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Westin, Gunnar
Publications (10 of 74) Show all publications
Barazeghi, E., Hellman, P., Westin, G. & Stålberg, P. (2019). PTPRM, a candidate tumor suppressor gene in small intestinal neuroendocrine tumors. Endocrine Connections, 8(8), 1126-1135
Open this publication in new window or tab >>PTPRM, a candidate tumor suppressor gene in small intestinal neuroendocrine tumors
2019 (English)In: Endocrine Connections, ISSN 2049-3614, E-ISSN 2049-3614, Vol. 8, no 8, p. 1126-1135Article in journal (Refereed) Published
Abstract [en]

Small intestinal neuroendocrine tumors (SI-NETs) are small, slow growing neoplasms with loss of one copy of chromosome 18 as a common event. Frequently mutated genes on chromosome 18 or elsewhere have not been found so far. The aim of this study was to investigate a possible tumor suppressor role of the transmembrane receptor type tyrosine phosphatase PTP mu (PTPRM at 18p11) in SI-NETs. Immunohistochemistry, quantitative RT-PCR, colony formation assay and quantitative CpG methylation analysis by pyrosequencing were performed. Undetectable/very low levels of PTPRM or aberrant pattern of immunostaining, with both negative and positive areas, were detected in the majority of tumors (33/40), and a significantly reduced mRNA expression in metastases compared to primary tumors was observed. Both the DNA methylation inhibitor 5-aza-2'deoxycytidine and the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) induced PTPRM expression in CNDT2.5 and KRJ-I SI-NET cells. CpG methylation of upstream regulatory regions, the promoter region and the exon 1/intron 1 boundary was detected by pyrosequencing analysis of the two cell lines and not in the analyzed SI-NETs. Overexpression of PTPRM in the SI-NET cell lines reduced cell growth and cell proliferation and induced apoptosis. The tyrosine phosphatase activity of PTPRM was not involved in cell growth inhibition. The results support a role for PTPRM as a dysregulated candidate tumor suppressor gene in SI-NETs and further analyses of the involved mechanisms are warranted.

Place, publisher, year, edition, pages
BIOSCIENTIFICA LTD, 2019
Keywords
DNA methylation, neuroendocrine tumors, epigenetic, SI-NETs, PTPRM
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-393899 (URN)10.1530/EC-19-0279 (DOI)000483142900007 ()31349215 (PubMedID)
Funder
Swedish Cancer SocietyErik, Karin och Gösta Selanders Foundation
Available from: 2019-10-18 Created: 2019-10-18 Last updated: 2019-10-18Bibliographically approved
Barazeghi, E., Prabhawa, S., Norlén, O., Hellman, P., Stålberg, P. & Westin, G. (2018). Decrease of 5-hydroxymethylcytosine and TET1 with nuclear exclusion of TET2 in small intestinal neuroendocrine tumors.. BMC Cancer, 18(1), Article ID 764.
Open this publication in new window or tab >>Decrease of 5-hydroxymethylcytosine and TET1 with nuclear exclusion of TET2 in small intestinal neuroendocrine tumors.
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2018 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 18, no 1, article id 764Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) originate from enterochromaffin cells scattered in the intestinal mucosa of the ileum and jejunum. Loss of one copy of chromosome 18 is the most frequent observed aberration in primary tumors and metastases. The aim of this study was to investigate possible involvement of 5-hydroxymethylcytosine (5hmC), TET1 and TET2 in SI-NETs.

METHODS: The analysis was conducted using 40 primary tumors and corresponding 47 metastases. The level of 5hmC, TET1 and TET2 was analyzed by DNA immune-dot blot assay and immunohistochemistry. Other methods included a colony forming assay, western blotting analysis, and quantitative bisulfite pyrosequencing analysis. The effect of the exportin-1 nuclear transport machinery inhibitors on cell proliferation and apoptosis was also explored using two SI-NET cell lines.

