Catharina Larsson

Karolinska Institutet, Solna, Stockholm, Sweden

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Publications (231)1187.78 Total impact

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    ABSTRACT: Recurrent mutations in the promoter of the human telomerase reverse transcriptase (TERT) gene have been described in several types of endocrine tumors. These mutations lead to TERT over-expression allowing tumor cells to escape senescence. Parathyroid carcinomas are rare, but constitute a diagnostic challenge for the pathologist and lack effective oncological treatment regimes. Previously, telomerase activation and TERT expression has been reported in parathyroid carcinomas but not in parathyroid adenomas, suggesting an important role in parathyroid malignancy. We investigated 88 parathyroid adenomas, 22 carcinomas and 10 atypical adenomas from two clinical institutions for TERT promoter mutations. TERT gene expression was quantified in a subset of cases. All tumors revealed the wild-type sequence only at the common mutation sites -124C and -146C, and in 39/40 tumors TERT expression was not detectable. A constitutional TERT promoter variant -145C>T was detected in an atypical adenoma and an adenoma from a single patient. TERT expression was detected in 1/6 atypical adenomas and in 0/34 adenomas. In conclusion, TERT promoter mutations are rare in parathyroid tumors and the previously reported telomerase activity in parathyroid carcinomas is expected to stem from other molecular genetic mechanisms.
    Endocrine Related Cancer 04/2015; DOI:10.1530/ERC-15-0121 · 4.91 Impact Factor
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    ABSTRACT: Primary hyperparathyroidism is a common endocrinopathy, frequently caused by a parathyroid adenoma, rarely by a parathyroid carcinoma that lacks effective oncological treatment. Since the majority of cases present in postmenopausal women, oestrogen signalling was implicated in the tumourigenesis. Oestrogen receptors beta (ERB) 1 and 2 were recently identified in parathyroid adenomas, the former inducing genes coupled to tumour apoptosis. We applied immunohistochemistry and slide digitalisation to quantify nuclear ERB1 and ERB2 in 172 parathyroid adenomas, atypical adenomas and carcinomas, and 10 normal parathyroid glands. All the normal parathyroid glands expressed ERB1 and ERB2. The majority of tumours expressed ERB1 (70.6%) at varying intensities, and ERB2 (96.5%) at strong intensities. Parathyroid carcinomas expressed ERB1 in 3/6 cases and ERB2 in 5/6 cases. Tumour nuclear ERB1 intensity significantly correlated inversely to tumour weight (p = 0.011); and patients whose tumours were classified as ERB1-negative had significantly larger tumour weight as well as higher serum calcium (p = 0.002) and parathyroid hormone levels (p = 0.003). Additionally, tumour nuclear ERB1 was not expressed differentially to patient gender or age. Tumour nuclear ERB2 did not correlate to clinical characteristics. In conclusion, decreased ERB1 immunoreactivity is associated with increased tumour weight in parathyroid adenomas. Given the previously reported correlation to tumour suppressive signalling, oestrogen receptor modulation (SERMs) may play a role in the treatment of parathyroid carcinomas. Future studies of SERMs and oestrogen treatment in primary hyperparathyroidism should consider tumour weight as a potential factor in pharmacological responsiveness.
