Noninvasive detection of any-stage cancer using free glycosaminoglycans.

Cancer mortality is exacerbated by late-stage diagnosis. Liquid biopsies based on genomic biomarkers can noninvasively diagnose cancers. However, validation studies have reported ~10% sensitivity to detect stage I cancer in a screening population and specific types, such as brain or genitourinary tumors, remain undetectable. We investigated urine and plasma free glycosaminoglycan profiles (GAGomes) as tumor metabolism biomarkers for multi-cancer early detection (MCED) of 14 cancer types using 2,064 samples from 1,260 cancer or healthy subjects. We observed widespread cancer-specific changes in biofluidic GAGomes recapitulated in an in vivo cancer progression model. We developed three machine learning models based on urine (Nurine = 220 cancer vs. 360 healthy) and plasma (Nplasma = 517 vs. 425) GAGomes that can detect any cancer with an area under the receiver operating characteristic curve of 0.83-0.93 with up to 62% sensitivity to stage I disease at 95% specificity. Undetected patients had a 39 to 50% lower risk of death. GAGomes predicted the putative cancer location with 89% accuracy. In a validation study on a screening-like population requiring ≥ 99% specificity, combined GAGomes predicted any cancer type with poor prognosis within 18 months with 43% sensitivity (21% in stage I; N = 121 and 49 cases). Overall, GAGomes appeared to be powerful MCED metabolic biomarkers, potentially doubling the number of stage I cancers detectable using genomic biomarkers.

Surgery for advanced neuroendocrine tumours of the small bowel: recommendations based on a consensus meeting of the European Society of Endocrine Surgeons (ESES).

BACKGROUND: Small bowel neuroendocrine tumours often present with locally advanced or metastatic disease. The aim of this paper is to provide evidence-based recommendations regarding (controversial) topics in the surgical management of advanced small bowel neuroendocrine tumours. METHODS: A working group of experts was formed by the European Society of Endocrine Surgeons. The group addressed 11 clinically relevant questions regarding surgery for advanced disease, including the benefit of primary tumour resection, the role of cytoreduction, the extent of lymph node clearance, and the management of an unknown primary tumour. A systematic literature search was performed in MEDLINE to identify papers addressing the research questions. Final recommendations were presented and voted upon by European Society of Endocrine Surgeons members at the European Society of Endocrine Surgeons Conference in Mainz in 2023. RESULTS: The literature review yielded 1223 papers, of which 84 were included. There were no randomized controlled trials to address any of the research questions and therefore conclusions were based on the available case series, cohort studies, and systematic reviews/meta-analyses of the available non-randomized studies. The proposed recommendations were scored by 38-51 members and rated 'strongly agree' or 'agree' by 64-96% of participants. CONCLUSION: This paper provides recommendations based on the best available evidence and expert opinion on the surgical management of locally advanced and metastatic small bowel neuroendocrine tumours.

Overall Survival in Patients with Stage IV Pan-NET Eligible for Liver Transplantation.

BACKGROUND: The use of liver transplantation (LT) in patients with stage IV neuroendocrine pancreatic tumors (pan-NET) is under debate. Previous studies report a 5-year survival of 27-53% after LT in pan-NET and up to 92.7% in patients with mixed NETs. This study aimed to determine survival rates of patients with stage IV pan-NET meeting criteria for LT while only subjected to multimodal treatment. METHODS: Medical records of patients with pan-NET diagnosed from 2000 to 2021 at a tertiary referral center were evaluated for eligibility. Patients without liver metastases, who did not undergo primary tumor surgery, age > 75 years and with grade 3 tumors were excluded. The patients were divided into groups; all included patients, patients meeting the Milan, the United Network for Organ Sharing (UNOS) or the European Neuroendocrine Tumor Society (ENETS) criteria for LT. Kaplan-Meier survival analysis was used to calculate overall survival. RESULTS: Out of 519 patients with pan-NET, 41 patients were included. Mean follow-up time was 5.4 years. Overall survival was 9.3 years (95% Cl 6.8-11.7), and 5-year survival was 64.7% (95% CI 48.2-81.2). Patients meeting the Milan, ENETS and UNOS criteria for LT had a 5-year survival of 64.9% (95% CI 32.2-97.6), 85.7% (95% CI 59.8-100.0) and 55.4% (95% CI 26.0-84.8), respectively. CONCLUSIONS: In patients with stage IV pan-NET, grade 1 and 2, with no extra abdominal disease, 5-year survival was 64.7% (95% CI 48.2-81.2). As these survival rates exceed previously published series of LT for pan-NET, the evidence base for this treatment is very weak.

Risk of Permanent Hypoparathyroidism After Total Thyroidectomy for Benign Disease: A Nationwide Population-based Cohort Study From Sweden.

