Trisha Dwight

Silencing of the PTEN tumor-suppressor gene in anaplastic thyroid cancer

Germline mutations in the tumor‐suppressor gene PTEN (MMAC1, TEP1) are found in Cowden syndrome, which predisposes to hamartomas, breast cancer, trichilemmomas, and thyroid tumors of follicular epithelium. PTEN has also been found to be somatically deleted, mutated, and/or silenced in various sporadically occurring cancers such as glioblastoma, breast cancer, kidney cancer, malignant melanoma, and endometrial cancer. Loss or reduction of PTEN protein expression as well as inappropriate subcellular compartmentalization is seen in non‐medullary thyroid cancers. However, although allelic loss of the PTEN locus in 10q23.3 is frequently seen, this is not coupled with mutations in the PTEN gene. To approach further the frequency and mechanism behind PTEN silencing, we screened a panel of 87 sporadic thyroid tumors for PTEN mRNA expression, including 14 anaplastic carcinomas, 37 follicular carcinomas, 21 atypical adenomas, and 15 ordinary adenomas. Complete loss of PTEN mRNA expression was evident in six of the tumors, including four anaplastic carcinomas, one widely invasive carcinoma, and one ordinary adenoma. The transcriptional silencing of PTEN was significantly associated with the anaplastic subtype, suggesting that PTEN is involved in the carcinogenesis of highly malignant or late‐stage thyroid cancers, whereas this particular mechanism appears to be of minor importance in differentiated follicular thyroid tumors. No association was observed between the expression, loss of heterozygosity, and mutation status in the 33 cases in which these parameters were compared. This indicates that PTEN silencing is a result of a wide variety of epigenetic and/or structural silencing mechanisms rather than a consequence of structural biallelic inactivation of the classical type. Furthermore, the high rate of alterations in the 10q23 region might indicate the presence of an as‐yet unknown tumor‐suppressor gene with an important role in the development of thyroid tumors.

Loss of SDHA expression identifies SDHA mutations in succinate dehydrogenase-deficient gastrointestinal stromal tumors.

Succinate dehydrogenase–deficient gastrointestinal stromal tumors (SDH-deficient GISTs) are a unique class of GIST defined by negative immunohistochemical staining for succinate dehydrogenase B (SDHB). SDH-deficient GISTs show distinctive clinical and pathologic features including absence of KIT and PDGFRA mutations, exclusive gastric location, common lymph node metastasis, a prognosis not predicted by size and mitotic rate, and indolent behavior of metastases. They may be syndromal with some being associated with the Carney Triad or germline SDHA, SDHB, SDHC, or SDHD mutations (Carney-Stratakis syndrome). It is normally recommended that genetic testing for SDHA, SDHB, SDHC, and SDHD be offered whenever an SDH-deficient GIST is encountered. However, testing for all 4 genes is burdensome and beyond the means of most centers. In this study we performed SDHA mutation and immunohistochemical analyses for SDHA on 10 SDH-deficient GISTs. Three showed negative staining for SDHA, and all of these were associated with germline SDHA mutations. In 2 tumors, 3 novel mutations were identified (p.Gln54X, p.Thr267Met, and c.1663+3G>C), none of which have previously been reported in GISTs or other SDH-associated tumors. Seven showed positive staining for SDHA and were not associated with SDHA mutation. In conclusion, 30% of SDH-deficient GISTs in this study were associated with germline SDHA mutation. Negative staining for SDHA can be used to triage formal genetic testing for SDHA when an SDH-deficient GIST is encountered.

Familial SDHA Mutation Associated With Pituitary Adenoma and Pheochromocytoma/Paraganglioma

CONTEXT Reports of the coexistence of pituitary adenomas and pheochromocytoma/paraganglioma are uncommon. Recently germline mutations in 2 of the genes encoding succinate dehydrogenase, SDHC and SDHD, were associated with pituitary tumors. OBJECTIVE Our aim was to determine whether the development of a pituitary adenoma was associated with SDHA mutation. PATIENTS A 46-year-old female presented with carotid body paraganglioma (proband). Subsequently the probands son was diagnosed with a nonfunctioning pituitary macroadenoma at age 30 years. RESULTS An immunohistochemical analysis of the resected paraganglioma and pituitary adenoma revealed the loss of succinate dehydrogenase subunit B and succinate dehydrogenase subunit A (SDHA) expression in both tumors, with the preservation of staining in nonneoplastic tissue. Mutation analysis showed a novel SDHA mutation (c.1873C>T, p.His625Tyr) in the germline of the proband as well as in the probands son. In the paraganglioma of the proband, in addition to the germline mutation, a somatic mutation was observed (c.1865G>A, p.Trp622*). In the pituitary adenoma of the probands son, loss of SDHA immunoreactivity was paradoxically accompanied by loss of the mutant allele. CONCLUSIONS This is the first report of a pituitary adenoma arising in the setting of germline SDHA mutation. The loss of SDHA protein expression in both the paraganglioma (proband) and pituitary adenoma (probands son) argues strongly for a causative role of SDHA mutation. This report further strengthens the link between pituitary neoplasia and germline SDH mutation. Although pituitary adenomas appear rare among patients carrying SDH subunit mutations, they may have been underrecognized due to the low penetrance of disease and lack of systematic surveillance.

