The Cooperative Human Tissue Network of the National Cancer Institute: Supporting Cancer Research for 35 Years.

The Cooperative Human Tissue Network was created by the NCI in 1987 to support a coordinated national effort to collect and distribute high quality, pathologist-validated human tissues for cancer research. Since then, the network has expanded to provide different types of tissue samples, blood and body fluid samples, immunohistologic and molecular sample preparations, tissue microarrays, and clinical datasets inclusive of biomarkers and molecular testing. From inception through the end of 2021, the network has distributed 1,375,041 biospecimens. It served 889 active investigators in 2021. The network has also taken steps to begin to optimize the representation of diverse communities among the distributed biospecimens. In this article, the authors review the 35-year history of this network, describe changes to the program over the last 15 years, and provide operational and scientific highlights from each of the divisions. Readers will learn how to engage with the network and about the continued evolution of the program for the future.

von Hippel-Lindau disease-associated hemangioblastomas are derived from embryologic multipotent cells.

BACKGROUND: To determine the origin of the neoplastic cell in central nervous system (CNS) hemangioblastomas in von Hippel-Lindau disease (VHL) and its role in tumor formation and distribution, we characterized and differentiated neoplastic cells from hemangioblastomas removed from VHL patients. METHODS AND FINDINGS: A total of 31 CNS hemangioblastomas from 25 VHL patients were resected and analyzed. Tumor cells from the hemangioblastomas were characterized, grown, and differentiated into multiple lineages. Resected hemangioblastomas were located in the cerebellum (11 tumors), brainstem (five tumors), and spinal cord (15 tumors). Consistent with an embryologically derived hemangioblast, the neoplastic cells demonstrated coexpression of the mesodermal markers brachyury, Flk-1 (vascular endothelial growth factor-2), and stem cell leukemia (Scl). The neoplastic cells also expressed hematopoietic stem cell antigens and receptors including CD133, CD34, c-kit, Scl, erythropoietin, and erythropoietin receptor. Under specific microenvironments, neoplastic cells (hemangioblasts) were expanded and differentiated into erythrocytic, granulocytic, and endothelial progenitors. Deletion of the wild-type VHL allele in the hematopoietic and endothelial progeny confirmed their neoplastic origin. CONCLUSIONS: The neoplastic cell of origin for CNS hemangioblastomas in VHL patients is the mesoderm-derived, embryologically arrested hemangioblast. The hematopoietic and endothelial differentiation potential of these cells can be reactivated under suitable conditions. These findings may also explain the unique tissue distribution of tumor involvement.

Proteomic comparison of oligodendrogliomas with and without 1pLOH.

OBJECTIVE: Chemoresistance is a widespread therapeutic challenge in glial tumors. The molecular basis of chemoresistance is poorly understood, precluding advances in glioma treatment and leaving gliomas among the most lethal tumors. Oligodendrogliomas provide a unique model to study the molecular basis of chemoresistance, as there are two distinct genetic subtypes with significant differences in chemosensitivity. Despite a high morphological similarity, tumors with allelic loss on the short arm of chromosome 1 (1pLOH) are more chemosensitive than those without 1pLOH. METHODS: In order to identify candidate proteins potentially responsible for glioma chemosensitivity, we compared the proteome of four oligodendrogliomas with and five without 1pLOH using comparative proteomic profiling. Proteomic analysis was performed by two-dimensional protein gel electrophoresis and subsequent computerized gel analysis for detection of distinguishing patterns of protein expression. Differentially expressed proteins were identified using Liquid Chromatography/Mass Spectrometry. Differential expression of select proteins was confirmed by Western blotting. RESULTS: We identified seven candidate proteins that are overexpressed in oligodendrogliomas without 1pLOH. Two of these proteins (glyoxalase I and Rho GDP dissociation inhibitor) have previously been shown to enhance chemoresistance in other tumors. In turn, we identified twelve overexpressed proteins in tumors with 1pLOH that have previously been reported to induce chemosensitivity in other forms of human neoplasia. CONCLUSIONS: These identified proteins are potential targets for pharmacological therapy and may also be useful as biomarkers for differentiation of chemoresistant and chemosensitive oligodendroglioma.