RESULTS: Variable levels of 5hmC and a mosaic staining appearance with a mixture of positive and negative cell nuclei, regardless of cell number and staining strength, was observed overall both in primary tumors and metastases. Similarly aberrant staining pattern was observed for TET1 and TET2. In a number of tumors (15/32) mosaic pattern together with areas of negative staining was also observed for TET1. Abolished expression of TET1 in the tumors did not seem to involve hypermethylation of the TET1 promoter region. Overexpression of TET1 in a colony forming assay supported a function as cell growth regulator. In contrast to 5hmC and TET1, TET2 was also observed in the cytoplasm of all the analyzed SI-NETs regardless of nuclear localization. Treatment of CNDT2.5 and KRJ-I cells with the exportin-1 (XPO1/CRM1) inhibitor, leptomycin B, induced reduction in the cytoplasm and nuclear retention of TET2. Aberrant partitioning of TET2 from the nucleus to the cytoplasm seemed therefore to involve the exportin-1 nuclear transport machinery. Reduced cell proliferation and induction of apoptosis were observed after treatment of CNDT2.5 and KRJ-I cells with leptomycin B or KPT-330 (selinexor).

CONCLUSIONS: SI-NETs are epigenetically dysregulated at the level of 5-hydroxymethylcytosine/ TET1/TET2. We suggest that KPT-330/selinexor or future developments should be considered and evaluated for single treatment of patients with SI-NET disease and also in combinations with somatostatin analogues, peptide receptor radiotherapy, or everolimus.

Keywords
5-hydroxymethylcytosine, Epigenetic, Neuroendocrine tumors, SI-NET, TET1, TET2
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-365219 (URN)10.1186/s12885-018-4579-z (DOI)000439700400002 ()30045709 (PubMedID)
Funder
Swedish Cancer SocietyErik, Karin och Gösta Selanders Foundation
Note

Peter Stålberg and Gunnar Westin contributed equally to this work

Available from: 2018-11-12 Created: 2018-11-12 Last updated: 2019-06-26Bibliographically approved
Barazeghi, E., Gill, A. J., Sidhu, S., Norlen, O., Dina, R., Palazzo, F. F., . . . Westin, G. (2017). A role for TET2 in parathyroid carcinoma. Endocrine-Related Cancer, 24(7), 329-338
Open this publication in new window or tab >>A role for TET2 in parathyroid carcinoma
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2017 (English)In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 24, no 7, p. 329-338Article in journal (Refereed) Published
Abstract [en]

Primary hyperparathyroidism (pHPT) is rarely caused by parathyroid carcinoma (PC, <1-5% of pHPT cases). The TET proteins oxidize the epigenetic mark 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and inactivation by mutation or epigenetic deregulation of TET1 and TET2 play important roles in various cancers. Recently, we found that 5hmC was severely reduced in all of the analyzed PCs and with deranged expression of TET1 for the majority of PCs. Here, we have examined the expression of the TET2 protein in 15 5hmC-negative PCs from patients who had local invasion or metastases. Cell growth and cell migratory roles for TET2 as well as epigenetic deregulated expression were addressed. Immunohistochemistry revealed very low/undetectable expression of TET2 in all PCs and verified for two PCs that were available for western blotting analysis. Knockdown of TET2 in the parathyroid cell line sHPT-1 resulted in increased cell growth and increased cell migration. DNA sequencing of TET2 in PCs revealed two common variants and no obvious inactivating mutations. Quantitative bisulfite pyrosequencing analysis of the TET2 promoter CpG island revealed higher CpG methylation level in the PCs compared to that in normal tissues and treatment of a PC primary cell culture with the DNA methylation inhibitor 5-aza-2'-deoxycytidine caused increased expression of the methylated TET2 gene. Hence, the data suggest that deregulated expression of TET2 by DNA hypermethylation may contribute to the aberrantly low level of 5hmC in PCs and further that TET2 plays a cell growth and cell migratory regulatory role and may constitute a parathyroid tumor suppressor gene.

