Charles F. Timmons

Primary Renal Neoplasms with the ASPL-TFE3 Gene Fusion of Alveolar Soft Part Sarcoma A Distinctive Tumor Entity Previously Included among Renal Cell Carcinomas of Children and Adolescents

The unbalanced translocation, der(17)t(X;17)(p11.2;q25), is characteristic of alveolar soft part sarcoma (ASPS). We have recently shown that this translocation fuses the TFE3 transcription factor gene at Xp11.2 to ASPL, a novel gene at 17q25. We describe herein eight morphologically distinctive renal tumors occurring in young people that bear the identical ASPL-TFE3 fusion transcript as ASPS, with the distinction that the t(X;17) translocation is cytogenetically balanced in these renal tumors. A relationship between these renal tumors and ASPS was initially suggested by the cytogenetic finding of a balanced t(X;17)(p11.2;q25) in two of the cases, and the ASPL-TFE3 fusion transcripts were then confirmed by reverse transcriptase-polymerase chain reaction. The morphology of these eight ASPL-TFE3 fusion-positive renal tumors, although overlapping in some aspects that of classic ASPS, more closely resembles renal cell carcinoma (RCC), which was the a priori diagnosis in all cases. These tumors demonstrate nested and pseudopapillary patterns of growth, psammomatous calcifications, and epithelioid cells with abundant clear cytoplasm and well-defined cell borders. By immunohistochemistry, four tumors were negative for all epithelial markers tested, whereas four were focally positive for cytokeratin and two were reactive for epithelial membrane antigen (EMA) (one diffusely, one focally). Electron microscopy of six tumors demonstrated a combination of ASPS-like features (dense granules in four cases, rhomboid crystals in two cases) and epithelial features (cell junctions in six cases, microvilli and true glandular lumens in three cases). Overall, although seven of eight tumors demonstrated at least focal epithelial features by electron microscopy or immunohistochemistry, the degree and extent of epithelial differentiation was notably less than expected for typical RCC. We confirmed the balanced nature of the t(X;17) translocation by fluorescence in situ hybridization in all seven renal tumors thus analyzed, which contrasts sharply with the unbalanced nature of the translocation in ASPS. In summary, a subset of tumors previously considered to be RCC in young people are in fact genetically related to ASPS, although their distinctive morphological and genetic features justify their classification as a distinctive neoplastic entity. Finally, the finding of distinctive tumors being associated with balanced and unbalanced forms of the same translocation is to our knowledge, unprecedented.

PRCC-TFE3 renal carcinomas: morphologic, immunohistochemical, ultrastructural, and molecular analysis of an entity associated with the t(X;1)(p11.2;q21).

The reappraisal of genetically defined subsets of renal tumors can help to highlight the key pathologic features of specific neoplastic entities. We report the morphologic, immunophenotypic, ultrastructural, and molecular features of 11 renal carcinomas bearing a t(X;1)(p11.2;q21) and/or the resulting PRCC-TFE3 gene fusion. The male/female ratio was 4:7. Ten patients were in the age range of 9–29 years and one was 64 years old (mean 21.3 years, median 15 years). The predominant histologic pattern was nested, with islands of tumor cells compartmentalized by thin-walled capillary vasculature. Minor variations on this pattern yielded solid, acinar, alveolar, and tubular architecture. Papillary architecture was seen in nine cases, usually as a minor component. Neoplastic cells were typically characterized by irregularly shaped nuclei with vesicular chromatin and small nucleoli not visible with a 10× objective, and cytoplasm that ranged from clear to densely granular and eosinophilic. Mitoses were extremely rare; 5 were found in 900 high power fields examined from the 11 neoplasms. The most distinctive immunohistochemical feature of these neoplasms was moderate to intense nuclear labeling for TFE3 protein. These tumors were also consistently immunoreactive for the RCC antigen (10 of 11) and CD10 (9 of 9), whereas cytokeratin and epithelial membrane antigen were negative in four cases and were positive focally in the others. Ultrastructurally, all of the six neoplasms examined showed features consistent with conventional-type (clear cell) renal carcinoma, although two demonstrated distinctive intracisternal microtubules. Both tumors tested contained PRCC-TFE3 fusion transcripts. The differential diagnosis includes conventional-type papillary renal cell carcinoma, conventional-type (clear cell) renal carcinoma, and the ASPL-TFE3 renal carcinomas associated with the t(X;17)(p11.2;q25), with the latter two being morphologically the most similar to the t(X;1) renal carcinomas. Aside from their distinctive clinicopathologic features described here, there is experimental evidence suggesting that these tumors may show differential sensitivity to certain chemotherapeutic agents.