    02/2015; DOI:10.1530/EC-14-0109
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    ABSTRACT: Anaplastic thyroid carcinoma (ATC) is a frequently lethal malignancy that is often unresponsive to available therapeutic strategies. The tumorigenesis of ATC and its relationship to the widely prevalent well-differentiated thyroid carcinomas are unclear. We have analyzed 22 cases of ATC as well as 4 established ATC cell lines using whole-exome sequencing. A total of 2,674 somatic mutations (121/sample) were detected. Ontology analysis revealed that the majority of variants aggregated in the MAPK, ErbB, and RAS signaling pathways. Mutations in genes related to malignancy not previously associated with thyroid tumorigenesis were observed, including mTOR, NF1, NF2, MLH1, MLH3, MSH5, MSH6, ERBB2, EIF1AX and USH2A; some of which were recurrent and were investigated in 24 additional ATC cases and 8 ATC cell lines. Somatic mutations in established thyroid cancer genes were detected in 14 of 22 (64%) tumors and included recurrent mutations in BRAF, TP53, and RAS-family genes (6 cases each), as well as PIK3CA (2 cases) and single cases of CDKN1B, CDKN2C, CTNNB1 and RET mutations. BRAF V600E and RAS mutations were mutually exclusive; all ATC cell lines exhibited a combination of mutations in either BRAF and TP53 or NRAS and TP53. A hypermutator phenotype in two cases with >8 times higher mutational burden than the remaining mean was identified; both cases harbored unique somatic mutations in MLH mismatch-repair genes. This first comprehensive exome-wide analysis of the mutational landscape of ATC identifies novel genes potentially associated with ATC tumorigenesis, some of which may be targets for future therapeutic intervention. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
    Human Molecular Genetics 01/2015; DOI:10.1093/hmg/ddu749 · 6.68 Impact Factor
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    ABSTRACT: Context: Adrenocortical carcinoma (ACC) is a rare and lethal malignancy with a poorly defined etiology, and the molecular genetics of ACC are incompletely understood. Objective: Utilize whole-exome sequencing for genetic characterization of the underlying somatic mutations and copy number alterations (CNA) present in ACC. Design: Screening for somatic mutation events and CNAs by comparative analysis of tumors and matched normal samples from 41 patients with ACC. Results: In total, 966 non-synonymous somatic mutations were detected, including 40 tumors with a mean of 16 mutations per sample and one tumor with 314 mutations. Somatic mutations in ACC-associated genes included TP53 (8/41 tumors, 19.5%) and CTNNB1 (4/41, 9.8%). Genes with potential disease-causing mutations included GNAS, NF2 and RB1, and recurrently mutated genes with unknown roles in tumorigenesis comprised CDC27, SCN7A and SDK1. Recurrent CNAs included amplification at 5p15.33 including TERT (6/41, 14.6%) and homozygous deletion at 22q12.1 including the Wnt repressors ZNRF3 and KREMEN1 (4/41; 9.8% and 3/41; 7.3% respectively). Somatic mutations in ACC established genes and recurrent ZNRF3 and TERT loci CNAs were mutually exclusive in the majority of cases. Moreover, gene ontology identified Wnt signaling as the most frequently mutated pathway in ACCs. Conclusions: These findings highlight the importance of Wnt pathway dysregulation in ACC and corroborate the finding of homozygous deletion of Wnt repressors ZNRF3 and KREMEN1. Overall, mutations in either TP53 or CTNNB1 as well as focal CNAs at the ZNRF3 or TERT loci denote mutually exclusive events, suggesting separate mechanisms underlying the development of these tumors.
    Journal of Clinical Endocrinology &amp Metabolism 12/2014; 100(3):jc20143282. DOI:10.1210/jc.2014-3282 · 6.31 Impact Factor
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    ABSTRACT: The p53 target gene WIG-1 (ZMAT3) is located in chromosomal region 3q26, that is frequently amplified in human tumors, including cervical cancer. We have examined the status of WIG-1 and the encoded Wig-1 protein in cervical carcinoma cell lines and tumor tissue samples. Our analysis of eight cervical cancer lines (Ca Ski, ME-180, MS751, SiHa, SW756, C-4I, C-33A, and HT-3) by spectral karyotype, comparative genomic hybridization and Southern blotting revealed WIG-1 is not the primary target for chromosome 3 gains. However, WIG-1/Wig-1 were readily expressed and WIG-1 mRNA expression was higher in the two HPV-negative cervical cell lines (C33-A, HT-3) than in HPV-positive lines. We then assessed Wig-1 expression by immunohistochemistry in 38 cervical tumor samples. We found higher nuclear Wig-1 expression levels in HPV-negative compared to HPV positive cases (p = 0.002) and in adenocarcinomas as compared to squamous cell lesions (p<0.0001). Cases with moderate nuclear Wig-1 staining and positive cytoplasmic Wig-1 staining showed longer survival than patients with strong nuclear and negative cytoplasmic staining (p = 0.042). Nuclear Wig-1 expression levels were positively associated with age at diagnosis (p = 0.023) and histologic grade (p = 0.034). These results are consistent with a growth-promoting and/or anti-cell death function of nuclear Wig-1 and suggest that Wig-1 expression can serve as a prognostic marker in cervical carcinoma.
