histiocytoma and review the clinical and histological features, immunohistochemical profile, and molecular genetics of this uncommon variant. We also discuss the diagnostic mimics of this condition, including benign myxoid soft tissue tumors and sarcomas, to illustrate the potential pitfalls in arriving at the diagnosis.

Can there be a place for intraoperative salvaged blood in spine tumor surgery?

BACKGROUND: Intraoperative cell salvage (IOCS) has been used in musculoskeletal surgery extensively. However, it has never found its place in musculoskeletal oncologic surgery. We have conducted the first-ever study to evaluate the feasibility of IOCS in combination with a leucocyte-depletion filter (LDF) in metastatic spine tumor surgery. This was to pave the path for use of IOCS-LDF in musculoskeletal oncologic surgery. METHODS: Patients with a known primary epithelial tumor, who were offered surgery for metastatic spinal disease, were recruited. Blood samples were collected at three different stages during the surgery: from the operative field before IOCS processing, after IOCS processing, and after IOCS-LDF processing. Three separate samples (5 mL each) were taken at each stage. Samples were examined using immunohistochemical monoclonal antibodies to identify tumor cells of epithelial origin. RESULTS: Of 30 patients in the study, 6 were excluded for not fulfilling the inclusion criteria, leaving 24 patients. Malignant tumor cells were detected in the samples from the operative field before IOCS processing in eight patients and in the samples from the transfusion bag after IOCS processing in three patients. No viable malignant cell was detectable in any of the blood samples after passage through both IOCS and LDF. CONCLUSIONS: The findings support the notion that the IOCS-LDF combination works effectively in eliminating tumor cells from salvaged blood, so this technique can be applied successfully in spine tumor surgery. This concept can then further be extended to whole musculoskeletal tumor surgery and other oncologic surgeries with further appropriate clinical studies.

The scar sign: a useful finding on FDG PET/CT to distinguish sarcoidosis from other causes of lymphadenopathy.

Scar involvement is a rare but characteristic cutaneous manifestation of sarcoidosis. The concurrent presence of FDG-avid lymphadenopathy and scar involvement (the "scar sign") is a useful finding on FDG PET/CT to suggest sarcoidosis, especially when biopsy specimens are difficult to obtain. A 46-year-old woman who presented with fever, cough, and weight loss was found to have mediastinal and hilar lymphadenopathy on chest radiography and CT scan. FDG PET/CT scan showed FDG-avid lower cervical, mediastinal, hilar, and inguinal lymphadenopathy. There was also increased FDG uptake along an old hysterectomy scar. Mediastinoscopy and nodal biopsy revealed noncaseating granulomas compatible with sarcoidosis.

Atypical cellular unguioblastic fibroma-A rare case with more atypical histological features than previously reported.

We herein present the clinicopathological features of an atypical cellular unguioblastic fibroma, a rare onychomatrical tumor, in an adult woman of Southeast Asian origin. The tumor displayed both diffuse hypercellularity and atypia in the mesenchymal component and the presence of large multinucleated cells with a ring-/floret-like arrangement of irregular and hyperchromatic nuclei. The mesenchymal cells displayed a patchy weak positive immunereactivity for CD34 (including atypical and wreath-like multinucleated cells) and for CD10 but were negative for low molecular weight cytokeratins, epithelial membrane antigen, CD68, factor XIIIa, smooth muscle actin, desmin, and HMB-45. The proliferative index as measured immunohistochemically (Mib-1/Ki-67) was 5%. The extent of these atypical histological features has not previously been described. One-year follow-up has been uneventful.

Inflammatory tumour microenvironment is associated with superior survival in hepatocellular carcinoma patients.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is an aggressive malignancy with few treatment options. As the status of the tumour immune microenvironment can affect progression of established tumours, we evaluated potential immune mechanisms associated with survival in HCC. METHODS: Immune gene expression profiles were analyzed in tumour and non-tumour liver tissues from resected HCC patients using quantitative PCR and immunohistochemistry. Tumour-infiltrating leukocytes (TILs) were isolated to verify the expression of immune genes and to identify proliferating TILs. These parameters were analyzed statistically in relation with patient survival and tumour phenotype (apoptosis and proliferation). RESULTS: The immune microenvironment within tumours was found to be heterogeneous, although globally more inert compared to the adjacent non-tumour liver tissue. Univariate analysis in 61 patients identified a group of innate immune genes whose expression within tumours is positively associated with patient survival. TNF, IL6 and CCL2 are the most significant genes, with TNF being an independent predictor of survival in multivariate analysis. The gene set includes macrophage and NK-associated molecules such as TLR4, TLR3, CCR2, NCR3. Most of these molecules are expressed by TILs. Importantly, proliferating immune cells, predominantly NK and T cells, are present in tumours of patients with longer survival, and exclusively in areas devoid of proliferating tumour cells. NK and CD8(+) T cell densities are correlated positively with tumour apoptosis, and negatively with tumour proliferation. CONCLUSIONS: Hence, an inflammatory immune microenvironment within HCC tumours could be an important means to control tumour progression via TIL activation and proliferation.