Molecular genetic events in gastrointestinal and pancreatic neuroendocrine tumors.

Gastrointestinal and pancreatic neuroendocrine tumors originate from the cells of the diffuse endocrine system. Their molecular genetic mechanism of development and progression is complex and remains largely unknown, and they are different in genetic composition from the gastrointestinal epithelial tumors. The current literature suggests that multiple genes are involved in their tumorigenesis with significant differences for tumors of different embryological derivatives: foregut, midgut and hindgut. The MEN1 gene is involved in initiation of 33% of foregut gastrointestinal neuroendocrine tumors. 18q defects are present almost exclusively in mid/hindgut neuroendocrine tumors. X-chromosome markers are associated with malignant behavior in foregut tumors only. Analysis of poorly differentiated neuroendocrine carcinomas of any site demonstrates high chromosomal instability and frequent p53 alterations similar to other poorly differentiated carcinomas. Several factors played a limiting role in the molecular studies published to date: the tumors are rare and heterogeneous, it is difficult to predict their behavior and prognosis, and several different tumor classifications are used by the investigators in the studies. Future studies need to evaluate molecular genetic composition of large series of gastrointestinal and pancreatic neuroendocrine tumors of each specific tumor type. Understanding of specific genetic alterations characteristic for gastrointestinal and pancreatic neuroendocrine tumors might lead to their improved diagnosis, morphologic and molecular characterization and treatment.

Proteomic analysis of hemangioblastoma cyst fluid.

OBJECTIVE: Hemangioblastomas are benign CNS tumors that occur sporadically or in patients with von Hippel-Lindau (VHL) disease. These tumors are characteristically associated with formation of intra- or peritumoral cysts. Hemangioblastoma cyst formation is a major cause of morbidity and mortality with these tumors. While peritumoral cysts have been suggested to result from vascular leakage, the mechanism of intratumoral cyst formation is not understood. METHODS: To elucidate the origin of intratumoral hemangioblastoma cyst fluid, we characterized its biochemical composition by two-dimensional (2D) proteomic profiling followed by sequencing of several proteins. The proteomic pattern of intratumoral cyst fluid was furthermore compared to the proteomic pattern of serum, hemangioblastoma tumor tissue, and hemangioblastoma peritumoral cyst fluid. RESULTS: We show that proteomic patterns of intra- and peritumoral cyst fluid are identical Both are highly similar to serum and not to tumor. CONCLUSIONS: Intratumoral hemangioblastoma cyst fluid originates from serum. Cyst formation associated with hemangioblastoma, whether peri- or intratumoral, is a consequence of vascular leakage. Anti-VEGF therapy may effectively control hemangioblastoma cyst formation.

From tissue phenotype to proteotype: sensitive protein identification in microdissected tumor tissue.

Correlation of disease phenotype with protein profile (proteotype) is a significant challenge for biomedical research. The main obstacles have been the need to insure sufficient quantities of pure protein sample, the reproducibility of protein display, and rapid and accurate protein identification. We present a modified approach that combines enhanced detection sensitivity with tissue microdissection from frozen primary renal cancer tissues of different histological subtypes, followed by 2D gel analysis and protein identification with MALDI mass spectrometry. We obtained reliable and highly consistent results in phenotypically similar tumors of each individual subtype by performing strict morphological control of the analyzed tumor cells without physical or chemical alteration of the frozen tissue samples. By application of non-oxidizing silver staining, proteins were resolved and identified with high levels of specificity and sensitivity. This new combination of techniques allows not only for sensitive identification of specific protein patterns that correspond to a histological tumor phenotype, but also for identification of specific disease-associated protein targets.

Coexpression of erythropoietin and erythropoietin receptor in von Hippel-Lindau disease-associated renal cysts and renal cell carcinoma.

Von Hippel-Lindau (VHL) disease is characterized by multiple tumors in specific target organs. The tumors at different sites share distinct morphologic and genetic characteristics but their cell of origin is unknown. We show that VHL disease-associated renal clear cell carcinomas (RCC) consistently coexpress erythropoietin (Epo) and Epo receptor (EpoR). In addition, coexpression of Epo and EpoR is detected in many renal cysts, providing further evidence that renal cysts are potential precursors for RCC. In conjunction with VHL gene deficiency, coexpression of Epo and EpoR in renal cysts and tumors may reflect a developmental arrest in immature mesenchymal cells. Such arrest may lead to autocrine stimulation, cell proliferation, and renal tumor development, similar to tumorigenesis of VHL disease-associated hemangioblastomas.