    PLoS ONE 11/2014; 9(11):e111125. DOI:10.1371/journal.pone.0111125 · 3.53 Impact Factor
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    ABSTRACT: BACKGROUND: The telomerase reverse transcriptase (TERT) promoter mutations C228T and C250T have been found in many malignancies, including in thyroid carcinomas. However, it is unclear how early these mutations occur in thyroid tumorigenesis. METHODS: The study included primary tumors from 58 patients initially diagnosed with follicular thyroid adenoma (FTA), a benign entity, 18 with atypical FTA (AFTA) having an uncertain malignant potential, and 52 with follicular thyroid carcinoma (FTC). Sanger sequencing was used to investigate the mutational status of the TERT promoter. Telomere length and TERT messenger RNA (mRNA) expression were determined using quantitative polymerase chain reaction (PCR). Telomerase activity was assessed using a Telomerase PCR enzymelinked immunosorbent assay kit. RESULTS: The C228T mutation was identified in 1 of 58 FTA (2%) and 3 of 18 AFTA (17%) samples. These 4 tumors all expressed TERT mRNA and telomerase activity, whereas the majority of C228T-negative adenomas lacked TERT expression (C228T versus wild-type, P=.008). The C228T mutation was associated with NRAS gene mutations (P=.016). The patient with C228T-mutated FTA later developed a scar recurrence and died of FTC, whereas none of the remaining 57 patients with FTA had recurrence. No recurrence occurred in 3 patients with AFTA who carried C228T during the follow-up period (36-285 months). Nine of the 52 FTCs (17%) exhibited the TERT mutation (8 of 9 C228T and 1 of 9 C250T), and the presence of the mutation was associated with shorter patient survival. CONCLUSIONS: TERT promoter mutations may occur as an early genetic event in thyroid follicular tumors that have not developed malignant features on routine histopathological workup. (C) 2014 American Cancer Society.
    Cancer 10/2014; 120(19). DOI:10.1002/cncr.28800 · 4.90 Impact Factor
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    ABSTRACT: Telomerase activation through the induction of its catalytic component TERT is essential in carcinogenesis. The regulatory mechanism and clinical significance underlying cancer-specific TERT expression have been extensively investigated in various human malignancies, but little is known about these in Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor. Here we addressed these issues by determining TERT promoter mutations, gene amplification, mRNA expression and association with clinical variables in MCC. TERT mRNA was expressed in 6/6 MCC cell lines and 41 of 43 tumors derived from 35 MCC patients. Telomerase activity was detectable in all 6 cell lines and 11 tumors analyzed. TERT promoter mutations were identified in 1/6 cell lines and 4/35 (11.4%) MCC cases. The mutation exhibited UV signature and occurred in sun-exposed areas. Increased TERT gene copy numbers were observed in 1/6 cell lines and 11/14 (79%) tumors, and highly correlated with its mRNA expression (r = 0.7419, P = 0.0024). Shorter overall survival was significantly associated with higher TERT mRNA levels in MCC patients (P = 0.032). Collectively, TERT expression and telomerase activity is widespread in MCC, and may be attributable to TERT promoter mutations and gene amplification. Higher TERT expression predicts poor patient outcomes.
    Oncotarget 09/2014; · 6.63 Impact Factor
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    ABSTRACT: Purpose: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive forms of cancer with no curative therapies available. To date, strategies to target ATC by immunotherapy have not been evaluated. We investigated whether ATC would be a suitable target for NK-cell-based immunotherapy. Experimental Design: We first established 7 new cell lines from ATC tumors, 3 from PTC tumors and analyzed them together with 8 additional ATC cell lines. Cells were analyzed for sensitivity to lysis by NK cells and their ability to chemoattract and regulate the activity of NK cells. In addition, fresh tumor samples and peripheral blood from 6 ATC patients were analyzed for NK cell infiltration and phenotype. Results: We observed that ATC cell lines are sensitive to lysis by ex vivo expanded NK cells and that the lysis was abrogated upon blockade of NKG2D. Sensitivity of thyroid cancer cell lines to NK-cell-mediated lysis correlated with surface expression of UL16-binding protein 2 on tumor cells. Moreover, ATC cell lines produced high levels of CXCL10 and stimulated migration of expanded NK cells and ATC tumors were enriched for NK cells expressing the cognate chemokine receptor CXCR3. However, compared with NK cells in peripheral blood, ATC-tumor-derived NK cells displayed a suppressed phenotype with a down-regulated expression of NKG2D. In vitro, suppression of NK-cell-mediated lysis and NKG2D expression by ATC cells was restored upon neutralization of prostaglandin-E2. Conclusions: ATC cell lines are sensitive to NK-cell-mediated lysis via ULBP2/5/6 and chemoattract CXCR3-positive NK-cells. Patients with ATC may benefit from NK-cell-based immunotherapy.