Keywords
5-hydroxymethylcytosine, TET2, primary hyperparathyroidism, parathyroid carcinoma, promoter hypermethylation, tumor suppressor
National Category
Endocrinology and Diabetes Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-330022 (URN)10.1530/ERC-17-0009 (DOI)000404978400007 ()
Funder
Swedish Cancer Society
Available from: 2017-10-09 Created: 2017-10-09 Last updated: 2018-04-03Bibliographically approved
Barazeghi, E., Prabhawa, S., Hellman, P., Norlén, O., Stålberg, P. & Westin, G. (2017). A Role of TETs and 5-Hydroxymethylcytosine in SI-NETs. Paper presented at 14th Annual ENETS Conference for the Diagnosis and Treatment of Neuroendocrine Tumor Disease, MAR 08-10, 2017, Barcelona, SPAIN. Neuroendocrinology, 105, 18-18
Open this publication in new window or tab >>A Role of TETs and 5-Hydroxymethylcytosine in SI-NETs
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2017 (English)In: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 105, p. 18-18Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
KARGER, 2017
Keywords
si-net, 5-hydroxymethylcytosine, tets
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-346588 (URN)000413957900019 ()
Conference
14th Annual ENETS Conference for the Diagnosis and Treatment of Neuroendocrine Tumor Disease, MAR 08-10, 2017, Barcelona, SPAIN
Available from: 2018-03-20 Created: 2018-03-20 Last updated: 2018-03-20Bibliographically approved
Edfeldt, K., Daskalakis, K., Bäcklin, C., Norlén, O., Tiensuu Janson, E., Westin, G., . . . Stålberg, P. (2017). DcR3, TFF3 and Midkine are Novel Serum Biomarkers in Small Intestinal Neuroendocrine Tumors. Neuroendocrinology, 105(2), 170-181
Open this publication in new window or tab >>DcR3, TFF3 and Midkine are Novel Serum Biomarkers in Small Intestinal Neuroendocrine Tumors
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2017 (English)In: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 105, no 2, p. 170-181Article in journal (Refereed) Published
Abstract [en]

Small intestinal neuroendocrine tumors (SI-NETs) are amine- and peptide producing neoplasms. Most patients display metastases at the time of diagnosis, they have an unpredictable individual disease course and the tumors are often therapy resistant. Chromogranin A (CgA) and 5-hydroxyindoleacetic acid (5-HIAA) are the clinically most used biomarkers today, but there is a great need for novel diagnostic and prognostic biomarkers and new therapeutic targets. Sixty-nine biomarkers were screened in serum from 23 SI-NET patients and 23 healthy controls using multiplex PLA (proximity ligation assay). A refined method, PEA (proximity extension assay), was used to analyze 76 additional biomarkers. Statistical testing and multivariate classification were performed. Immunohistochemistry and ELISA assays were performed in an extended cohort. Using PLA, 19 biomarkers showed a significant difference in serum concentrations between patients and controls, and PEA revealed difference in concentrations in 13 proteins. Multivariate classification analysis revealed decoy receptor 3 (DcR3), trefoil factor 3 (TFF3) and Midkine to be good biomarkers for disease, which was confirmed by ELISA analysis. All three biomarkers were expressed in tumor tissue. DcR3 concentrations were elevated in patients with stage IV disease. High concentrations of DcR3 and TFF3 were correlated to poor survival. DcR3, TFF3 and Midkine exhibited elevated serum concentrations in SI-NET patients compared to healthy controls, and DcR3 and TFF3 were associated with poor survival. DcR3 seems to be a marker for liver metastases while TFF3 and Midkine may be new diagnostic biomarkers for SI-NETs.