Somatic point mutation of the wild-type allele detected in tumors of patients with VHL germline deletion.

The majority of patients with Von Hippel-Lindau (VHL) disease are affected by a VHL germline mutation involving one copy of the VHL gene. Loss of heterozygosity of the second VHL allele can be consistently demonstrated in tumor tissue from these patients, suggesting that allelic deletion is a very early or even initiating event for tumorigenesis. Approximately 20% of VHL disease patients, however, exhibit germline deletion of one entire copy or at least a substantial part of the VHL gene. To investigate the nature of the "second genetic hit" in this patient population, we analysed two renal cell carcinomas and one CNS hemangioblastoma from three unrelated patients for genetic changes of the second copy of the VHL gene. All three tumors showed retention of one VHL allele by FISH. Single-strand conformation polymorphism and mutation analysis of microdissected tumor DNA revealed somatic point mutations of the wild-type VHL copies in each of the three tumors. The results indicate that the "two hit model" is equally applicable to patients with VHL germline mutation and VHL germline deletion. In contrast to tumors from patients with VHL germline mutation, however, point mutations of the wild-type allele can be detected in tumors from patients with VHL germline deletion.

Protein patterns and proteins that identify subtypes of glioblastoma multiforme.

Glioblastoma multiforme (GBM) has been subdivided into two types based on clinical and genetic findings: primary tumors, which arise de novo, and secondary tumors, which progress from lower grade gliomas to GBMs. To analyse this dichotomy at the protein level, we employed selective tissue microdissection to obtain pure populations of tumor cells, which we studied using two-dimensional protein gel electrophoresis (2-DGE) and protein sequencing of select target proteins. Protein patterns were analysed in a blinded manner from the clinical and genetic data. 2-DGE clearly identified two distinct populations of tumors. 2-DGE was reproducible and reliable, as multiple samples analysed from the same patient gave identical results. In addition, we isolated and sequenced 11 proteins that were uniquely expressed in either the primary or the secondary GBMs, but not both. We demonstrate that specific proteomic patterns can be reproducibly identified by two-dimensional gel electrophoresis from limited numbers of selectively procured, microdissected tumor cells and that two patterns of GBMs, primary versus secondary, previously distinguished by clinical and genetic differences, can be recognized at the protein level. Proteins that are expressed distinctively may have important implications for the diagnosis, prognosis, and treatment of patients with GBM.

Differential expression of erythropoietin and its receptor in von hippel-lindau-associated and multiple endocrine neoplasia type 2-associated pheochromocytomas.

Pheochromocytoma is a neuroendocrine tumor associated with a variety of genetic disorders, which include von Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN 2), neurofibromatosis type 1, hereditary paraganglioma, and succinate dehydrogenase gene-related tumors. Previous studies of VHL-associated and MEN 2-associated pheochromocytomas suggest morphological, biochemical, and clinical differences exist among the tumors, but the process by which they develop remains unclear. Studies in other VHL-associated tumors suggest that VHL gene deficiency causes coexpression of erythropoietin (Epo) and its receptor (Epo-R), which facilitates tumor growth. The objective of this study was to understand the different process of tumorigenesis for VHL and MEN 2-associated pheochromocytomas. Ten pheochromocytomas (VHL patients n = 5, MEN 2 patients n = 5) were examined for the presence or absence of Epo and Epo-R using Western blot, immunohistochemistry, and RT-PCR analyses. Coexpression of Epo and Epo-R was found in all five VHL-associated pheochromocytomas; in contrast, expression of Epo-R, but not Epo, was documented in all five MEN 2-associated pheochromocytomas. Expression of Epo appears to be a result of VHL gene deficiency, possibly through activation of the hypoxia inducible factor-1 pathway, whereas Epo-R is an embryonal marker whose sustained expression in both VHL- and MEN 2-associated pheochromocytomas reflects an arrest or defect in development. These findings suggest an alternative process of tumorigenesis in VHL- and MEN 2-associated pheochromocytomas and implicate Epo as a clinical biomarker to differentiate these tumors.