    Clinical Cancer Research 09/2014; DOI:10.1158/1078-0432.CCR-14-0291 · 8.19 Impact Factor
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    ABSTRACT: Altered expression of specific microRNAs (miRNAs) has been observed in human cervical cancer. However, the biological functions of many of these miRNAs are yet to be discovered. We previously showed that miR-944 is significantly more abundant in cervical cancer tissues than their normal counterparts. In this study, we investigated the functions and targets of miR-944 in human cervical cancer cells. MiR-944 is located in the intron of the tumor protein p63 (TP63) gene, which is frequently overexpressed in cervical carcinomas. Using gain- and loss-of-function experiments in vitro, we demonstrate that miR-944 promotes cell proliferation, migration and invasion, but has no effect on apoptosis, in human cervical cancer cells. To identify the targets of miR-944, we performed photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation followed by deep sequencing. Among the candidate targets, we validated HECW2 (HECT domain ligase W2) and S100PBP (S100P binding protein) as direct targets of miR-944 using luciferase reporter assays and western blot analysis. Our findings reveal novel functions and targets of miR-944 in human cervical cancer cells, which may provide new insights of its role in cervical carcinogenesis. What's new? While miR-944 has been shown to be associated with tumor development and progression in several tumor types, its functions and targets remain undetermined. This study stands out as the first report of miR-944 functions and targets in human cancer. The authors demonstrate that miR-944 functions as an oncogene in human cervical cancer cells by promoting cell proliferation, migration, and invasion. In addition, they identified and validated HECW2 and S100PBP as direct targets of miR-944 in human cervical cancer cells. The findings provide new insights into the biological roles of miR-944 in human cervical cancer cells.
    International Journal of Cancer 09/2014; 136(5). DOI:10.1002/ijc.29160 · 5.01 Impact Factor
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    ABSTRACT: To outline further genetic mechanisms of transformation from follicular lymphoma (FL) to diffuse large B-cell lymphoma (DLBCL), we have performed whole genome array-CGH in 81 tumors from 60 patients [29 de novo DLBCL (dnDLBCL), 31 transformed DLBCL (tDLBCL), and 21 antecedent FL]. In 15 patients, paired tumor samples (primary FL and a subsequent tDLBCL) were available, among which three possessed more than two subsequent tumors, allowing us to follow specific genetic alterations acquired before, during, and after the transformation. Gain of 2p15–16.1 encompassing, among others, the REL, BCL11A, USP34, COMMD1, and OTX1 genes was found to be more common in the tDLBCL compared with dnDLBCL (P < 0.001). Furthermore, a high-level amplification of 2p15–16.1 was also detected in the FL stage prior to transformation, indicating its importance during the transformation event. Quantitative real-time PCR showed a higher level of amplification of REL, USP34, and COMMD1 (all involved in the NFκΒ-pathway) compared with BCL11A, which indicates that the altered genes disrupting the NFκΒ pathway may be the driver genes of transformation rather than the previously suggested BCL11A. Moreover, a 17q21.33 amplification was exclusively found in tDLBCL, never in FL (P < 0.04) or dnDLBCL, indicating an upregulation of genes of importance during the later phase of transformation. Taken together, our study demonstrates potential genomic markers for disease progression to clinically more aggressive forms. We also confirm the importance of the TP53-, CDKN2A-, and NFκΒ-pathways for the transformation from FL to DLBCL. © 2014 Wiley Periodicals, Inc.
    Genes Chromosomes and Cancer 09/2014; 53(9). DOI:10.1002/gcc.22184 · 3.84 Impact Factor
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    ABSTRACT: DOG1, a Ca(2+)-activated Cl(-) channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 hours, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl(-) currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.