Ovarian carcinoma presenting with isolated contralateral inguinal lymph node metastasis: a case report.

INTRODUCTION: Ovarian carcinoma usually presents at an advanced stage with diffuse intraabdominal manifestations. We report a patient who presented with a right groin swelling. CLINICAL PICTURE: The only clinical abnormality was an enlarged right inguinal lymph node (3 x 2 cm), for which excision biopsy revealed metastatic adenocarcinoma. A computed tomography (CT) scan showed an enlarged left ovarian lesion (9.0 x 6.4 cm). TREATMENT AND OUTCOME: Laparotomy with total hysterectomy, bilateral salpingo-oophrectomy and partial omentectomy were performed. Histology confirmed left ovarian adenocarcinoma, consistent with the earlier histology of the right inguinal lymph node. There were no other sites of involvement. Postoperatively, the patient received adjuvant chemotherapy for treatment of FIGO Stage IIIc ovarian carcinoma and is clinically disease free 13 months after surgery. CONCLUSIONS: Ovarian cancer presenting with inguinal lymph node metastases is uncommon. Ovarian cancer which manifests solely as a contralateral inguinal lymph node metastasis has not been previously reported. This case illustrates a rare presentation of ovarian carcinoma, and underscores the need to consider ovarian carcinoma in the differential diagnosis of women with inguinal lymphadenopathy.

Epstein-Barr virus-associated smooth muscle tumors are distinctive mesenchymal tumors reflecting multiple infection events: a clinicopathologic and molecular analysis of 29 tumors from 19 patients.

Epstein-Barr virus-associated smooth muscle tumors (EBV-SMT) are rare lesions that occur in immunocompromised patients. Because they have not been fully characterized pathologically or at the molecular level, we have studied 29 tumors from 19 patients, the largest series to date. Cases coded as EBV-SMT were identified in 19 patients from consultation files and from the renal transplant database at Singapore General Hospital. EBV-SMT occurred in adults (mean age 39 years; range, 21-57 years) and predominantly affected males (12 male, 7 female). Causes of immunocompromise were renal transplantation (10), AIDS (8), and steroid therapy (1). Tumors were located in soft tissue (5), lung (5), liver (4), and miscellaneous sites (15). In 13 patients (68%), the tumors were multiple. Infection with EBV was confirmed in all cases by in situ hybridization for EBV early RNAs (EBER). EBV-SMT were typically well-differentiated smooth muscle tumors with little atypia and usually a low level of mitotic activity. Unlike classic leiomyosarcomas, they lacked significant pleomorphism but frequently displayed primitive round cell areas and prominent intratumoral T lymphocytes. No consistent relationship between histologic features and clinical outcome was noted. All expressed actin (29 of 29) and less frequently desmin (14 of 26). Multiple tumors in a given patient were clonally distinct as assessed by the long terminal repeat region of the virus, supporting the view that multifocal tumors arise from multiple infection events rather than from metastasis. Strain typing by analysis of the EBNA-3C gene confirmed the presence of EBV type 2. Two of four tumors assessed were positive for a 30-bp deletion in the LMP1 gene. EBV copy number per cell ranged greatly between patients and between tumors from the same patient. Follow-up information was available in 18 of 19 patients (mean, 25 months; range, 1-105 months). Fifteen patients were alive: 11 with disease and 4 without. Three patients died, 1 due to disease. We conclude that EBV-SMT are histologically distinct from classic soft tissue smooth muscle tumors, are not readily evaluated by means of conventional histologic criteria, and in the case of multifocal tumors are the result of multiple infection events rather than metastasis. EBV-2 can transform smooth muscle cells, independent of the presence of the LMP1 deletion associated with greater virulence.

Polymorphous low-grade adenocarcinoma in the lung: a case report.

Although uncommon, it is well recognized that salivary gland-type tumors can occur as primary lung tumors, probably arising from minor salivary-type glands lining the bronchial tree. Polymorphous low-grade adenocarcinoma (PLGA) is a rare tumor that usually originates from oral minor salivary glands. There are only 2 reported cases showing metastasis to the lung; however, a primary lung tumor has not been reported so far. In this report we describe the clinical and pathological features of another case of PLGA involving the lung, but in a patient with no evidence of a previous oropharyngeal primary. While our case probably represents another example of metastatic PLGA to the lung, to our knowledge, it is the first description of a PLGA involving the lung in the absence of a history of a previous primary oral salivary gland tumor.