OBJECTIVE: To investigate the prevalence and risk factors for permanent hypoparathyroidism after total thyroidectomy for benign disease in a population-based setting with data independent of input of complication data. SUMMARY OF BACKGROUND DATA: The reported rate of permanent hypoparathyroidism is highly variable and mostly rely on reported complication data from national or institutional registries. METHODS: All patients who underwent total thyroidectomy in Sweden from 2005 to 2015 were identified through Scandinavian Quality Register for Thyroid, Parathyroid and Adrenal Surgery and the Swedish National Patient Register. Patients were matched to outcome data from the Swedish Prescribed Drug Register. Permanent hypoparathyroidism was defined as treatment with calcium and/or active vitamin D more than 1 year after surgery. RESULTS: Seven thousand eight hundred fifty-two patients were included and 938 (12.5%) developed permanent hypoparathyroidism. The risk was lower in patients registered in the quality register (11.0% vs 16%, P < 0.001). In a multivariable analysis there was a higher risk of permanent hypoparathyroidism in patients with parathyroid autotransplantation [Odds ratio (OR) 1.72; 95% confidence interval 1.47-2.01], center-volume <100 thyroidectomies per year (OR 1.22; 1.03-1.44), age above 60 year (OR 1.64; 1.36-1.98) and female sex (OR 1.27; 1.05-1.54). Reported data from the quality register only identified 178 of all 938 patients with permanent hypoparathyroidism. CONCLUSION: The risk of permanent hypoparathyroidism after total thyroidectomy was high and associated with parathyroid autotransplantation, higher age, female sex and surgery at a low volume center. Reported follow-up data might underestimate the rate of permanent hypoparathyroidism.

Nordic guidelines 2021 for diagnosis and treatment of gastroenteropancreatic neuroendocrine neoplasms.

BACKGROUND: The diagnostic work-up and treatment of patients with gastroenteropancreatic (GEP) neuroendocrine neoplasms (NEN) has undergone major advances and new methods are introduced. Furthermore, an update of the WHO classification has resulted in a new nomenclature for GEP-NEN that is implemented in the clinic. AIM: These Nordic guidelines summarise the Nordic Neuroendocrine Tumour Group's current view on how to diagnose and treat GEP-NEN patients and aims to be useful in the daily practice for clinicians.

Whole-genome sequencing of synchronous thyroid carcinomas identifies aberrant DNA repair in thyroid cancer dedifferentiation.

The genetics underlying thyroid cancer dedifferentiation is only partly understood and has not yet been characterised using comprehensive pan-genomic analyses. We investigated a unique case with synchronous follicular thyroid carcinoma (FTC), poorly differentiated thyroid carcinoma (PDTC), and anaplastic thyroid carcinoma (ATC), as well as regional lymph node metastases from the PDTC and ATC from a single patient using whole-genome sequencing (WGS). The FTC displayed mutations in CALR, RB1, and MSH2, and the PDTC exhibited mutations in TP53, DROSHA, APC, TERT, and additional DNA repair genes - associated with an immense increase in sub-clonal somatic mutations. All components displayed an overrepresentation of C>T transitions with associated microsatellite instability (MSI) in the PDTC and ATC, with borderline MSI in the FTC. Clonality analyses pinpointed a shared ancestral clone enriched for mutations in TP53-associated regulation of DNA repair and identified important sub-clones for each tumour component already present in the corresponding preceding lesion. This genomic characterisation of the natural progression of thyroid cancer reveals several novel genes of interest for future studies. Moreover, the findings support the theory of a stepwise dedifferentiation process and suggest that defects in DNA repair could play an important role in the clonal evolution of thyroid cancer. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Characterization of Glycosphingolipids in the Human Parathyroid and Thyroid Glands.

As part of a systematic investigation of the glycosphingolipids in human tissues, acid and non-acid glycosphingolipids from human thyroid and parathyroid glands were isolated and characterized with mass spectrometry and binding of carbohydrate-recognizing ligands, with a focus on complex compounds. The glycosphingolipid patterns of the human parathyroid and thyroid glands were very similar. The major acid glycosphingolipids were sulfatide and the gangliosides GM3, GD3, GD1a, GD1b, GT1b and Neu5Ac-neolactotetraosylceramide, and the major non-acid glycosphingolipids were globotriaosylceramide and globoside. We also found neolactotetra- and neolactohexaosylceramide, the x2 glycosphingolipid, and complex glycosphingolipids with terminal blood group O and A determinants in both tissues. A glycosphingolipid with blood group Leb determinant was identified in the thyroid gland, and the parathyroid sample had a glycosphingolipid with terminal blood group B determinant. Immunohistochemistry demonstrated the expression of blood group A antigens in both the thyroid and parathyroid glands. A weak cytoplasmatic expression of the GD1a ganglioside was present in the thyroid, while the parathyroid gland had a strong GD1a expression on the cell surface. Thus, the glycosylation of human thyroid and parathyroid glands is more complex than previously appreciated. Our findings provide a platform for further studies of alterations of cell surface glycosphingolipids in thyroid and parathyroid cancers.