Aberrant promoter methylation and silencing of the RASSF1A gene in pediatric tumors and cell lines

Aberrant promoter methylation of tumor suppressor genes has not been fully investigated in pediatric tumors. Therefore, we examined the methylation status of nine genes (p16INK4A, MGMT, GSTP1, RASSF1A, APC, DAPK, RARβ, CDH1 and CDH13) in 175 primary pediatric tumors and 23 tumor cell lines using methylation-specific PCR. We studied the major forms of pediatric tumors – Wilms tumor, neuroblastoma, hepatoblastoma, medulloblastoma, rhabdomyosarcoma, osteosarcoma, Ewings sarcoma, retinoblastoma and acute leukemia. The most frequently methylated gene in both primary tumors and cell lines was RASSF1A (40, 86%, respectively). However, the rates of RASSF1A methylation in individual tumor types varied from 0 to 88%. RASSF1A methylation was tumor specific and was absent in adjacent non-malignant tissues. Methylation of the other genes was relatively rare in tumors and non-malignant tissues (less than 5%). Neuroblastoma patients with methylation of RASSF1A were significantly older than patients without methylation (P=0.008). There was no relationship between methylation status and other clinico-pathologic parameters. We treated six cell lines lacking RASSF1A mRNA with 5-aza-2′deoxycytidine to examine the relationship between methylation and transcriptional silencing. In five of six cell lines, restoration of RASSF1A mRNA was confirmed by RT–PCR. Our findings indicate that aberrant promoter methylation of RASSF1A may contribute to the pathogenesis of many different forms of pediatric tumors.

Absence of expression of SMARCB1/INI1 in malignant rhabdoid tumors of the central nervous system, kidneys and soft tissue: an immunohistochemical study with implications for diagnosis

Malignant rhabdoid tumors are high-grade neoplasms of the central nervous system (CNS), kidneys and soft tissue that usually occur in children. The histologic diagnosis of malignant rhabdoid tumor depends on identification of characteristic rhabdoid cells—large cells with eccentrically located nuclei and abundant, eosinophilic cytoplasm—and immunohistochemistry with antibodies to vimentin, keratin and epithelial membrane antigen. In most malignant rhabdoid tumors, the SMARCB1/INI1 gene, located in chromosome band 22q11.2, is inactivated by deletions and/or mutations, so genetic diagnosis is often possible. However, tissue may not be available for genetic analysis or studies not confirmatory. We assessed SMARCB1/INI1 expression in 17 rhabdoid tumors and 57 other tumors of the CNS, kidney or soft tissue using immunohistochemistry. In total, 12 brain, three renal and two soft tissue rhabdoid tumors were examined along with four glioblastomas, four pilocytic astrocytomas, four oligodendrogliomas, two ependymomas, two choroid plexus papillomas, five pituitary adenomas, four germinomas, four renal carcinomas with Xp11.2 translocations, two clear cell sarcomas, two Wilms tumors, one renal medullary carcinoma, two desmoplastic small round cell tumors, two alveolar rhabdomyosarcomas, two embryonal rhabdomyosarcomas, one low-grade chondrosarcoma, two extraskeletal myxoid chondrosarcomas, one mesenchymal chondrosarcoma, four malignant peripheral nerve sheath tumors, five metastatic carcinomas and four epithelioid sarcomas, two primary and two metastatic. The neoplastic cells of all rhabdoid tumors, the four epithelioid sarcomas and the renal medullary carcinoma did not express SMARCB1/INI1 by immunohistochemistry; neoplastic cells of all other tumors expressed SMARCB1/INI1. Immunohistochemistry to assess expression of SMARCB1/INI1 may be useful in the diagnosis of rhabdoid tumors of the CNS, kidneys and soft tissue.

Imaging Characteristics of Atypical Teratoid–Rhabdoid Tumor in Children Compared with Medulloblastoma