National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-307769 (URN)10.1159/000452891 (DOI)000407672700008 ()27829249 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2016-11-21 Created: 2016-11-21 Last updated: 2018-02-20Bibliographically approved
Barazeghi, E., Gill, A. J., Sidhu, S., Norlen, O., Dina, R., Palazzo, F. F., . . . Westin, G. (2016). 5-Hydroxymethylcytosine discriminates between parathyroid adenoma and carcinoma. Clinical Epigenetics, 8, Article ID 31.
Open this publication in new window or tab >>5-Hydroxymethylcytosine discriminates between parathyroid adenoma and carcinoma
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2016 (English)In: Clinical Epigenetics, E-ISSN 1868-7083, Vol. 8, article id 31Article in journal (Refereed) Published
Abstract [en]

Background: Primary hyperparathyroidism is characterized by enlarged parathyroid glands due to an adenoma (80-85 %) or multiglandular disease (similar to 15 %) causing hypersecretion of parathyroid hormone (PTH) and generally hypercalcemia. Parathyroid cancer is rare (<1-5 %). The epigenetic mark 5-hydroxymethylcytosine (5hmC) is reduced in various cancers, and this may involve reduced expression of the ten-eleven translocation 1 (TET1) enzyme. Here, we have performed novel experiments to determine the 5hmC level and TET1 protein expression in 43 parathyroid adenomas (PAs) and 17 parathyroid carcinomas (PCs) from patients who had local invasion or metastases and to address a potential growth regulatory role of TET1. Results: The global 5hmC level was determined by a semi-quantitative DNA immune-dot blot assay in a smaller number of tumors. The global 5hmC level was reduced in nine PCs and 15 PAs compared to four normal tissue samples (p < 0.05), and it was most severely reduced in the PCs. By immunohistochemistry, all 17 PCs stained negatively for 5hmC and TET1 showed negative or variably heterogeneous staining for the majority. All 43 PAs displayed positive 5hmC staining, and a similar aberrant staining pattern of 5hmC and TET1 was seen in about half of the PAs. Western blotting analysis of two PCs and nine PAs showed variable TET1 protein expression levels. A significantly higher tumor weight was associated to PAs displaying a more severe aberrant staining pattern of 5hmC and TET1. Overexpression of TET1 in a colony forming assay inhibited parathyroid tumor cell growth. Conclusions: 5hmC can discriminate between PAs and PCs. Whether 5hmC represents a novel marker for malignancy warrants further analysis in additional parathyroid tumor cohorts. The results support a growth regulatory role of TET1 in parathyroid tissue.

Keywords
5-hydroxymethylcytosine, 5hmC, Parathyroid cancer, Primary hyperparathyroidism, TET1
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-282795 (URN)10.1186/s13148-016-0197-2 (DOI)000371782000002 ()26973719 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2016-04-14 Created: 2016-04-07 Last updated: 2017-11-30Bibliographically approved
Edfeldt, K., Hellman, P., Westin, G. & Stålberg, P. (2016). A plausible role for actin gamma smooth muscle 2 (ACTG2) in small intestinal neuroendocrine tumorigenesis. BMC Endocrine Disorders, 16(19)
Open this publication in new window or tab >>A plausible role for actin gamma smooth muscle 2 (ACTG2) in small intestinal neuroendocrine tumorigenesis
2016 (English)In: BMC Endocrine Disorders, ISSN 1472-6823, E-ISSN 1472-6823, Vol. 16, no 19Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) originate from the enterochromaffin cells in the ileum and jejunum. The knowledge about genetic and epigenetic abnormalities is limited. Low mRNA expression levels of actin gamma smooth muscle 2 (ACTG2) have been demonstrated in metastases relative to primary SI-NETs. ACTG2 and microRNA-145 (miR-145) are aberrantly expressed in other cancers and ACTG2 can be induced by miR-145. The aim of this study was to investigate the role of ACTG2 in small intestinal neuroendocrine tumorigenesis.

