Genetic characterization of large parathyroid adenomas

In this study, we genetically characterized parathyroid adenomas with large glandular weights, for which independent observations suggest pronounced clinical manifestations. Large parathyroid adenomas (LPTAs) were defined as the 5% largest sporadic parathyroid adenomas identified among the 590 cases operated in our institution during 2005–2009. The LPTA group showed a higher relative number of male cases and significantly higher levels of total plasma and ionized serum calcium (P<0.001). Further analysis of 21 LPTAs revealed low MIB1 proliferation index (0.1–1.5%), MEN1 mutations in five cases, and one HRPT2 (CDC73) mutation. Total or partial loss of parafibromin expression was observed in ten tumors, two of which also showed loss of APC expression. Using array CGH, we demonstrated recurrent copy number alterations most frequently involving loss in 1p (29%), gain in 5 (38%), and loss in 11q (33%). Totally, 21 minimal overlapping regions were defined for losses in 1p, 7q, 9p, 11, and 15q and gains in 3q, 5, 7p, 8p, 16q, 17p, and 19q. In addition, 12 tumors showed gross alterations of entire or almost entire chromosomes most frequently gain of 5 and loss of chromosome 11. While gain of 5 was the most frequent alteration observed in LPTAs, it was only detected in a small proportion (4/58 cases, 7%) of parathyroid adenomas. A significant positive correlation was observed between parathyroid hormone level and total copy number gain (r=0.48, P=0.031). These results support that LPTAs represent a group of patients with pronounced parathyroid hyperfunction and associated with specific genomic features

Germline CDKN2A/ARF alterations in human melanoma

Approximately 10% of cases of human cutaneous malignant melanoma (CMM) have been estimated to occur in individuals with a familial predisposition, frequently in association with dysplastic nevus syndrome (DNS). The genetics of familial melanoma is complex and heterogeneous. To date only two melanoma predisposing genes have been identified. The CDKN2A/ARF locus on human chromosome 9p21 encodes two distinct cell cycle regulatory proteins, p16 and p14ARF. Germline alterations in the CDKN2A gene have been detected in approximately 20% of CMM families. Germline mutations have also been reported in the CDK4 gene on chromosome 12q15, in a few families. The primary aim of this thesis was to investigate alterations in the CDKN2A/ARF locus among individuals belonging to Swedish families with melanoma heredity and to study their biological consequences. The relationships between germline CDKN2A mutations, melanoma and DNS phenotype were studied in five Swedish melanoma families with the mutations: a proline to leucine substitution (P48L), and an arginine insertion (113insR). We found significant correlations between CDKN2A mutations and CMM/DNS. Our results are consistent with the hypothesis that germline CDKN2A mutations and DNS both contribute to melanoma predisposition and may lead to early onset melanoma when present in the same individual. The 113insR germline mutation was detected in 17 Swedish melanoma families. Haplotype analysis, using microsatellite markers in the 9p21 region, showed that these families share a common allele at markers close to CDKN2A, suggesting that 113insR is a founder mutation. Statistical analysis of meiotic recombination events in the 9p21 region indicated that the mutation arose approximately 2000 years ago. We screened 80 individuals with multiple primary melanomas (MPM) for germline CDKN2A mutations. Mutations were detected in 11 % of these patients and the majority of them had a family history of melanoma. A novel 24 base pair deletion, which included codons 62-69 (delta-62- 69) was detected in one individual belonging to a family with melanoma heredity. CDKN2A mutation screening of individuals with MPM may thus identify high-risk families. We confirmed the role of the P48L and A62-69 CDKN2A germline mutations in the development of melanoma tumors by demonstrating that the mutant p 16 proteins are functionally abnormal and do not bind to CDK4 or CDK6. Due to overlapping open reading frames in exon 2 of CDKN2A, the p16-delta-62-69 also causes an in frame deletion of residues 77-84 in p14ARF. Our studies in cultured tumor cells showed that partial functional loss in p14ARF-delta-77-84 may complement the defective p16-delta-62-69 mutant and may contribute to melanoma development in patients carrying the 24 bp deletion in CDKN2A. The high rate of CDKN2A inactivation in human cancer encouraged us to address the mechanisms and frequency of Cdkn2a/b gene loss during immortalization of Ras-transformed rat embryo fibroblast clones. We found homozygous deletions of Cdkn2a/b in all established cell lines studied. The mechanisms for loss of heterozygosity (LOH) were mitotic non-disjunction, deletion/rearrangements and, rarely, mitotic recombination. The frequency of Cdkn2a/b gene deletions was estimated to be approximately 2 x 10-8 /cell/generation