OBJECTIVEnThe purpose of our study was to compare the imaging characteristics of atypical teratoid-rhabdoid tumor with medulloblastoma and seek distinguishing features that can aid in preoperative diagnosis.nnnMATERIALS AND METHODSnPreoperative MRI examinations of 55 patients (36 medulloblastomas and 19 atypical teratoid-rhabdoid tumors) were analyzed retrospectively. Imaging characteristics of atypical teratoid-rhabdoid tumor and medulloblastoma were assessed with conventional MRI and CT. Diffusion-weighted imaging (DWI) was available in 27 patients (19 medulloblastomas and eight atypical teratoid-rhabdoid tumors). Apparent diffusion coefficient (ADC) values were calculated for 14 medulloblastomas and six atypical teratoid-rhabdoid tumors.nnnRESULTSnBoth atypical teratoid-rhabdoid tumors in general and infratentorial atypical teratoid-rhabdoid tumors presented at a younger age than medulloblastomas. Eleven of 19 atypical teratoid-rhabdoid tumors were infratentorial. Cerebellopontine angle (CPA) involvement was more frequent (8/11, 72.7%) in atypical teratoid-rhabdoid tumor than in medulloblastoma (4/36, 11.1%) (p < 0.001). Intratumoral hemorrhage was more common in atypical teratoid-rhabdoid tumor (9/19, 47.4%) than in medulloblastoma (2/36, 5.6%) (p < 0.0001). All atypical teratoid-rhabdoid tumors and all medulloblastomas for which DWI was available displayed increased signal intensity on DWI compared with normal brain parenchyma. The mean ADC values for tumor types were not significantly different.nnnCONCLUSIONnAtypical teratoid-rhabdoid tumor presents at a younger age than medulloblastoma. Although atypical teratoid-rhabdoid tumor and medulloblastoma display similar imaging characteristics on conventional MRI, CPA involvement and intratumoral hemorrhage are more common in atypical teratoid-rhabdoid tumor. If a pediatric posterior fossa mass that displays restricted diffusion is involving the CPA, atypical teratoid-rhabdoid tumor is a more likely consideration than medulloblastoma.

DNA Methylation Profiles of Lymphoid and Hematopoietic Malignancies

Purpose: Aberrant methylation of the 5′ gene promoter regions is an epigenetic phenomenon that is the major mechanism for silencing of tumor suppressor genes in many cancer types. The aims of our study were (a) to compare the methylation profiles of the major forms of hematological malignancies and (b) to determine the methylation profile of monoclonal gammopathy of undetermined significance (MGUS) and compare it with that of multiple myeloma (MM). Experimental Design: We compared the aberrant promoter methylation profile of 14 known or suspected tumor suppressor genes in leukemias (n = 48), lymphomas (n = 42), and MMs (n = 40). We also examined the methylation profile of MGUS (n = 20), a premalignant plasma cell dyscrasia. The genes studied represent five of the six “hallmarks of cancer.” Results: Peripheral blood lymphocytes (n = 14) from healthy volunteers were negative for methylation of all genes, and methylation percentages in 41 nonmalignant tissues (peripheral blood mononuclear cells, bone marrows, and lymph nodes) from hematological patients were low (0–9%) for all 14 genes, confirming that methylation was tumor specific. Ten of the genes were methylated at frequencies of 29–68% in one or more tumor types, and the methylation indices (an indicator of overall methylation) varied from 0.25 to 0.34. With two exceptions, the methylation patterns of leukemias and lymphomas were similar. However, the pattern of MMs varied from the other tumor types for six genes. In general, the methylation pattern of MGUS was similar to that of MM, although the methylation frequencies were lower (the methylation index of MGUS was 0.15, and that of MM was 0.3). However, the methylation frequencies of six genes were significantly higher in MGUS than in control tissues. The relatively high frequencies of methylation in MGUS are consistent with it being a premalignant condition. Conclusions: The three major forms of lymphoid/hematopoietic malignancies show overlapping but individual patterns of methylation.

Presence of Simian Virus 40 DNA Sequences in Human Lymphoid and Hematopoietic Malignancies and Their Relationship to Aberrant Promoter Methylation of Multiple Genes

The simian polyoma virus SV40 has been detected in specific human tumors including non-Hodgkin’s lymphomas, although a causative role for the virus has not been convincingly demonstrated. Aberrant methylation of CpG islands in promoter regions is a frequent method of silencing tumor suppressor genes (TSGs) in cancers and may be induced by oncogenic viruses. We investigated the relationship between the presence of SV40 or EBV DNA sequences and the methylation profiles for 10 TSGs in 90 cases of non-Hodgkin’s lymphomas/leukemias and 56 control tissues. SV40 sequences were present in 33/90 (37%) non-Hodgkin’s lymphomas/leukemias, and EBV was present in 11/42 (26%) of non-Hodgkin’s lymphomas. We found a highly significant correlation between the presence of SV40 and methylation of seven genes (P values, 0.006 to <0.0001). In lymphomas, there was no relationship between EBV and methylation. Oncogenic viruses and methylation were rarely present in control tissues. We investigated methylation of the same 10 TSGs in peripheral blood mononuclear cells (PBMC) from a healthy volunteer infected with EBV or EBV and SV40. Promoter methylation of CDH1 and CDH13 were noted in dual SV40- and EBV-infected PBMC, and these two genes were also highly significantly correlated to the presence of SV40 sequences in tumors. SV40 infection also resulted in appearance of the lymphoma/leukemia-specific marker, methylated SHP1. Methylation was completely absent in uninfected and EBV-infected PBMC. Our results demonstrate that the presence of SV40 in hematological malignancies is associated with promoter methylation of TSGs and that in all probability, the virus plays a role in tumor pathogenesis.