METHODS: Protein expression was analyzed in SI-NETs (n = 24) and in enterochromaffin cells by immunohistochemistry. The cell line CNDT2.5 was treated with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep), the selective EZH2 inhibitor EPZ-6438, or 5-aza-2'-deoxycytidine, a DNA hypomethylating agent. Cells were transfected with ACTG2 expression plasmid or miR-145. Western blotting analysis, quantitative RT-PCR, colony formation- and viability assays were performed. miR-145 expression levels were measured in tumors.

RESULTS: Eight primary tumors and two lymph node metastases displayed variable levels of positive staining. Fourteen SI-NETs and normal enterochromaffin cells stained negatively. Overexpression of ACTG2 significantly inhibited CNDT2.5 cell growth. Treatment with DZNep or transfection with miR-145 induced ACTG2 expression (>10-fold), but no effects were detected after treatment with EPZ-6438 or 5-aza-2'-deoxycytidine. DZNep also induced miR-145 expression. SI-NETs expressed relatively low levels of miR-145, with reduced expression in metastases compared to primary tumors.

CONCLUSIONS: ACTG2 is expressed in a fraction of SI-NETs, can inhibit cell growth in vitro, and is positively regulated by miR-145. Theoretical therapeutic strategies based on these results are discussed.

Keywords
SI NET; ACTG2; miR 145; Epigenetic regulation
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-287949 (URN)10.1186/s12902-016-0100-3 (DOI)000374739800001 ()27107594 (PubMedID)
Available from: 2016-04-27 Created: 2016-04-27 Last updated: 2017-11-30Bibliographically approved
Stålberg, P., Westin, G. & Thirlwell, C. (2016). Genetics and epigenetics in small intestinal neuroendocrine tumours. Journal of Internal Medicine, 280(6), 584-594
Open this publication in new window or tab >>Genetics and epigenetics in small intestinal neuroendocrine tumours
2016 (English)In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 280, no 6, p. 584-594Article in journal (Refereed) Published
Abstract [en]

Neuroendocrine tumour of the small intestine (SI-NET), formerly known as midgut carcinoid tumour, is the most common small intestinal malignancy. The incidence is rising, with recent reports of 0.67 per 100 000 in the USA and 1.12 per 100 000 in Sweden. SI-NETs often present a challenge in terms of diagnosis and treatment, as patients often have widespread disease and are beyond cure by surgery. Somatostatin analogues provide the mainstay of medical treatment to control hormonal excess and increase the time to progression. Despite overall favourable prognosis (5-year overall survival of 65%), there is a need to find markers to identify both patients with worse outcome and new targets for therapy. Loss on chromosome 18 has been reported in 60-90% of SI-NETs, but mutated genes on this chromosome have failed detection. Recently, a putative tumour suppressor role has been suggested for TCEB3C occurring at 18q21 (encoding elongin A3), which may undergo epigenetic repression. CDKN1B has recently been revealed as the only recurrently mutated gene in SI-NETs but, with a frequency as low as 8%, its role as a driver in SI-NET development may be questioned. Integrated genomewide analysis including exome and whole-genome sequencing, gene expression, DNA methylation and copy number analysis has identified three novel molecular subtypes of SI-NET with differing clinical outcome. DNA methylation analysis has demonstrated that SI-NETs have significant epigenetic dysregulation in 70-80% of tumours. In this review, we focus on understanding of the genetic, epigenetic and molecular events that lead to development and progression of SI-NETs.