Genome-wide and locus specific alterations in CDC73/HRPT2-mutated parathyroid tumors.

Mutations in the hyperparathyroidism type 2 (HRPT2/CDC73) gene and alterations in the parafibromin protein have been established in the majority of parathyroid carcinomas and in subsets of parathyroid adenomas. While it is known that CDC73-mutated parathyroid tumors display specific gene expression changes compared to CDC73 wild-type cases, the molecular cytogenetic profile in CDC73-mutated cases compared to unselected adenomas (with an expected very low frequency of CDC73 mutations) remains unknown. For this purpose, nine parathyroid tumors with established CDC73 gene inactivating mutations (three carcinomas, one atypical adenoma and five adenomas) were analyzed for copy number alterations and loss of heterozygosity using array-comparative genomic hybridization (a-CGH) and single nucleotide polymorphism (SNP) microarrays, respectively. Furthermore, CDC73 gene promoter methylation levels were assessed using bisulfite Pyrosequencing. The panel included seven tumors with single mutation and three with double mutations of the CDC73 gene. The carcinomas displayed copy number alterations in agreement with previous studies, whereas the CDC73-mutated adenomas did not display the same pattern of alterations at loci frequently deleted in unselected parathyroid tumors. Furthermore, gross losses of chromosomal material at 1p and 13 were significantly (p = 0.012) associated with parathyroid carcinomas as opposed to adenomas. Quantitative PCR-based copy number loss regarding CDC73 was observed in three adenomas, while all the carcinomas were diploid or showed copy number gain for CDC73 gene. Hypermethylation of the CDC73 gene promoter was not observed. Our data could suggest that CDC73-mutated parathyroid adenomas exhibit a partly unique cytogenetic profile in addition to that of carcinomas and unselected adenomas. Furthermore, CDC73-mutated carcinomas displayed losses at 1p and 13 which are not seen in CDC73-mutated adenomas, making these regions of interest for further studies regarding malignant properties in tumors from CDC73-mutated cases. However, due to the small sample size, validation of the results in a larger cohort is warranted

Plasma proteome profiling of cardiotoxicity in patients with diffuse large B-cell lymphoma

Background Cardiovascular toxicity is a notorious complication of doxorubicin (DXR) therapy for diffuse large B-cell lymphoma (DLBCL). Although surveillance of well-known biological markers for cardiovascular disease (CVD) as NTproBNP and Troponins may be helpful, there are no established markers to monitor for evolving CVD during treatment. New possibilities have arisen with the emergence of newer techniques allowing for analysis of plasma proteins that can be associated with cardiovascular disease. Proximity Extension Assay is one of them. Objectives We aimed to illustrate the incidence of CVD in DLBCL patients treated with DXR and to establish whether there are plasma proteins associated with pre-existing or emerging CVD. Methods In 95 patients, 182 different proteins from OLINK panels, NTproBNP, Troponin I and CRP were assessed prior to, during and after treatment. For comparison, samples from controls were analyzed. Results In the DLBCL cohort, 33.3% had pre-treatment CVD compared to 5.0% in the controls and 23.2% developed new CVD. Of the 32.6% who died during follow up, CVD was the cause in 4 patients. Spondin-1 (SPON-1) correlated to pre-treatment CVD (1.22 fold change, 95% CI 1.10-1.35, p = 0.00025, q = 0.045). Interleukin-1 receptor type 1 (IL-1RT1) was associated to emerging CVD (1.24 fold change, 95% CI 1.10-1.39, p = 0.00044, q = 0.082). Conclusion We observed a higher prevalence of CVD in DLBCL patients compared to controls prior to DXR therapy. Two proteins, SPON-1 and IL-1RT1, were related to pre-existing and emerging CVD in DXR treated patients. If confirmed in larger cohorts, IL-1RT1 may emerge as a reliable biomarker for unfolding CVD in DLBCL