Decrease of 5-hydroxymethylcytosine and TET1 with nuclear exclusion of TET2 in small intestinal neuroendocrine tumors.

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.

U-CAN: a prospective longitudinal collection of biomaterials and clinical information from adult cancer patients in Sweden.

BACKGROUND: Progress in cancer biomarker discovery is dependent on access to high-quality biological materials and high-resolution clinical data from the same cases. To overcome current limitations, a systematic prospective longitudinal sampling of multidisciplinary clinical data, blood and tissue from cancer patients was therefore initiated in 2010 by Uppsala and Umeå Universities and involving their corresponding University Hospitals, which are referral centers for one third of the Swedish population. MATERIAL AND METHODS: Patients with cancer of selected types who are treated at one of the participating hospitals are eligible for inclusion. The healthcare-integrated sampling scheme encompasses clinical data, questionnaires, blood, fresh frozen and formalin-fixed paraffin-embedded tissue specimens, diagnostic slides and radiology bioimaging data. RESULTS: In this ongoing effort, 12,265 patients with brain tumors, breast cancers, colorectal cancers, gynecological cancers, hematological malignancies, lung cancers, neuroendocrine tumors or prostate cancers have been included until the end of 2016. From the 6914 patients included during the first five years, 98% were sampled for blood at diagnosis, 83% had paraffin-embedded and 58% had fresh frozen tissues collected. For Uppsala County, 55% of all cancer patients were included in the cohort. CONCLUSIONS: Close collaboration between participating hospitals and universities enabled prospective, longitudinal biobanking of blood and tissues and collection of multidisciplinary clinical data from cancer patients in the U-CAN cohort. Here, we summarize the first five years of operations, present U-CAN as a highly valuable cohort that will contribute to enhanced cancer research and describe the procedures to access samples and data.

Preoperative 68Ga-DOTA-Somatostatin Analog-PET/CT Hybrid Imaging Increases Detection Rate of Intra-abdominal Small Intestinal Neuroendocrine Tumor Lesions.

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) are the most common form of neoplasm in the small bowel. Radiological identification of primary tumors (PT), which may be multiple, is difficult, and therefore palpation of the entire small bowel is routinely performed during laparotomy. The aim was to determine detection rates of PT and peritoneal carcinomatosis (PC) with 68Ga-DOTATOC/TATE-PET/CT in comparison with i.v. contrast-enhanced computed tomography (CE-CT) and thus to clarify whether modern functional imaging can mitigate the need for palpation of bowel during surgery enabling oncologically adequate laparoscopic resection. METHODS: A total of 28 patients with SI-NET who preoperatively underwent both 68Ga-DOTATOC/TATE-PET/CT and CE-CT were included. The detection rates of PT and PC for PET/CT and CE-CT were compared to the findings in the surgical and histopathological reports. Appropriate statistical tests were used, and significance was set to p < 0.05. RESULTS: Out of 82 PT, 43 PT were not detected by any imaging modality. More PT lesions were detected with PET/CT (n = 39 [47.5%]) than with CE-CT (n = 10 [12.2%], p < 0.001). Also, PET/CT identified significantly more PC lesions than CE-CT (78 and 38%, p = 0.004, respectively). CONCLUSION: PET/CT detected more PT and PC lesions than CE-CT. Some PTs and PC lesions were only detected by one of the modalities, and CT performed in conjunction with PET/CT should therefore be performed as a fully diagnostic CE-CT for optimal results. Palpation of the small bowel remains crucial during surgery in these patients because several PTs escaped detection by both PET/CT and CE-CT.

Neomorphic effects of recurrent somatic mutations in Yin Yang 1 in insulin-producing adenomas.

Insulinomas are pancreatic islet tumors that inappropriately secrete insulin, producing hypoglycemia. Exome and targeted sequencing revealed that 14 of 43 insulinomas harbored the identical somatic mutation in the DNA-binding zinc finger of the transcription factor Yin Yang 1 (YY1). Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that this T372R substitution changes the DNA motif bound by YY1. Global analysis of gene expression demonstrated distinct clustering of tumors with and without YY1(T372R) mutations. Genes showing large increases in expression in YY1(T372R) tumors included ADCY1 (an adenylyl cyclase) and CACNA2D2 (a Ca(2+) channel); both are expressed at very low levels in normal ß-cells and show mutation-specific YY1 binding sites. Both gene products are involved in key pathways regulating insulin secretion. Expression of these genes in rat INS-1 cells demonstrated markedly increased insulin secretion. These findings indicate that YY1(T372R) mutations are neomorphic, resulting in constitutive activation of cAMP and Ca(2+) signaling pathways involved in insulin secretion.