    Experimental Cell Research 05/2014; DOI:10.1016/j.yexcr.2014.05.003 · 3.37 Impact Factor
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    ABSTRACT: The telomerase reverse transcriptase gene (TERT) encodes the reverse transcriptase component of the telomerase complex, which is essential for telomere stabilization and cell immortalization. Recent studies have demonstrated a transcriptional activation role for the TERT promoter mutations C228T and C250T in many human cancers, as well as a role in aggressive disease with potential clinical applications. Although telomerase activation is known in adrenal tumors, the underlying mechanisms are not established. We assessed C228T and C250T TERT mutations by direct Sanger sequencing in tumors of the adrenal gland, and further evaluated potential associations with clinical parameters and telomerase activation. A total of 199 tumors were evaluated, including 34 adrenocortical carcinomas (ACC), 47 adrenocortical adenomas (ACA), 105 pheochromocytomas (PCC; ten malignant and 95 benign), and 13 abdominal paragangliomas (PGL; nine malignant and four benign). TERT expression levels were determined by quantitative RT-PCR. The C228T mutation was detected in 4/34 ACCs (12%), but not in any ACA (P=0.028). C228T was also observed in one benign PCC and in one metastatic PGL. The C250T mutation was not observed in any case. In the ACC and PGL groups, TERT mutation-positive cases exhibited TERT expression, indicating telomerase activation; however, since expression was also revealed in TERT WT cases, this could denote additional mechanisms of TERT activation. To conclude, the TERT promoter mutation C228T is a recurrent event associated with TERT expression in ACCs, but rarely occurs in PGL and PCC. The involvement of the TERT gene in ACC represents a novel mutated gene in this entity.
    Endocrine Related Cancer 04/2014; 21(3):427-34. DOI:10.1530/ERC-14-0016 · 4.91 Impact Factor
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    ABSTRACT: Aberrant DNA methylation is a feature of human cancer affecting gene expression and tumor phenotype. Here, we quantified promoter methylation of candidate genes and global methylation in 44 small intestinal-neuroendocrine tumors (SI-NETs) from 33 patients by pyrosequencing. Findings were compared with gene expression, patient outcome and known tumor copy number alterations. Promoter methylation was observed for WIF1, RASSF1A, CTNNB1, CXCL14, NKX2-3, P16, LAMA1, and CDH1. By contrast APC, CDH3, HIC1, P14, SMAD2, and SMAD4 only had low levels of methylation. WIF1 methylation was significantly increased (P = 0.001) and WIF1 expression was reduced in SI-NETs vs. normal references (P = 0.003). WIF1, NKX2-3, and CXCL14 expression was reduced in metastases vs. primary tumors (P<0.02). Low expression of RASSF1A and P16 were associated with poor overall survival (P = 0.045 and P = 0.011, respectively). Global methylation determined by pyrosequencing of LINE1 repeats was reduced in tumors vs. normal references, and was associated with loss in chromosome 18. The tumors fell into three clusters with enrichment of WIF1 methylation and LINE1 hypomethylation in Cluster I and RASSF1A and CTNNB1 methylation and loss in 16q in Cluster II. In Cluster III, these alterations were low-abundant and NKX2-3 methylation was low. Similar analyses in the SI-NET cell lines HC45 and CNDT2 showed methylation for CDH1 and WIF1 and/or P16, CXCL14, NKX2-3, LAMA1, and CTNNB1. Treatment with the demethylating agent 5-azacytidine reduced DNA methylation and increased expression of these genes in vitro. In conclusion, promoter methylation of tumor suppressor genes is associated with suppressed gene expression and DNA copy number alterations in SI-NETs, and may be restored in vitro.
    Epigenetics: official journal of the DNA Methylation Society 04/2014; 9(7). DOI:10.4161/epi.28936 · 5.11 Impact Factor
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    ABSTRACT: Background: Telomere maintenance via telomerase activation and the alternative lengthening of telomeres (ALT) mechanism was assessed in medullary thyroid carcinoma (MTC). Methods: Totally 42 MTCs were studied including 24 RET mutated cases. Relative telomerase reverse transcriptase (TERT) expression, splice forms and telomere length were determined by PCR-based methods, and telomerase activity by ELISA. The ALT mechanism was detected by Southern blot analysis and immunofluorescence. Results: TERT expression and telomerase activity were detected in 21/42 tumors (50%), and was independent of the common somatic M918T RET mutation. Mean telomere length was shorter in MTCs compared to thyroids. Telomerase activation was associated with large tumor size (P = 0.027), advanced clinical stage (P = 0.0001), and short survival (P = 0.0001). Full-length TERT and the α(-) and β(-)-deletion forms were revealed, and the full-length form was associated with short survival (P = 0.04). A subset of cases without telomerase activation showed involvement of the ALT mechanism, which was associated with a low MIB-1 proliferation index (P = 0.024). Conclusion: Stabilization of telomeres by telomerase activation occurs in half of the MTCs and by the ALT mechanism in a subset of cases. Telomerase activation may be used as an additional prognostic marker in MTC.