The RNA Component of Telomerase as a Marker of Biologic Potential and Clinical Outcome in Childhood Neuroblastic Tumors

Telomerase is a ribonucleoprotein enzyme associated with cellular immortality that may be useful in determining the biologic potential of a tumor. Neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN) are neuroblastic tumors (NTs) that exhibit a spectrum of histologic features and are often associated with unpredictable behavior and clinical outcome.

Cyclooxygenase-2 expression does not correlate with outcome in osteosarcoma or rhabdomyosarcoma

Purpose To determine if expression of cyclooxygenase (COX)-2, an inducible enzyme with known tumor-promoting activity, correlates with outcome in pediatric sarcomas. COX-2 overexpression correlates with more aggressive disease in a variety of adult solid tumors. Methods Archived human osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma tumors were retrospectively evaluated, blinded to outcome, for COX-2 expression by immunohistochemistry and correlated with patient characteristics and survival. Results COX-2 expression was detected in 94 of 142 (66%) tumors (osteosarcoma, 66/99 [67%]; rhabdomyosarcoma, 21/35 [60%]; Ewing sarcoma, 7/8 [88%]) and 51 of 85 (60%) diagnostic biopsies (osteosarcoma, 26/45 [58%]; rhabdomyosarcoma, 21/35 [60%]; Ewing sarcoma, 4/5 [80%]). COX-2 expression did not vary with clinicopathologic features and was not predictive of prognosis in these cases. Conclusions This study does not support the use of COX-2 expression as an upfront prognostic variable in patients with osteosarcoma or rhabdomyosarcoma.Results from the small number of patients studied with Ewing sarcoma suggest a similar lack of predictive value for COX-2 expression. However, COX-2 inhibitors are not entirely dependent upon enzyme expression for their antitumor effects; this study does not necessarily preclude the use of COX-2 inhibitors for the treatment of pediatric sarcomas.

Effects of Orchiopexy on Congenitally Cryptorchid Insulin-3 Knockout Mice

PURPOSEnInsulin-3 (Insl3) knockout mice exhibit isolated, bilateral, high intra-abdominal cryptorchidism. If left in this position until adulthood, these testes will deteriorate to the Sertoli-cell-only state, leading to infertility in 100%. We examined the effect of orchiopexy in this genetically engineered animal model.nnnMATERIALS AND METHODSnA total of 160 testes from 80 male offspring of Insl3 F crosses underwent either no orchiopexy (29 testes), sham surgery (25 testes), 1-stage Fowler-Stephens scrotal orchiopexy (57 testes) or primary orchiopexy into a low abdominal, subcutaneous pouch (49 testes). A group of postoperative mice underwent fertility testing 3 months postoperatively. At 6 to 9 months postoperatively the testes were harvested and histologically analyzed.nnnRESULTSnTestes were atrophic in 100% (25 of 25) of the sham group, 91% (52 of 57) of the Fowler-Stephens orchiopexy group and 33% (16 of 49) of the subcutaneous pouch group (testis weight less than 50 mg.). Fertility testing was done in 30 mice (sham 5, Fowler-Stephens orchiopexy 17 and subcutaneous pouch 8). Infertility was secondary to bilateral testicular atrophy (sham 5 of 5, Fowler-Stephens orchiopexy 13 of 17), spermatogenic maturation arrest (subcutaneous pouch 8 of 8) and ductal obstruction [normal spermatogenesis with epididymis devoid of sperm] (Fowler-Stephens orchiopexy 2 of 17). Fertility with normal spermatogenesis was observed in 2 of 17 Fowler-Stephens orchiopexy mice, both of which were Insl3 knockout mice.nnnCONCLUSIONSnIntrascrotal orchiopexy can rescue these congenitally cryptorchid Insl3 knockout testes from their intra-abdominal fate of Sertoli-cell-only and lead to fertility. The results suggest that orchiopexy has a crucial and central role in preservation of spermatogenesis.