DcR3, TFF3, and Midkine Are Novel Serum Biomarkers in Small Intestinal Neuroendocrine Tumors.

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 and 5-hydroxyindoleacetic acid are the biomarkers clinically used most often 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 the multiplex proximity ligation assay (PLA). A refined method, the proximity extension assay (PEA), was used to analyze 76 additional biomarkers. Statistical testing and multivariate classification were performed. Immunohistochemistry and ELISA were performed in an extended cohort. Using PLA, 19 biomarkers showed a significant difference in serum concentrations between patients and controls, and PEA revealed a difference in the concentrations of 17 proteins. Multivariate classification analysis revealed decoy receptor 3 (DcR3), trefoil factor 3 (TFF3), and midkine to be good biomarkers for the disease, which was confirmed by ELISA analysis. All 3 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.

A plausible role for actin gamma smooth muscle 2 (ACTG2) in small intestinal neuroendocrine tumorigenesis.

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.

An Integrative Genomic Analysis of Formalin Fixed Paraffin-Embedded Archived Serous Ovarian Carcinoma Comparing Long-term and Short-term Survivors.

OBJECTIVE: This study aimed to perform an integrative genetic analysis of patients with matched serous ovarian cancer having long-term or short-term survival using formalin fixed paraffin-embedded (FFPE) tissue samples. METHODS: All patients with serous ovarian carcinoma who underwent surgery between 1998 and 2007 at the Department of Gynaecology, Uppsala University Hospital, Sweden were considered. From this cohort, we selected biomaterial from 2 groups of patients with long-term and short-term survival matched for age, stage, histologic grade, and outcome of surgery. Genomic DNA from FFPE sample was analyzed with SNP array and targeted next-generation sequencing of 26 genes. RESULTS: Forty-three samples (primary tumors and metastases) from 23 patients were selected for genomic profiling, the survival in the subgroups were 134 and 36 months, respectively. We observed a tendency toward increased genomic instability in those with long-term survival with higher proportion of somatic copy number alterations (P = 0.083) and higher average ploidy (P = 0.037). TP53 mutations were found in 50% of the patients. Frequency of TP53 mutations did not differ between the survival groups (P = 0.629). CONCLUSIONS: We validated both previous genomic findings in ovarian cancer and the proposed association between increased genomic instability and better survival. These results exemplify that analysis of genomic biomarkers is feasible on archived FFPE tissue.

Long-Term Surveillance of Treated Hyperparathyroidism for Multiple Endocrine Neoplasia Type 1: Recurrence or Hypoparathyroidism?

BACKGROUND: Primary hyperparathyroidism (HPT) in multiple endocrine neoplasia type 1 (MEN1) is surgically treated with either a subtotal parathyroidectomy removing 3 or 3,5 glands (SPX), less than 3 glands (LSPX), or a total parathyroidectomy with autotransplantation (TPX). Previous studies with shorter follow-up have shown that LSPX and SPX are associated with recurrent HPT, and TPX with hypocalcemia and substitution therapy. We examined the situation after long-term follow-up (median 20,6 years). METHODS: Sixty-nine patients with MEN1 HPT underwent 110 operations, the first operation being 31 LSPX, 30 SPX, and 8 TPX. Thirty patients underwent reoperative surgery in median 120 months later, as completion to TPX (n = 12), completion of LSPX to SPX (n = 9), extirpation of single glands (n = 3) still resulting in LSPX, and resection of forearm grafts (n = 3). Nine patients underwent a second, and 2 a third reoperation. In 24 patients genetic testing confirmed MEN1, and in the remaining heredity and phenotype led to the diagnosis. RESULTS: TPX had higher risk for hypoparathyroidism necessitating substitution therapy, at latest follow-up 50%, compared to SPX (16% after 3-6 months; none at latest follow-up). Recurrent HPT was common after LSPX, leading to 24 reoperations in 17 patients. No need for substitution therapy after SPX indicated forthcoming recurrent disease. Not having hypocalcemia in the postoperative period and less radical surgery than TPX were significantly associated to risk for recurrence. Further, mutation in exon 3 in the MEN1 gene may eventually be linked to risk of recurrence. CONCLUSION: LSPX is highly associated with recurrence and TPX with continuous hypoparathyroidism, also after long-term follow-up. SPX should be the chosen method in the majority of patients with MEN1 HPT.