Joshua D. Schiffman

SDH5, a Gene Required for Flavination of Succinate Dehydrogenase, Is Mutated in Paraganglioma

Tapping the Mitochondrial Proteome Mitochondria produce the energy that cells need to survive, function, and divide. A growing list of human disorders has been traced to defects in mitochondrial function. About 300 mammalian mitochondrial proteins are functionally uncharacterized, and Hao et al. (p. 1139, published online 23 July) reasoned that the most highly conserved proteins within this group might provide insights into human disease. A combination of bioinformatics, yeast genetics, biochemistry, and human genetics was used to show that a previously uncharacterized mitochondrial protein (Sdh5) is required for the activity of respiratory complex II. Inactivating mutations in the human gene encoding SDH5 were found in individuals with hereditary paraganglioma, a rare neuroendocrine tumor. Thus, analysis of a mitochondrial protein in yeast has revealed a human tumor susceptibility gene. Analysis of a yeast mitochondrial protein reveals a human tumor susceptibility gene. Mammalian mitochondria contain about 1100 proteins, nearly 300 of which are uncharacterized. Given the well-established role of mitochondrial defects in human disease, functional characterization of these proteins may shed new light on disease mechanisms. Starting with yeast as a model system, we investigated an uncharacterized but highly conserved mitochondrial protein (named here Sdh5). Both yeast and human Sdh5 interact with the catalytic subunit of the succinate dehydrogenase (SDH) complex, a component of both the electron transport chain and the tricarboxylic acid cycle. Sdh5 is required for SDH-dependent respiration and for Sdh1 flavination (incorporation of the flavin adenine dinucleotide cofactor). Germline loss-of-function mutations in the human SDH5 gene, located on chromosome 11q13.1, segregate with disease in a family with hereditary paraganglioma, a neuroendocrine tumor previously linked to mutations in genes encoding SDH subunits. Thus, a mitochondrial proteomics analysis in yeast has led to the discovery of a human tumor susceptibility gene.

Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: a prospective observational study

BACKGROUND Individuals with Li-Fraumeni syndrome have a high lifetime risk of developing cancer. We assessed the feasibility and potential clinical effect of a comprehensive surveillance protocol in asymptomatic TP53 mutation carriers in families with this syndrome. METHODS We implemented a clinical surveillance protocol, using frequent biochemical and imaging studies, for asymptomatic TP53 mutation carriers on Jan 1, 2004, and did a prospective observational study of members of eight families with Li-Fraumeni syndrome who either chose to undergo surveillance or chose not to undergo surveillance. The primary outcome measure was detection of new cancers. The secondary outcome measure was overall survival. FINDINGS As of Nov 1, 2010, 33 TP53 mutation carriers were identified, 18 of whom underwent surveillance. The surveillance protocol detected ten asymptomatic tumours in seven patients, including small, high-grade tumours and low-grade or premalignant tumours. All seven mutation carriers were alive after a median follow-up of 24 months (IQR 22-65 months). 12 high-grade, high-stage tumours developed in 10 individuals in the non-surveillance group, two of whom (20%) were alive at the end of follow-up (p=0·0417 for comparison with survival in the surveillance group). 3-year overall survival was 100% in the surveillance group and 21% (95% CI 4-48%) in the non-surveillance group (p=0·0155). INTERPRETATION Our findings show the feasibility of a clinical surveillance protocol for the detection of asymptomatic neoplasms in individuals with germline TP53 mutations. This strategy offers a management option for affected individuals, and its benefits lend support to the use of early genetic testing of at-risk individuals and families. FUNDING Canadian Cancer Society Research Institute, Canadian Institutes of Health Research, SickKids Foundation, and Soccer for Hope.

Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas.

Malignant astrocytomas are a deadly solid tumor in children. Limited understanding of their underlying genetic basis has contributed to modest progress in developing more effective therapies. In an effort to identify such alterations, we performed a genome-wide search for DNA copy number aberrations (CNA) in a panel of 33 tumors encompassing grade 1 through grade 4 tumors. Genomic amplifications of 10-fold or greater were restricted to grade 3 and 4 astrocytomas and included the MDM4 (1q32), PDGFRA (4q12), MET (7q21), CMYC (8q24), PVT1 (8q24), WNT5B (12p13), and IGF1R (15q26) genes. Homozygous deletions of CDKN2A (9p21), PTEN (10q26), and TP53 (17p3.1) were evident among grade 2 to 4 tumors. BRAF gene rearrangements that were indicated in three tumors prompted the discovery of KIAA1549-BRAF fusion transcripts expressed in 10 of 10 grade 1 astrocytomas and in none of the grade 2 to 4 tumors. In contrast, an oncogenic missense BRAF mutation (BRAF(V600E)) was detected in 7 of 31 grade 2 to 4 tumors but in none of the grade 1 tumors. BRAF(V600E) mutation seems to define a subset of malignant astrocytomas in children, in which there is frequent concomitant homozygous deletion of CDKN2A (five of seven cases). Taken together, these findings highlight BRAF as a frequent mutation target in pediatric astrocytomas, with distinct types of BRAF alteration occurring in grade 1 versus grade 2 to 4 tumors.