    The Journal of Clinical Endocrinology and Metabolism 04/2014; 99(8):jc20141158. DOI:10.1210/jc.2014-1158 · 6.31 Impact Factor
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    ABSTRACT: Pheochromocytomas are neuroendocrine tumors arising from the adrenal medulla. While heritable mutations are frequently described, less is known about the genetics of sporadic pheochromocytoma. Mutations in genes involved in the cellular hypoxia response have been identified in the tumors, and recently EPAS1, encoding HIF-2α, was revealed as a new gene involved in the pathogenesis of pheochromocytoma and abdominal paraganglioma. The aim of this study was to further characterize EPAS1 alterations in non-familial pheochromocytomas. Tumor DNA from 42 adrenal pheochromocytoma cases with apparently sporadic presentation, without known hereditary mutations in predisposing genes, were analyzed for mutations in EPAS1 by sequencing of exons 9 and 12, which contain the two hydroxylation sites involved in HIF-2α degradation, and also exon 2. In addition, copy number at the EPAS1 locus as well as transcriptome-wide gene expression was studied by DNA and RNA microarray analysis, respectively. We identified six missense EPAS1 mutations, three in exon 9 and three in exon 12, in five of 42 pheochromocytomas (12%). The mutations were both somatic and constitutional, and had no overlap to 11 cases (26%) with somatic mutations in NF1 or RET. One sample had two different EPAS1 mutations, shown by cloning to occur in cis, possibly suggesting a novel mechanism of HIF-2α stabilization through inactivation of both hydroxylation sites. One of the tumors with EPAS1 mutation also had DNA copy number gain at the EPAS1 locus. All EPAS1-mutated tumors displayed a pseudo-hypoxic gene expression pattern, suggestive of an oncogenic role of the identified mutations.
    Endocrine Related Cancer 04/2014; DOI:10.1530/ERC-13-0384 · 4.91 Impact Factor
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    ABSTRACT: Context: Pheochromocytomas and paragangliomas have a highly diverse genetic background, with a third of the cases carrying a germline mutation in one of 14 identified genes. Objective: This study aimed to evaluate next-generation sequencing for more efficient genetic testing of pheochromocytoma and paraganglioma and to establish germline and somatic mutation frequencies for all known susceptibility genes. Design: A targeted next-generation sequencing approach on an Illumina MiSeq instrument was used for mutation analysis in 86 unselected pheochromocytoma and paraganglioma tumor samples. The study included the genes EGLN1, EPAS1, KIF1Bβ, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127 and VHL. Results were verified in tumor and constitutional DNA with Sanger sequencing. Results: In all cases with clinical syndromes or known germline mutations, a mutation was detected in the expected gene. Among 68 non-familial tumors, 32 mutations were identified in 28 (41%) of the samples, including germline mutations in EGLN1, KIF1Bβ, SDHA, SDHB and TMEM127 and somatic mutations in EPAS1, KIF1Bβ, MAX, NF1, RET and VHL, including one double mono-allelic EPAS1 mutation. Conclusions: Targeted next-generation sequencing proved to be fast and cost-effective for genetic analysis of pheochromocytoma and paraganglioma. More than half of the tumors harboured mutations in the investigated genes. Notably, 7% of the apparently sporadic cases carried germline mutations, highlighting the importance of comprehensive genetic testing. KIF1Bβ, which previously has not been investigated in a large cohort, appears to be an equally important tumor suppressor as MAX and TMEM127, and could be considered for genetic testing of these patients.