A report of a national mutation testing service for the MEN1 gene: clinical presentations and implications for mutation testing

Introduction: Mutation testing for the MEN1 gene is a useful method to diagnose and predict individuals who either have or will develop multiple endocrine neoplasia type 1 (MEN 1). Clinical selection criteria to identify patients who should be tested are needed, as mutation analysis is costly and time consuming. This study is a report of an Australian national mutation testing service for the MEN1 gene from referred patients with classical MEN 1 and various MEN 1-like conditions. Results: All 55 MEN1 mutation positive patients had a family history of hyperparathyroidism, had hyperparathyroidism with one other MEN1 related tumour, or had hyperparathyroidism with multiglandular hyperplasia at a young age. We found 42 separate mutations and six recurring mutations from unrelated families, and evidence for a founder effect in five families with the same mutation. Discussion: Our results indicate that mutations in genes other than MEN1 may cause familial isolated hyperparathyroidism and familial isolated pituitary tumours. Conclusions: We therefore suggest that routine germline MEN1 mutation testing of all cases of “classical” MEN1, familial hyperparathyroidism, and sporadic hyperparathyroidism with one other MEN1 related condition is justified by national testing services. We do not recommend routine sequencing of the promoter region between nucleotides 1234 and 1758 (Genbank accession no. U93237) as we could not detect any sequence variations within this region in any familial or sporadic cases of MEN1 related conditions lacking a MEN1 mutation. We also suggest that testing be considered for patients <30 years old with sporadic hyperparathyroidism and multigland hyperplasia.

Intratumoral heterogeneity identified at the epigenetic, genetic and transcriptional level in glioblastoma.

Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. Promoter methylation status of the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is the most important clinical biomarker in glioblastoma, predicting for therapeutic response. However, it does not always correlate with response. This may be due to intratumoral heterogeneity, with a single biopsy unlikely to represent the entire lesion. Aberrations in other DNA repair mechanisms may also contribute. This study investigated intratumoral heterogeneity in multiple glioblastoma tumors with a particular focus on the DNA repair pathways. Transcriptional intratumoral heterogeneity was identified in 40% of cases with variability in MGMT methylation status found in 14% of cases. As well as identifying intratumoral heterogeneity at the transcriptional and epigenetic levels, targeted next generation sequencing identified between 1 and 37 unique sequence variants per specimen. In-silico tools were then able to identify deleterious variants in both the base excision repair and the mismatch repair pathways that may contribute to therapeutic response. As these pathways have roles in temozolomide response, these findings may confound patient management and highlight the importance of assessing multiple tumor biopsies.

Succinate dehydrogenase deficiency is rare in pituitary adenomas.

Germline mutations in the succinate dehydrogenase genes (SDHA, SDHB, SDHC, and SDHD) are established as causes of pheochromocytoma/paraganglioma, renal carcinoma, and gastrointestinal stromal tumor. It has recently been suggested that pituitary adenomas may also be a component of this syndrome. We sought to determine the incidence of SDH mutation in pituitary adenomas. We performed screening immunohistochemistry for SDHB and SDHA on all available pituitary adenomas resected at our institution from 1998 to 2012. In those patients with an abnormal pattern of staining, we then performed SDH mutation analysis on DNA extracted from paraffin-embedded tissue, fresh frozen tissue, and peripheral blood. One of 309 adenomas (0.3%) demonstrated an abnormal pattern of staining, a 30 mm prolactin-producing tumor from a 62-year-old man showing loss of staining for both SDHA and SDHB. Examination of paraffin-embedded and frozen tissues confirmed double-hit inactivating somatic SDHA mutations (c.725_736del and c.989_990insTA). Neither of these mutations was present in the germline. We conclude that, although pathogenic SDH mutation may occur in pituitary adenomas and can be identified by immunohistochemistry, it appears to be a very rare event and can occur in the absence of germline mutation. SDH-deficient pituitary adenomas may be larger and more likely to produce prolactin than other pituitary adenomas. Unless suggested by family history and physical examination, it is difficult to justify screening for SDH mutations in pituitary adenomas. Surveillance programs for patients with SDH mutation may be tailored to include the possibility of pituitary neoplasia; however, this is likely to be a low-yield strategy.

Loss of heterozygosity in sporadic parathyroid tumours : involvement of chromosome 1 and the MEN1 gene locus in 11q13

Hyperparathyroidism (HPT) is a common endocrine disorder. Several loci of genetic interest have been identified in parathyroid tumours, including the MEN1 gene locus at 11q13; the HPT‐JT region at 1q21‐q32; and a putative tumour suppressor gene on 1p. We analysed these intervals, which harbour known genes or putative loci associated with familial hyperparathyroidism, in order to clarify the involvement of the respective regions in parathyroid tumourigenesis.