Haplotype analysis and age estimation of the 113insR CDKN2A founder mutation in Swedish melanoma families

Germline mutations in the CDKN2A tumor suppressor gene located on 9p21 have been linked to development of melanomas in some families. A germline 3-bp insertion in exon 2 of CDKN2A, leading to an extra arginine at codon 113 (113insR), has been identified in 17 Swedish melanoma families. Analysis of 10 microsatellite markers, spanning approximately 1 Mbp in the 9p21 region, showed that all families share a common allele for at least one of the markers closest to the CDKN2A gene, suggesting that the 113insR mutation is an ancestral founder mutation. Differences in the segregating haplotypes, due to meiotic recombinations and/or mutations in the short-tandem-repeat markers, were analyzed further to estimate the age of the mutation. Statistical analysis using a maximum likelihood approach indicated that the mutation arose 98 generations (90% confidence interval: 52-167 generations), or approximately 2,000 years, ago. Thus, 113insR would be expected to have a more widespread geographic distribution in European and North American regions with ancestral connections to Sweden. Alternatively, CDKN2A may lie in a recombination hot spot region, as suggested by the many meiotic recombinations in this narrow approximately 1-cM region on 9p21

Checkpoint CD47 expression in classical Hodgkin lymphoma

The glycoprotein CD47 regulates antiphagocytic activity via signal regulatory protein alpha (SIRPa). This study investigated CD47 expression on Hodgkin and Reed–Sternberg (HRS) cells in the classical Hodgkin lymphoma (cHL) tumour microenvironment and its correlation with prognosis, programmed-death (PD) immune markers, and SIRPa+ leukocytes. We conducted immunohistochemistry with CD47 and SIRPa antibodies on diagnostic biopsies (tissue microarrays) from cHL patients from two cohorts (n = 178). In cohort I (n = 136) patients with high expression of CD47 on HRS cells (n = 48) had a significantly inferior event-free survival [hazard ratio (HR) = 5.57; 95% confidence interval (CI), 2.78–11.20; p &lt; 0.001] and overall survival (OS) (HR = 8.54; 95% CI, 3.19–22.90; p &lt; 0.001) compared with patients with low expression (n = 88). The survival results remained statistically significant in multivariable Cox regression adjusted for known prognostic factors. In cohort II (n = 42) high HRS cell CD47 expression also carried shorter event-free survival (EFS) (HR = 5.96; 95% CI, 1.20–29.59; p = 0.029) and OS (HR = 5.61; 95% CI, 0.58–54.15; p = 0.136), although it did not retain statistical significance in the multivariable analysis. Further, high CD47 expression did not correlate with SIRPa+ leukocytes or PD-1, PD-L1 and PD-L2 expression. This study provides a deeper understanding of the role of CD47 in cHL during an era of emerging CD47 therapies.Authors in thesis list of papers: Gholiha, A.R., Hollander, P., Hedstrom, G., Molin, D., Hjalgrim, H., Smedby, K.E., Glimelius, I., Hashemi, J., Amini, R-M., Enblad, G.Title in thesis list of papers: Checkpoint CD47 Expression Predicts Inferior Survival in classical Hodgkin Lymphoma</p

Sequencing chromatograms of <i>CDC73</i> mutations in parathyroid tumors: T1 (IVS1+1 G>C), T5 constitutional (c128-IVS1+1 delG) and T5a (c.71del 72–90).

<p>Wild-type (WT) sequences are indicated above the mutated sequences.</p