Coexpression of erythropoietin and its receptor in endolymphatic sac tumors.

OBJECT: Von Hippel-Lindau (VHL) disease is characterized by multiple tumors in specific organs. The cell of origin and the reason for the particular organ distribution of the tumors remains unknown. Endolymphatic sac tumor (ELST) is one of the lesions associated with VHL disease. Data from previous studies of VHL disease-associated hemangioblastomas (HBs) and renal cell carcinomas (RCCs) have indicated that VHL gene deficiency causes coexpression of erythropoietin (Epo) and its receptor (Epo-R), which facilitates tumor growth. METHODS: The authors studied ELSTs from five patients with VHL germline mutations. Analysis of the five ELST samples revealed loss of the wild-type allele, consistent with Knudson's two-hit hypothesis for tumorigenesis. All five ELST specimens were characterized microscopically and by immunohistochemical analysis. Coexpression of Epo and Epo-R was found in all five tumors on immunohistochemical studies and confirmed through reverse transcription-polymerase chain reaction and Western blot analysis. CONCLUSIONS: Expression of Epo appears to be a result of VHL gene deficiency, whereas the simultaneous coexpression of Epo-R may reflect a developmental mechanism of tumorigenesis. Coexpression of Epo and Epo-R in ELSTs together with the morphological and genetic similarities of these lesions with other VHL disease-associated tumors indicates that VHL disease-associated tumors in different organs share common pathogenetic pathways.

Amplification and overexpression of mutant RET in multiple endocrine neoplasia type 2-associated medullary thyroid carcinoma.

We have previously identified two second hit mechanisms involved in the development of multiple endocrine neoplasia type 2 (MEN 2)-associated tumors: trisomy 10 with duplication of the mutant RET allele and loss of the wild-type RET allele. However, some of the MEN 2-associated tumors investigated did not demonstrate either mechanism. Here, we studied the TT cell line derived from MEN 2-associated medullary thyroid carcinoma with a RET germline mutation in codon 634, for alternative mechanisms of tumorigenesis. Although we observed a 2:1 ratio between mutant and wild-type RET at the genomic DNA level in this cell line, fluorescence in situ hybridization analysis revealed neither trisomy 10 nor loss of the normal chromosome 10. Instead, a tandem duplication event was responsible for amplification of mutant RET. In further studies we could for the first time demonstrate that the genomic chromosome 10 abnormalities in this cell line cause an increased production of mutant RET mRNA. These findings provide evidence for a third second hit mechanism resulting in overrepresentation and overexpression of mutant RET in MEN 2-associated tumors.

Prospective study of thymic carcinoids in patients with multiple endocrine neoplasia type 1.

Little is known of the natural history of thymic carcinoids in multiple endocrine neoplasia type 1 (MEN1). This is important because in 1993 they were identified as a frequent cause of death, yet only small retrospective studies and case reports exist. We report results of a prospective study of 85 patients with MEN1 evaluated for pancreatic endocrine tumors and followed over a mean of 8 yr with serial chest computed tomography, magnetic resonance imaging (MRI), chest x-ray, and, since 1994, octreoscans [somatostatin receptor scintigraphy (SRS)]. Seven patients (8%) developed thymic carcinoids. Patients with and without carcinoids did not differ in clinical, laboratory, or MEN1 tumor features, except for male gender and the presence of a gastric carcinoid. All thymic tumors were hormonally inactive. Four thymic carcinoids lacked 11q loss of heterozygosity, although it was found in three pancreatic endocrine tumors. Computed tomography and/or MRI were more sensitive than SRS or chest x-ray in detecting tumors initially or with recurrence. All patients underwent resection of the thymic carcinoid, and in all patients followed more than 1 yr, the tumor recurred. Bone metastases developed in two patients and were detected early only on MRI, not SRS. This study provides information on early thymic carcinoids and allows modifications of existing guidelines to be recommended for their diagnosis, surveillance, and treatment.

