Familial rhabdoid tumour 'avant la lettre'--from pathology review to exome sequencing and back again.

Here we provide compelling evidence that next-generation sequencing will revolutionize diagnostics. We reappraised a case from 1991, published in 1993, describing the unique occurrence of an ovarian immature teratoma arising in a young woman and a clonally distinct intracerebral immature teratoma developing in her daughter. We conducted whole-exome sequencing on constitutional DNA from the mother and her daughter and identified a previously unreported nonsense mutation (c.3533G>A; p.Trp1178*) in the chromatin remodelling gene, SMARCA4, that was present in both individuals and was subject to nonsense-mediated decay. Tumour analysis by Sanger sequencing revealed a somatic SMARCA4 mutation in both the mother (c.2438+1G>T) and her daughter (c.3229C>T; p.Arg1077*), which are predicted to be truncating. As immature teratomas are classified as germ cell tumours, we performed a comprehensive mutation survey of 106 apparently sporadic germ cell tumours, but did not find any other clearly deleterious SMARCA4 mutations. Recently, inactivating mutations in SMARCA4 have been found in two cases of rhabdoid tumour predisposition syndrome type 2. In the light of these findings, renewed efforts to locate previously unobtainable tumour samples were successfully undertaken. Histopathological and immunohistochemical re-analysis of the daughter's tumour revealed that it was indeed a rhabdoid tumour (atypical teratoid/rhabdoid tumour). In this context, the original pathology report of the mother's ovarian tumour was re-interpreted as describing a malignant rhabdoid tumour of the ovary. This report raises the question as to whether molecular genetic analysis should be included in tumour classification, alongside more traditional microscopy-based methods. The use of new sequencing technologies, particularly when applied to archived samples, will lead to many more 'molecular rediagnoses'. This is the earliest known case of rhabdoid tumour predisposition syndrome type 2 and the first described case with an autosomal dominant pattern of inheritance, only discovered through an exome sequencing project.

Three-year financial analysis of minimally invasive radio-guided parathyroidectomy.

Minimally invasive radio-guided parathyroidectomy (MIRP) has had a high success rate in correcting hypercalcemia, along with a low morbidity rate and high patient satisfaction. Our study was conducted in an attempt to analyze the cost-effectiveness of MIRP in patients treated for primary hyperparathyroidism. We conducted a retrospective study of the total charges of three groups of patients undergoing surgery for previously untreated hyperparathyroidism in a single health care system. The three study groups included patients undergoing traditional bilateral neck exploration, MIRP, and neck exploration guided by intraoperative parathormone (PTH) assay. Charges were stratified into preoperative, intraoperative, and postoperative categories. The average total charge was $8,512 for MIRP, $12,723 for traditional neck exploration, and $13,011 for bilateral neck exploration with PTH assay. The decreased charge for MIRP was due to reduced operating room time, anesthesia costs, length of hospitalization, and an avoidance of the use of intraoperative tissue analysis and PTH assay. There was a greater than $4,000 savings with MIRP as compared with the more extensive neck exploration. These savings more than compensate for the cost of technology (preoperative sestamibi scan and intraoperative gamma probe) necessary to perform radio-guided parathyroidectomy.

Hormone production andc-myc protein labeling in plurihormonal pituitary adenomas.

The biological activity of plurihormonal pituitary adenomas was compared with that of tumors producing only one hormone by evaluating the percentage ofc- myc protein-labeled cells and ultrastructural characteristics. Twenty-five pituitary adenomas producing 3 or more hormones and 14 adenomas producing only I hormone were studied. Tissue sections were stained immunohistochemically using antibodies for pituitary hormones andc- myc protein, and they were examined by electronmicroscopy. DNA extracted from ethanol-fixed, paraffinembedded tissue was analyzed for p53 mutations by polymerase chain reaction and singlestrand conformation polymorphism analysis. The percentage ofc- myc protein-labeled cells in adenomas producing 4 or 5 pituitary hormones was significantly higher (p < 0.01 ) than in those producing 3 or 1 hormones. There were no p53 mutations in plurihormonal adenomas. Pituitary adenomas producing 4 or 5 pituitary hormones demonstrate biological aggressiveness; therefore, multihormone production reflects aggressive capacity rather than degree of differentiation.