Keywords
DNA methylation, epigenetics, genetics, midgut carcinoid, molecular profiling, small intestinal neuroendocrine tumour
National Category
Cancer and Oncology Surgery
Identifiers
urn:nbn:se:uu:diva-307770 (URN)10.1111/joim.12526 (DOI)000388573300006 ()27306880 (PubMedID)
Available from: 2016-11-21 Created: 2016-11-21 Last updated: 2017-11-29Bibliographically approved
Daskalakis, K., Edfeldt, K., Norlén, O., Karakatsanis, A., Backlin, C., Tiensuu Janson, E., . . . Stålberg, P. (2016). Midkine Is a New Novel Serum Biomarker in Small Intestinal Neuroendocrine Tumors (SI-NETs). Paper presented at 13th Annual ENETS Conference for the Diagnosis and Treatment of Neuroendocrine Tumor Disease, MAR 09-11, 2016, Barcelona, SPAIN. Neuroendocrinology, 103, 45-45
Open this publication in new window or tab >>Midkine Is a New Novel Serum Biomarker in Small Intestinal Neuroendocrine Tumors (SI-NETs)
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2016 (English)In: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 103, p. 45-45Article in journal (Refereed) Published
Keywords
Biomarker, SI-NET
National Category
Cancer and Oncology Endocrinology and Diabetes Neurology
Identifiers
urn:nbn:se:uu:diva-313702 (URN)000386481600119 ()
Conference
13th Annual ENETS Conference for the Diagnosis and Treatment of Neuroendocrine Tumor Disease, MAR 09-11, 2016, Barcelona, SPAIN
Available from: 2017-01-23 Created: 2017-01-23 Last updated: 2017-11-29Bibliographically approved
Westin, G. (2016). Molecular genetics and epigenetics of nonfamilial (sporadic) parathyroid tumours. Paper presented at Symposium on New Genetics with Impact on Treatment of Endocrine Tumour Disease, Uppsala, SWEDEN, JUN 04-05, 2015. Journal of Internal Medicine, 280(6), 551-558
Open this publication in new window or tab >>Molecular genetics and epigenetics of nonfamilial (sporadic) parathyroid tumours
2016 (English)In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 280, no 6, p. 551-558Article in journal, Meeting abstract (Refereed) Published
Abstract [en]

Primary hyperparathyroidism (pHPT) is a common endocrine disease characterized by excessive secretion of parathyroid hormone and an increased level of serum calcium. Overall, 80-85% of pHPT cases are due to a benign, single parathyroid adenoma (PA), and 15% to multiglandular disease (multiple adenomas/hyperplasia). Parathyroid carcinoma (PC) is rare, accounting for <0.5-1% of pHPT cases. Secondary hyperparathyroidism (sHPT) is a complication of renal failure, with the development of parathyroid tumours and hypercalcaemia. Recurrent mutations in the MEN1 gene have been confirmed by the whole-exome sequencing in 35% of PAs, suggesting that non-protein-coding genes, regulatory elements or epigenetic derangements may also have roles in the majority of PAs. DNA translocations with cyclin D1 overexpression occur in PAs (8%). In PCs, mutations in CDC73/HRPT2 are common. Activation of the WNT/β-catenin signalling pathway (accumulation of nonphosphorylated β-catenin) by an aberrantly truncated LRP5 receptor has been seen for the majority of investigated PAs and sHPT tumours, and possibly by APC inactivation through promoter methylation in PCs. Promoter methylation of several other genes and repressive histone H3 lysine 27 trimethylation by EZH2 of the HIC1 gene may also contribute to parathyroid tumorigenesis. It is possible that a common pathway exists for parathyroid tumour development. CCND1 (cyclin D1) and EZH2 overexpression, accumulation of nonphosphorylated β-catenin and repression of HIC1 have all been observed to occur in PAs, PCs and sHPT tumours. In addition, hypermethylation has been observed for the same genes in PAs and PCs (e.g. SFRP1, CDKN2A and WT1). Whether β-catenin represents a 'hub' in parathyroid tumour development will be discussed.

Keywords
adenoma, beta-catenin, cancer, H3K27me3, hypermethylation and hyperparathyroidism
National Category
Cancer and Oncology Surgery
Identifiers
urn:nbn:se:uu:diva-307772 (URN)10.1111/joim.12458 (DOI)000388573300003 ()27071708 (PubMedID)
Conference
Symposium on New Genetics with Impact on Treatment of Endocrine Tumour Disease, Uppsala, SWEDEN, JUN 04-05, 2015
Available from: 2016-11-21 Created: 2016-11-21 Last updated: 2017-11-29Bibliographically approved
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