Hurthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome ... Show moreHurthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome (NHG), a phenomenon recently attributed to reactive oxygen species (ROS) generated during mitosis by malfunctioning mitochondria. We studied shared metabolic traits during standard and glucose-depleted cell culture in thyroid cancer cell lines (TCCLs), with or without a NHG, using quantitative analysis of extra and intracellular metabolites and ROS production following inhibition of complex III with antimycin A. We found that the XTC.UC1 and FTC-236 cell lines (both NHG) are functionally impaired in complex I and produce significantly more superoxide radicals than SW579 and BHP 2-7 (non-NHG) after challenge with antimycin A. FTC-236 showed the lowest levels of glutathione and SOD2. XTC.UC1 and FTC-236 both exhibited reduced glycolytic activity and utilization of alternative sources to meet energy demands. Both cell lines also shared low levels of alpha-ketoglutarate and high levels of creatine, phosphocreatine, uridine diphosphate-N-acetylglucosamine, pyruvate and acetylcarnitine. Furthermore, the metabolism of XTC.UC1 was skewed towards the de novo synthesis of aspartate, an effect that persisted even in glucose-free media, pointing to reductive carboxylation. Our data suggests that metabolic reprogramming and a subtle balance between ROS generation and scavenging/conversion of intermediates may be involved in ROS-induced w-CIN in HCC and possibly also in rare cases of follicular thyroid cancer showing a NHG. Show less
Schubert, S.A.; Morreau, H.; Miranda, N.F.C.C. de; Wezel, T. van 2020
Pinpointing heritability factors is fundamental for the prevention and early detection of cancer. Up to one-quarter of colorectal cancers (CRCs) occur in the context of familial aggregation of this... Show morePinpointing heritability factors is fundamental for the prevention and early detection of cancer. Up to one-quarter of colorectal cancers (CRCs) occur in the context of familial aggregation of this disease, suggesting a strong genetic component. Currently, only less than half of the heritability of CRC can be attributed to hereditary syndromes or common risk loci. Part of the missing heritability of this disease may be explained by the inheritance of elusive high-risk variants, polygenic inheritance, somatic mosaicism, as well as shared environmental factors, among others. A great deal of the missing heritability in CRC is expected to be addressed in the coming years with the increased application of cutting-edge next-generation sequencing technologies, routine multigene panel testing and tumour-focussed germline predisposition screening approaches. On the other hand, it will be important to define the contribution of environmental factors to familial aggregation of CRC incidence. This review provides an overview of the known genetic causes of familial CRC and aims at providing clues that explain the missing heritability of this disease. Show less
Objectives: Targeted therapies in the management of patients with lung cancer provide significantly better outcome compared to chemotherapy. Detection of the anaplastic lymphoma kinase (ALK) gene... Show moreObjectives: Targeted therapies in the management of patients with lung cancer provide significantly better outcome compared to chemotherapy. Detection of the anaplastic lymphoma kinase (ALK) gene rearrangement has great predictive value for treatment with small molecule tyrosine kinase inhibitor (crizotinib and alectinib commonly). Fluorescent in situ hybridisation (FISH) assay is a basic diagnostic test designed for detecting ALK gene rearrangements. Although being considered as gold standard method by IASLC's guideline, it is often regarded as difficult and error prone. Our aim was to examine a unique atypical ALK FISH pattern, revealed during a systematic large-scale monitoring, which carries the great risk of misinterpretation, hence may result in loss of patients eligible for targeted therapy.Materials and Methods: Tissue and cytology samples from nearly one thousand patients with advanced stage non-small cell lung cancer (NSCLC, n = 996) were routinely examined by ALK FISH and immunohistochemistry (Ventana ALK-D5F3-CDx assay). Anchored Multiplex PCR based Next Generation Sequencing (AMP-NGS) was used to detect fusion gene transcripts in ambiguous cases.Results: Fifty-nine (5,9%) of the cases were positive with ALK FISH test. Three cases showed atypical pattern with a significantly