Detection of Heterozygous Mutation in the Retinoblastoma Gene in a Human Pituitary Adenoma Using PCR-SSCP Analysis and Direct Sequencing.

Genomic deoxyribonucleic acid from surgical specimens of 25 pituitary adenomas was screened for the presence of mutations in the tumor suppressor gene retinoblastoma gene, using polymerase chain reaction and single-strand conformation polymorphism analysis, followed by direct deoxyribonucleic acid sequencing. Mutation causing an amino acid change was found in one of the 25 pituitary adenomas. The mutation site was in exon 19 (codon 621) of the retinoblastoma gene. In addition, there were three types of silent mutations in introns of the gene. The patient in whom the retinoblastoma mutation was identified had a tumor with high clinical malignancy, a high percentage of c-myc protein-labeled cells, and a diagnosis of plurihormonal pituitary adenoma based on the presence of cells immunoreactive for five pituitary hormones. This article suggests that point mutation of retinoblastoma gene is rare in human pituitary adenomas but may provide a marker for aggressive pituitary adenoma.

Higher stromal expression of transforming growth factor-beta type II receptors is associated with poorer prognosis breast tumors.

Transforming growth factor-beta (TGFB) is a potent inhibitor of normal epithelial cell proliferation, and may be one of the regulatory factors that are perturbed during tumor development. While many tumor cell lines no longer respond to the inhibitory effects of TGFB due to a reduction or absence of the type II receptor (TGFBR2), the role of TGFBR2 in tumors from patients with breast cancer is less clear. The objective of this study was to screen human breast tumors to determine if there was a TGFBR2 mutation and/or altered expression of TGFBR2 protein. Using 10 unique primers, SSCP-PCR was used to detect heterozygosity in the complete coding sequence from 72 tumors and normal DNA from 20 individuals. One region of the promoter was also examined. Expression of TGFBR2 in the same breast tumors was examined by immunohistochemistry. Sequence variations were identified among normal and tumor tissue samples by SSCP-PCR within coding regions of exon 4 (1/72 samples) and within non-coding regions of intron 2 (1/72), intron 3 (72/72), and intron 6 (1/72). A new polymorphism was identified in intron 3. Observed allele frequencies were consistent with Hardy-Weinberg equilibrium in both the tumors and normal DNA. TGFBR2 was expressed in the epithelium and stroma of tumor tissue. The percentage of cells expressing TGFBR2 in stroma was higher in patients that had a positive lymph node status and/or negative estrogen and progesterone receptor expression. There was no relationship between TGFBR2 expression in the epithelium and these variables.

Mutational analyses of RB and BRCA2 as candidate tumour suppressor genes in parathyroid carcinoma.

OBJECTIVE: Strong evidence indicates that at least one key tumour suppressor gene important for the development of malignant parathyroid tumours is located on chromosome 13, but the critical target gene remains unknown. Importantly, the region of acquired DNA loss includes two established tumour suppressor genes, the retinoblastoma gene, RB (RB1) and BRCA2. Resolution of whether RB or BRCA2 is the critical 13q tumour suppressor gene in parathyroid cancer requires analysis of these genes' sequences for intragenic inactivating mutations. Therefore, RB and BRCA2 were analysed in a group of parathyroid carcinomas in which mutations of these genes should be most readily detectable. PATIENTS AND DESIGN: Six parathyroid carcinomas from four patients which showed loss of heterozygosity (LOH) at the RB locus and/or 13q loss by comparative genomic hybridazation (CGH) were selected from a CGH/LOH-screened panel of 16 carcinoma specimens from 10 patients. These tumours were examined for mutations by direct sequencing of the complete 27-exon coding region, intron-exon boundaries and promoter of RB. The 26 coding exons and intron-exon boundaries of BRCA2 were also directly sequenced in seven parathyroid carcinomas with loss in the BRCA2 region. RESULTS: No microdeletions, insertions, or point mutations were detected in either RB or BRCA2 in any of the carcinomas. CONCLUSION: The absence of tumour-specific somatic mutations in RB and BRCA2 suggests that they are unlikely to act as classic tumour suppressor genes in the pathogenesis of parathyroid carcinomas. While decreased expression of these genes might contribute to parathyroid carcinomatosis in a secondary fashion and 13q loss warrants further study as a diagnostic marker for parathyroid carcinoma, the putative 13q tumour suppressor awaits identification.