Succinate Dehydrogenase Mutation Underlies Global Epigenomic Divergence in Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway-mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH-mutant GIST with isocitrate dehydrogenase-mutant glioma, another Krebs cycle-defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance.

Succinate dehydrogenase - Assembly, regulation and role in human disease

Succinate dehydrogenase (or Electron Transport Chain Complex II) has been the subject of a focused but significant renaissance. This complex, which has been the least studied of the mitochondrial respiratory complexes has seen renewed interest due to the discovery of its role in human disease. Under this heightened scrutiny, the succinate dehydrogenase complex has proven to be a fascinating machine, whose regulation and assembly requires additional factors that are beginning to be discovered. Mutations in these factors and in the structural subunits of the complex itself cause a variety of human diseases. The mechanisms underlying the pathogenesis of SDH mutations is beginning to be understood.

The Epidemiology of Sarcoma

Sarcomas account for over 20% of all pediatric solid malignant cancers and less than 1% of all adult solid malignant cancers. The vast majority of diagnosed sarcomas will be soft tissue sarcomas, while malignant bone tumors make up just over 10% of sarcomas. The risks for sarcoma are not well-understood. We evaluated the existing literature on the epidemiology and etiology of sarcoma. Risks for sarcoma development can be divided into environmental exposures, genetic susceptibility, and an interaction between the two. HIV-positive individuals are at an increased risk for Kaposi’s sarcoma, even though HHV8 is the causative virus. Radiation exposure from radiotherapy has been strongly associated with secondary sarcoma development in certain cancer patients. In fact, the risk of malignant bone tumors increases as the cumulative dose of radiation to the bone increases (p for trend <0.001). A recent meta-analysis reported that children with a history of hernias have a greater risk of developing Ewing’s sarcoma (adjusted OR 3.2, 95% CI 1.9, 5.7). Bone development during pubertal growth spurts has been associated with osteosarcoma development. Occupational factors such as job type, industry, and exposures to chemicals such as herbicides and chlorophenols have been suggested as risk factors for sarcomas. A case-control study found a significant increase in soft tissue sarcoma risk among gardeners (adjusted OR 4.1, 95% CI 1.00, 14.00), but not among those strictly involved in farming. A European-based study reported an increased risk in bone tumors among blacksmiths, toolmakers, or machine-tool operators (adjusted OR 2.14, 95% CI 1.08, 4.26). Maternal and paternal characteristics such as occupation, age, smoking status, and health conditions experienced during pregnancy also have been suggested as sarcoma risk factors and would be important to assess in future studies. The limited studies we identified demonstrate significant relationships with sarcoma risk, but many of these results now require further validation on larger populations. Furthermore, little is known about the biologic mechanisms behind each epidemiologic association assessed in the literature. Future molecular epidemiology studies may increase our understanding of the genetic versus environmental contributions to tumorigenesis in this often deadly cancer in children and adults.

A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.

Recent Advances in the Molecular Pathogenesis of Ewing's Sarcoma

Tumor development is a complex process resulting from interplay between mutations in oncogenes and tumor suppressors, host susceptibility factors, and cellular context. Great advances have been made by studying rare tumors with unique clinical, genetic, or molecular features. Ewings sarcoma serves as an excellent paradigm for understanding tumorigenesis because it exhibits some very useful and important characteristics. For example, nearly all cases of Ewings sarcoma contain the (11;22)(q24;q12) chromosomal translocation that encodes the EWS/FLI oncoprotein. Besides the t(11;22), however, many cases have otherwise simple karyotypes with no other demonstrable abnormalities. Furthermore, it seems that an underlying genetic susceptibility to Ewings sarcoma, if it exists, must be rare. These two features suggest that EWS/FLI is the primary mutation that drives the development of this tumor. Finally, Ewings sarcoma is an aggressive tumor that requires aggressive treatment. Thus, improved understanding of the pathogenesis of this tumor will not only be of academic interest, but may also lead to new therapeutic approaches for individuals afflicted with this disease. The purpose of this review is to highlight recent advances in understanding the molecular pathogenesis of Ewings sarcoma, while considering the questions surrounding this disease that still remain and how this knowledge may be applied to developing new treatments for patients with this highly aggressive disease.