reduced sized red (3') signal and complete loss of green signals. Digital signal measurement confirmed this finding, showing consistent attenuation of 3' signals throughout the tumours. In all three cases AMP-NGS and ALK IHC verified the presence of a fusion gene and expressed oncoprotein, respectively.Conclusion: Approximately 5% of the 59 ALK positive cases exhibited atypical attenuated isolated 3' signal pattern. The immunohistochemistry and AMP-NGS examinations helped to clarify the presence of oncoprotein and the fusion gene, respectively. Our results emphasize the importance of extensive exploration of the genetic background of any unexpected FISH finding to avoid false diagnosis. This enables clinicians to indicate the adequate therapy with higher efficiency for patients suffering from NSCLC. Show less
Jansen, A.M.L.; Tops, C.M.J.; Ruano, D.; Eijk, R. van; Wijnen, J.T.; Broeke, S. ten; ... ; Morreau, H. 2020
Germline variants in the DNA mismatch repair (MMR) gene PMS2 cause 1-14% of all Lynch Syndrome cancers. Correct variant analysis of PMS2 is complex due to the presence of multiple pseudogenes and... Show moreGermline variants in the DNA mismatch repair (MMR) gene PMS2 cause 1-14% of all Lynch Syndrome cancers. Correct variant analysis of PMS2 is complex due to the presence of multiple pseudogenes and the occurrence of gene conversion. The analysis complexity increases in highly fragmented DNA from formalin-fixed paraffin-embedded (FFPE) tissue. Here we describe a reliable approach to detect true PMS2 variants in fragmented DNA. A custom NGS panel designed for FFPE tissue was used targeting four MMR genes, POLE and POLD1. Amplicon design for PMS2 was based on the position of paralogous sequence variants (PSVs) that distinguish PMS2 from its pseudogenes. PMS2 variants in exons 1-11 can be correctly curated based on this information. For exons 12-15 this is less reliable as these undergo gene conversion. Using this method, we screened PMS2 variants in 125 MMR-deficient tumors. Of the 125 tumors tested, six were unexplained MMR-deficient tumors with solitary PMS2 protein expression loss. In these six tumors two unclassified variants (class 3) and five variants likely affecting function (class 4/5) were detected in PMS2. One microsatellite unstable tumor with positive staining for all MMR proteins was found to carry a frameshift PMS2 variant (class 5). No class 4 or class 5 PMS2 variants were detected in tumors with other patterns of MMR protein expression loss. Show less
Terlouw, D.; Suerink, M.; Singh, S.S.; Gille, H.J.J.P.; Hes, F.J.; Langers, A.M.J.; ... ; Nielsen, M. 2020
This study aimed to determine the prevalence of APC-associated familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) in a large cohort, taking into account factors as adenoma... Show moreThis study aimed to determine the prevalence of APC-associated familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) in a large cohort, taking into account factors as adenoma count and year of diagnosis. All application forms used to send patients in for APC and MUTYH variant analysis between 1992 and 2017 were collected (n = 2082). Using the data provided on the application form, the APC and biallelic MUTYH prevalence was determined and possible predictive factors were examined using multivariate multinomial logistic regression analysis in SPSS. The prevalence of disease causing variants in the APC gene significantly increases with adenoma count while MAP shows a peak prevalence in individuals with 50-99 adenomas. Logistic regression analysis shows significant odds ratios for adenoma count, age at diagnosis, and, interestingly, a decline in the chance of finding a variant in either gene over time. Moreover, in 22% (43/200) of patients with FAP-related extracolonic manifestations a variant was identified. The overall detection rates are above 10% for patients with >10 adenomas aged 20 adenomas aged <70. Patients with variants outside these criteria had FAP-related extracolonic manifestations, colorectal cancer aged <40, somatic KRAS c.34G > T variant in the tumor or a first-degree relative with >10 adenomas. Therefore, APC and MUTYH testing in patients with >10 adenomas aged 20 adenomas aged <70 is advised. Almost all FAP and MAP patients not meeting these criteria showed other characteristics that can be used as an indication to prompt genetic testing. Show less
Aydemirli, M.D.; Tuin, K. van der; Hes, F.J.; Ouweland, A.M.W. van den; Wezel, T. van; Kapiteijn, E.; Morreau, H. 2020