Pheochromocytoma in von hippel-lindau disease: distinct histopathologic phenotype compared to pheochromocytoma in multiple endocrine neoplasia type 2.

Pheochromocytomas are rare neuroendocrine tumors that arise from chromaffin tissue. In a small subset of patients, pheochromocytomas occur as a manifestation of von Hippel- Lindau (VHL) disease. The histology of VHL-associated pheochromocytomas has not been reported in detail. In this article, we describe histopathologic features of 14 pheochromocytomas in eight patients with VHL disease and demonstrate that VHL-associated pheochromocytomas have a distinct histologic phenotype as compared with pheochromocytomas in patients with multiple endocrine neoplasia type 2 (MEN 2). VHL tumors are characterized by a thick vascular tumor capsule; myxoid and hyalinized stroma; round, small to medium tumor cells intermixed with small vessels; predominantly amphophilic and clear cytoplasm; absence of cytoplasmic hyaline globules; and lack of nuclear atypia or mitoses. In contrast to MEN 2, there is no extratumoral adrenomedullary hyperplasia in the VHL adrenal gland. Our findings of a distinct histologic phenotype of VHL pheochromocytoma may further help in subdividing patients who clinically present with multiple, bilateral pheochromocytomas.

A von Hippel-Lindau disease-associated microcystic adenoma of the ethmoid sinus: case report.

BACKGROUND AND IMPORTANCE: We present a unique case of an anterior cranial base von Hippel-Lindau disease (VHL)-associated microcystic neoplasm. To determine the lesion's relationship with VHL and its appropriate management, we discuss its salient clinical, pathological, and molecular features. CLINICAL PRESENTATION: A 36-year-old woman with VHL presented with a 3-month history of phantosmia. Serial magnetic resonance imaging studies revealed a lesion within the ethmoid and frontal sinus region that was first evident 18 months before symptom development and demonstrated progressive growth over the interval period. The lesion was resected via a transbasal approach. Histopathological and immunohistochemical analysis revealed a microcystic lesion composed of bland clear cells and underlying endothelial cells consistent with a VHL-associated microcystic neoplasm that are not known to metastasize. Molecular testing demonstrated loss of heterozygosity of the VHL locus, verifying the tumor as a VHL-related neoplasm. CONCLUSION: Because primary VHL-associated microcystic tumors in the anterior cranial base have not been described previously, the natural history of these tumors remains unclear. Based on the benign features of these lesions, they can be managed conservatively with close observation and surgical intervention reserved for those that produce symptoms.

Troglitazone reduces glyoxalase I protein expression in glioma and potentiates the effects of chemotherapeutic agents.

Despite resistance of most gliomas to chemotherapy, approximately 2/3 of oligodendrogliomas show sensitivity to such agents. This sensitivity has been associated with deletions on chromosome 1p alone or in combination with 19q. Higher expression of the enzyme glyoxalase I has been found in oligodendrogliomas with chromosome 1p intact compared to those with a deletion. Higher expression of this enzyme is also associated with tumor chemoresistance in other cancers. The present study tested whether the drug troglitazone would make a glioma cell line more sensitive to chemotherapeutic agents. This drug was chosen because it has been shown to decrease glyoxalase I enzyme activity in cells. Treatment with troglitazone decreased expression of glyoxalase I, and potentiated cell death when used in combination with chemotherapeutic agents. This decrease in glyoxalase I protein may be one mechanism by which this potentiation occurs, and troglitazone may be a candidate for use in glioma therapy.

Deciphering von Hippel-Lindau (VHL/Vhl)-associated pancreatic manifestations by inactivating Vhl in specific pancreatic cell populations.