Assessing mutant p53 in primary high-grade serous ovarian cancer using immunohistochemistry and massively parallel sequencing

The tumour suppressor p53 is mutated in cancer, including over 96% of high-grade serous ovarian cancer (HGSOC). Mutations cause loss of wild-type p53 function due to either gain of abnormal function of mutant p53 (mutp53), or absent to low mutp53. Massively parallel sequencing (MPS) enables increased accuracy of detection of somatic variants in heterogeneous tumours. We used MPS and immunohistochemistry (IHC) to characterise HGSOCs for TP53 mutation and p53 expression. TP53 mutation was identified in 94% (68/72) of HGSOCs, 62% of which were missense. Missense mutations demonstrated high p53 by IHC, as did 35% (9/26) of non-missense mutations. Low p53 was seen by IHC in 62% of HGSOC associated with non-missense mutations. Most wild-type TP53 tumours (75%, 6/8) displayed intermediate p53 levels. The overall sensitivity of detecting a TP53 mutation based on classification as ‘Low’, ‘Intermediate’ or ‘High’ for p53 IHC was 99%, with a specificity of 75%. We suggest p53 IHC can be used as a surrogate marker of TP53 mutation in HGSOC; however, this will result in misclassification of a proportion of TP53 wild-type and mutant tumours. Therapeutic targeting of mutp53 will require knowledge of both TP53 mutations and mutp53 expression.

Fumarate Hydratase-deficient Uterine Leiomyomas Occur in Both the Syndromic and Sporadic Settings.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome secondary to germline fumarate hydratase (FH) mutation presents with cutaneous and uterine leiomyomas, and a distinctive aggressive renal carcinoma. Identification of HLRCC patients presenting first with uterine leiomyomas may allow early intervention for renal carcinoma. We reviewed the morphology and immunohistochemical (IHC) findings in patients with uterine leiomyomas and confirmed or presumed HLRCC. IHC was also performed on a tissue microarray of unselected uterine leiomyomas and leiomyosarcomas. FH-deficient leiomyomas underwent Sanger and massively parallel sequencing on formalin-fixed paraffin-embedded tissue. All 5 patients with HLRCC had at least 1 FH-deficient leiomyoma: defined as completely negative FH staining with positive internal controls. One percent (12/1152) of unselected uterine leiomyomas but 0 of 88 leiomyosarcomas were FH deficient. FH-deficient leiomyoma patients were younger (42.7 vs. 48.8 y, P=0.024) and commonly demonstrated a distinctive hemangiopericytomatous vasculature. Other features reported to be associated with FH-deficient leiomyomas (hypercellularity, nuclear atypia, inclusion-like nucleoli, stromal edema) were inconstantly present. Somatic FH mutations were identified in 6 of 10 informative unselected FH-deficient leiomyomas. None of these mutations were found in the germline. We conclude that, while the great majority of patients with HLRCC will have FH-deficient leiomyomas, 1% of all uterine leiomyomas are FH deficient usually due to somatic inactivation. Although IHC screening for FH may have a role in confirming patients at high risk for hereditary disease before genetic testing, prospective identification of FH-deficient leiomyomas is of limited clinical benefit in screening unselected patients because of the relatively high incidence of somatic mutations.

Independent Genetic Events Associated with the Development of Multiple Parathyroid Tumors in Patients with Primary Hyperparathyroidism

Multiple parathyroid tumors, as opposed to hyperplasia, have been reported in a subset of patients with sporadic primary hyperparathyroidism (PHPT). It is not clear whether these multiple tumors are representative of a neoplastic process or whether they merely represent hyperplasia that has affected the parathyroid glands differentially and resulted in asynchronous growth. The molecular genetic techniques of comparative genomic hybridization (CGH), loss of heterozygosity (LOH), and MEN1 mutation analysis were performed on a series of five patients with multiglandular PHPT, each of which had two parathyroid tumors removed. Analysis of these multiple parathyroid tumors from patients with PHPT revealed that independent genetic events were associated with the development of a subset of these tumors. The DNA sequence copy number changes, identified by CGH analyses, either involved different chromosomal regions in the paired glands of a patient (two patients), or those regions implicated in one gland were not changed in a second gland from the same patient (two patients). Each of the three patients exhibiting LOH demonstrated different changes between the paired glands. Where LOH was detected in one gland from a patient, the other gland from the same patient either exhibited no allelic loss or the loss detected was in another region. Each of the three tumors exhibiting LOH at 11q13 was found to contain a somatic MEN1 mutation in the remaining allele, however these mutations were not present in the germline or in the paired gland from the same patient. Although it is possible that a separate series of genetic changes has arisen randomly in two separate glands within the same individual, it seems more likely that the development of these multiple tumors has arisen because of the involvement of other unknown factors. These factors may be genetic [such as the involvement of one or more germline mutations in an unknown low-penetrance gene(s), germline mosaicism or alterations in calcium-sensing receptor gene(s)], epigenetic, physiological, or environmental.