    The Journal of Clinical Endocrinology and Metabolism 04/2014; 99(7):jc20134375. DOI:10.1210/jc.2013-4375 · 6.31 Impact Factor
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    ABSTRACT: The adrenocortical adenoma (ACA) entity includes aldosterone-producing (APA), cortisol-producing (CPA) and non-hyperfunctioning (NHFA) phenotypes. While gene mutations and mRNA expression profiles have been partly characterized, less is known about alterations involving microRNA (miRNA) expression. To characterize miRNA expression profile in relation to the subtypes of ACAs. miRNA expression profiles were determined in 26 ACAs (9 APAs, 10 CPAs, 7 NHFAs) and 4 adrenal references using microarray based screening. Significance analysis of microarrays (SAM) was performed to identify differentially expressed miRNAs between ACA and adrenal cortices, or between tumor subtypes. Selected identified differentially expressed miRNAs were validated in an extended series of 43 ACAs and 10 adrenal references by quantitative RT-PCR. Hierarchical clustering revealed separate clusters for APAs and CPAs, while the NHFAs were found spread out within the APA/CPA clusters. When NHFA was excluded, the clustering analysis showed a better separation between APA and CPA. SAM analysis identified 40 over-expressed and 3 under-expressed miRNAs in adenomas as compared to adrenal references. Fourteen miRNAs were common among the three ACA subtypes. Furthermore, we found specific miRNAs associated with different tumor phenotypes. The results suggest that miRNA expression profiles can distinguish different subtypes of ACA, which may contribute to a deeper understanding of ACA development and potential therapeutics.
    European Journal of Endocrinology 01/2014; DOI:10.1530/EJE-13-0817 · 3.69 Impact Factor
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    ABSTRACT: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm in the gastrointestinal tract. In most GISTs, the underlying mechanism is a gain-of-function mutation in the KIT or the PDGFRA gene. Imatinib is a tyrosine kinase inhibitor that specifically blocks the intracellular ATP-binding sites of these receptors. A correlation exists between plasma levels of imatinib and progression-free survival, but it is not known whether the plasma concentration correlates with the intracellular drug concentration. We determined intracellular imatinib levels in two GIST cell lines: the imatinib-sensitive GIST882 and the imatinib-resistant GIST48. After exposing the GIST cells to imatinib, the intracellular concentrations were evaluated using LC-MS (TOF). The concentration of imatinib in clinical samples from three patients was also determined to assess the validity and reliability of the method in the clinical setting. Determination of imatinib uptake fits within detection levels and values are highly reproducible. The GIST48 cells showed significantly lower imatinib uptake compared with GIST882 in therapeutic doses, indicating a possible difference in uptake mechanisms. Furthermore, imatinib accumulated in the tumor tissues and showed intratumoral regional differences. These data show, for the first time, a feasible and reproducible technique to measure intracellular imatinib levels in experimental and clinical settings. The difference in the intracellular imatinib concentration between the cell lines and clinical samples indicates that drug transporters may contribute toward resistance mechanisms in GIST cells. This highlights the importance of further clinical studies to quantify drug transporter expression and measure intracellular imatinib levels in GIST patients.
    Anti-cancer drugs 12/2013; 25(4). DOI:10.1097/CAD.0000000000000069 · 1.89 Impact Factor
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    ABSTRACT: Pheochromocytomas (PCCs) develop from the adrenal medulla and are often part of a hereditary syndrome such as von Hippel-Lindau (VHL) syndrome. In VHL, only about 30 % of patients with a VHL missense mutation develop PCCs. Thus, additional genetic events leading to formation of such tumors in patients with VHL syndrome are sought. SDHAF2 (previously termed SDH5) and SDHD are both located on chromosome 11q and are required for the function of mitochondrial complex II. While SDHAF2 has been shown to be mutated in patients with paragangliomas (PGLs), SDHD mutations have been found both in patients with PCCs and in patients with PGLs. Because loss of 11q is a common event in VHL-associated PCCs, we aimed to investigate whether SDHAF2 and SDHD are targets. In the present study, 41 VHL-associated PCCs were screened for mutations and loss of heterozygosity (LOH) in SDHAF2 or SDHD. Promoter methylation, as well as mRNA expression of SDHAF2 and SDHD, was studied. In addition, immunohistochemistry (IHC) of SDHB, known to be a universal marker for loss of any part the SDH complex, was conducted. LOH was found in more than 50 % of the VHL-associated PCCs, and was correlated with a significant decrease (p < 0.05) in both SDHAF2 and SDHD mRNA expression, which may be suggestive of a pathogenic role. However, while SDHB protein expression as determined by IHC in a small cohort of tumors was lower in PCCs than in the surrounding adrenal cortex, there was no obvious correlation with LOH or the level of SDHAF2/SDHD mRNA expression. In addition, the lack of mutations and promoter methylation in the investigated samples indicates that other events on chromosome 11 might be involved in the development of PCCs in association with VHL syndrome.