Potential Mechanisms for Cancer Resistance in Elephants and Comparative Cellular Response to DNA Damage in Humans

IMPORTANCE Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. OBJECTIVE To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). DESIGN, SETTING, AND PARTICIPANTS A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015. EXPOSURES Ionizing radiation and doxorubicin. MAIN OUTCOMES AND MEASURES Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. RESULTS Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes underwent p53-mediated apoptosis at higher rates than human lymphocytes proportional to TP53 status (ionizing radiation exposure: patients with LFS, 2.71% [95% CI, 1.93%-3.48%] vs human controls, 7.17% [95% CI, 5.91%-8.44%] vs elephants, 14.64% [95% CI, 10.91%-18.37%]; P < .001; doxorubicin exposure: human controls, 8.10% [95% CI, 6.55%-9.66%] vs elephants, 24.77% [95% CI, 23.0%-26.53%]; P < .001). CONCLUSIONS AND RELEVANCE Compared with other mammalian species, elephants appeared to have a lower-than-expected rate of cancer, potentially related to multiple copies of TP53. Compared with human cells, elephant cells demonstrated increased apoptotic response following DNA damage. These findings, if replicated, could represent an evolutionary-based approach for understanding mechanisms related to cancer suppression.

Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: 11 year follow-up of a prospective observational study

BACKGROUND Carriers of a germline TP53 pathogenic variant have a substantial lifetime risk of developing cancer. In 2011, we did a prospective observational study of members of families who chose to either undergo a comprehensive surveillance protocol for individuals with Li-Fraumeni syndrome or not. We sought to update our assessment of and modify the surveillance protocol, so in this study we report both longer follow-up of these patients and additional patients who underwent surveillance, as well as update the originally presented surveillance protocol. METHODS A clinical surveillance protocol using physical examination and frequent biochemical and imaging studies (consisting of whole-body MRI, brain MRI, breast MRI, mammography, abdominal and pelvic ultrasound, and colonoscopy) was introduced at three tertiary care centres in Canada and the USA on Jan 1, 2004, for carriers of TP53 pathogenic variants. After confirmation of TP53 mutation, participants either chose to undergo surveillance or chose not to undergo surveillance. Patients could cross over between groups at any time. The primary outcome measure was detection of asymptomatic tumours by surveillance investigations. The secondary outcome measure was 5 year overall survival established from a tumour diagnosed symptomatically (in the non-surveillance group) versus one diagnosed by surveillance. We completed survival analyses using an as-treated approach. FINDINGS Between Jan 1, 2004, and July 1, 2015, we identified 89 carriers of TP53 pathogenic variants in 39 unrelated families, of whom 40 (45%) agreed to surveillance and 49 (55%) declined surveillance. 19 (21%) patients crossed over from the non-surveillance to the surveillance group, giving a total of 59 (66%) individuals undergoing surveillance for a median of 32 months (IQR 12-87). 40 asymptomatic tumours have been detected in 19 (32%) of 59 patients who underwent surveillance. Two additional cancers were diagnosed between surveillance assessments (false negatives) and two biopsied lesions were non-neoplastic entities on pathological review (false positives). Among the 49 individuals who initially declined surveillance, 61 symptomatic tumours were diagnosed in 43 (88%) patients. 21 (49%) of the 43 individuals not on surveillance who developed cancer were alive compared with 16 (84%) of the 19 individuals undergoing surveillance who developed cancer (p=0·012) after a median follow-up of 46 months (IQR 22-72) for those not on surveillance and 38 months (12-86) for those on surveillance. 5 year overall survival was 88·8% (95% CI 78·7-100) in the surveillance group and 59·6% (47·2-75·2) in the non-surveillance group (p=0·0132). INTERPRETATION Our findings show that long-term compliance with a comprehensive surveillance protocol for early tumour detection in individuals with pathogenic TP53 variants is feasible and that early tumour detection through surveillance is associated with improved long-term survival. Incorporation of this approach into clinical management of these patients should be considered. FUNDING Canadian Institutes for Heath Research, Canadian Cancer Society, Terry Fox Research Institute, SickKids Foundation, and Soccer for Hope Foundation.