The von Hippel-Lindau (VHL) syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75% of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system. Specifically, Vhl was inactivated in different pancreatic cell populations distinguished by their roles during embryonic organ development and their endocrine lineage commitment. With Cre recombinase expression directed by a glucagon promoter in alpha-cells or an insulin promoter in beta-cells, we showed that deletion of Vhl is dispensable for normal functions of the endocrine pancreas. In addition, deficiency of VHL protein (pVHL) in terminally differentiated alpha-cells or beta-cells is insufficient to induce pancreatic neuroendocrine tumorigenesis. Most significantly, we presented the first mouse model of VHL-associated pancreatic disease in mice lacking pVHL utilizing Pdx1-Cre transgenic mice to inactivate Vhl in pancreatic progenitor cells. The highly vascularized microcystic adenomas and hyperplastic islets that developed in Pdx1-Cre;Vhl f/f homozygous mice exhibited clinical features similar to VHL patients. Establishment of three different, cell-specific Vhl knockouts in the pancreas have allowed us to provide evidence suggesting that VHL is functionally important for postnatal ductal and exocrine pancreas, and that VHL-associated pancreatic lesions are likely to originate from progenitor cells, not mature endocrine cells. The novel model systems reported here will provide the basis for further functional and genetic studies to define molecular mechanisms involved in VHL-associated pancreatic diseases.

Overexpression of interleukin-13 receptor-alpha2 in neuroendocrine malignant pheochromocytoma: a novel target for receptor directed anti-cancer therapy.

CONTEXT: Pheochromocytomas and paragangliomas are rare catecholamine-secreting neuroendocrine tumors arising from the adrenal medulla and sympathetic tissues. When complete surgical resection is not an option, the treatment of pheochromocytoma is limited. OBJECTIVE: The objective of the study was to identify and characterize overexpression of IL-13 receptor-alpha2 (IL-13Ralpha2) gene expression in human and murine tumors and verify xenograft mouse pheochromocytoma cell (MPC)-derived tumor's response to a selective cytotoxin. DESIGN/SETTING/PATIENTS: Expression of IL-13Ralpha2 was evaluated in a panel of 25 human pheochromocytoma clinical samples by RT-PCR and eight MPC tumors by indirect immunofluorescence assay and RT-PCR. INTERVENTION: The function of IL-13Ralpha2 in these tumor cells was examined by evaluating tumor sensitivity to a recombinant IL-13-Pseudomonas exotoxin (IL-13PE). Subcutaneous small and large MPC tumors in athymic nude mice (n = 10) were treated intratumorally with IL-13PE (100 m icrog/kg). MAIN OUTCOME MEASURES: IC(50) and tumor size were measured. RESULTS: IL-13PE immunotoxin was highly cytotoxic to IL-13Ralpha2-overexpressing MPC cells (IC(50) <2.5 ng/ml) in vitro. Furthermore, IL-13PE was highly cytotoxic to sc tumors. Our results showed a statistically significant decrease in tumor size as early as 3 d after initial treatment and further suppressed growth of MPC tumors. All tumors displayed a histological evidence of necrosis in response to IL-13 immunotoxin without any adverse effects in host at this dose. CONCLUSIONS: Human and murine neuroendocrine pheochromocytoma overexpress the IL-13Ralpha2 chain, and an IL-13PE-based receptor-directed anticancer approach may prove useful in treatment for metastatic pheochromocytoma patients.

Characterization of an animal model of aggressive metastatic pheochromocytoma linked to a specific gene signature.

Pheochromocytomas are chromaffin cell-derived neuroendocrine tumors. There is presently no cure for metastatic pheochromocytoma and no reliable way to distinguish malignant from benign tumors before the development of metastases. In order to successfully manage pheochromocytoma, it is necessary to better understand the biological determinants of tumor behavior. For this purpose, we have recently established a mouse model of metastatic pheochromocytoma using tail vein injection of mouse pheochromocytoma (MPC) cells. We optimized this model modifying the number of cells injected, length of trypsin pre-treatment, and incubation temperature and duration for the MPC cells before injection, and by serial passage and re-selection of tumors exhibiting the metastatic phenotype. We evaluated the effect of these modifications on tumor growth using serial in vivo Magnetic Resonance Imaging studies. These results show that number of cells injected, the pre-injection incubation temperature, and duration of trypsin treatment are important factors to produce faster growing, more aggressive tumors that yielded secondary metastatic lesions. Serial harvest, culture and re-selection of metastatic liver lesions produced even more aggressive pheochromocytoma cells that retained their biochemical phenotype. Microarray gene expression comparison and quantitative real-time PCR of these more aggressive cells to the MPC-parental cell line identified genes that may be important for the metastatic process.