    World Journal of Surgery 12/2013; DOI:10.1007/s00268-013-2373-2 · 2.35 Impact Factor
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    ABSTRACT: Small intestinal neuroendocrine tumors (SI-NETs) are typically slow-growing tumors that have metastasized already at the time of diagnosis. The purpose of the present study was to further refine and define regions of recurrent copy number (CN) alterations (CNA) in SI-NETs. Genome-wide CNAs was determined by applying array CGH (a-CGH) on SI-NETs including 18 primary tumors and 12 metastases. Quantitative PCR analysis (qPCR) was used to confirm CNAs detected by a-CGH as well as to detect CNAs in an extended panel of SI-NETs. Unsupervised hierarchical clustering was used to detect tumor groups with similar patterns of chromosomal alterations based on recurrent regions of CN loss or gain. The log rank test was used to calculate overall survival. Mann--Whitney U test or Fisher's exact test were used to evaluate associations between tumor groups and recurrent CNAs or clinical parameters. The most frequent abnormality was loss of chromosome 18 observed in 70% of the cases. CN losses were also frequently found of chromosomes 11 (23%), 16 (20%), and 9 (20%), with regions of recurrent CN loss identified in 11q23.1-qter, 16q12.2-qter, 9pter-p13.2 and 9p13.1-11.2. Gains were most frequently detected in chromosomes 14 (43%), 20 (37%), 4 (27%), and 5 (23%) with recurrent regions of CN gain located to 14q11.2, 14q32.2-32.31, 20pter-p11.21, 20q11.1-11.21, 20q12-qter, 4 and 5. qPCR analysis confirmed most CNAs detected by a-CGH as well as revealed CNAs in an extended panel of SI-NETs. Unsupervised hierarchical clustering of recurrent regions of CNAs revealed two separate tumor groups and 5 chromosomal clusters. Loss of chromosomes 18, 16 and 11 and again of chromosome 20 were found in both tumor groups. Tumor group II was enriched for alterations in chromosome cluster-d, including gain of chromosomes 4, 5, 7, 14 and gain of 20 in chromosome cluster-b. Gain in 20pter-p11.21 was associated with short survival. Statistically significant differences were observed between primary tumors and metastases for loss of 16q and gain of 7. Our results revealed recurrent CNAs in several candidate regions with a potential role in SI-NET development. Distinct genetic alterations and pathways are involved in tumorigenesis of SI-NETs.
    BMC Cancer 10/2013; 13(1):505. DOI:10.1186/1471-2407-13-505 · 3.32 Impact Factor

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6k Citations
1,187.78 Total Impact Points

Institutions

  • 1991–2015
    • Karolinska Institutet
      • • Department of Oncology-Pathology
      • • Department of Molecular Medicine and Surgery
      • • Department of Cell and Molecular Biology
      • • Department of Clinical Genetics
      Solna, Stockholm, Sweden
  • 1988–2015
    • Karolinska University Hospital
      • • Cancer Center Karolinska (CCK)
      • • Department of Surgery
      • • Department of Clinical Genetics
      Tukholma, Stockholm, Sweden
  • 2012
    • City of Stockholm
      Tukholma, Stockholm, Sweden
  • 2004–2012
    • Laboratory for Molecular Infection Medicine Sweden
      Umeå, Västerbotten, Sweden
  • 2009
    • Tianjin Eye Hospital
      T’ien-ching-shih, Tianjin Shi, China
    • Johns Hopkins Bloomberg School of Public Health
      Baltimore, Maryland, United States
  • 2007
    • Uppsala University
      Uppsala, Uppsala, Sweden
  • 2003
    • Van Andel Research Institute
      Grand Rapids, Michigan, United States
  • 2002
    • University of Freiburg
      Freiburg, Baden-Württemberg, Germany
  • 2001
    • University of Tampere
      Tammerfors, Pirkanmaa, Finland
  • 1997
    • Princess Alexandra Hospital (Queensland Health)
      • Department of Urology
      Brisbane, Queensland, Australia
  • 1996
    • University of Gothenburg
      Goeteborg, Västra Götaland, Sweden
  • 1993
    • University of Antwerp
      Antwerpen, Flanders, Belgium
  • 1992
    • Baylor College of Medicine
      • Department of Pediatrics
      Houston, Texas, United States