We report a case of a 22-year-old female patient who was diagnosed with a cribriform-morular variant of papillary thyroid carcinoma (CMV-PTC). While at early ages this thyroid cancer variant is... Show moreWe report a case of a 22-year-old female patient who was diagnosed with a cribriform-morular variant of papillary thyroid carcinoma (CMV-PTC). While at early ages this thyroid cancer variant is highly suggestive for familial adenomatous polyposis (FAP), there was no family history of FAP. In the tumor biallelic, inactivating APC variants were identified. The patient tested negative for germline variants based on analysis of genomic DNA from peripheral blood leukocytes. Somatic mosaicism was excluded by subsequent deep sequencing of leukocyte and normal thyroid DNA using next generation sequencing (NGS). This report presents a rare sporadic case of CMV-PTC, and to the best of our knowledge the first featuring two somatic APC mutations underlying the disease, with an overview of CMV-PTC cases with detected APC and CTNNB1 pathogenic variants from the literature. Show less
PARAT, a new European Society of Endocrinology program, aims to identify unmet scientific and educational needs of parathyroid disorders, such as primary hyperparathyroidism (PHPT), including... Show morePARAT, a new European Society of Endocrinology program, aims to identify unmet scientific and educational needs of parathyroid disorders, such as primary hyperparathyroidism (PHPT), including parathyroid cancer (PC), and hypoparathyroidism (HypoPT). The discussions and consensus statements from the first PARAT workshop (September 2018) are reviewed. PHPT has a high prevalence in Western communities, yet evidence is sparse concerning the natural history and whether morbidity and long-term outcomes are related to hypercalcemia or plasma PTH concentrations or both. Cardiovascular mortality and prevalence of low energy fractures are increased, whereas quality of life is decreased, although their reversibility by treatment of PHPT has not been convincingly demonstrated. PC is a rare cause of PHPT, with increasing incidence, and international collaborative studies are required to advance knowledge of the genetic mechanisms, biomarkers for disease activity and optimal treatments. For example, similar to 20% of PCs demonstrate high mutational burden, and identifying targetable DNA variations, gene amplifications and gene fusions may facilitate personalized care, such as different forms of immunotherapy or targeted therapy. HypoPT, a designated orphan disease, is associated with a high risk of symptoms and complications. Most cases are secondary to neck surgery. However, there is a need to better understand the relation between disease biomarkers and intellectual function and to establish the role of PTH in target tissues, as these may facilitate the appropriate use of PTH substitution therapy. Management of parathyroid disorders is challenging, and PARAT has highlighted the need for international transdisciplinary scientific and educational studies in advancing in this field. Show less
Aydemirli, M.D.; Corver, W.; Beuk, R.; Roepman, P.; Solleveld-Westerink, N.; Wezel, T. van; ... ; Morreau, H. 2019
Objective: To evaluate the efficacy and treatment rationale of Hürthle cell carcinoma (HCC) following a patient with progressive and metastatic HCC. HCC was recently shown to harbor a distinct... Show moreObjective: To evaluate the efficacy and treatment rationale of Hürthle cell carcinoma (HCC) following a patient with progressive and metastatic HCC. HCC was recently shown to harbor a distinct genetic make-up and the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kiase (PI3K)/AKT signaling pathways are potential targets for anti-cancer agents in the management of recurrent HCC. The presence or absence of gene variants can give a rationale for targeted therapies that could be made available in the context of drug repurposing trials. Methods: Treatment included everolimus, sorafenib, nintedanib, lenvatinib, and panitumumab. Whole genome sequencing (WGS) of metastatic tumor material obtained before administration of the last drug, was performed. We subsequently evaluated the rationale and efficacy of panitumumab in thyroid cancer and control cell lines after epidermal growth factor (EGF) stimulation and treatment with panitumumab using immunofluorescent Western blot analysis. EGF receptor (EGFR) quantification was performed using flow cytometry. Results: WGS revealed a near-homozygous genome (NHG) and a somatic homozygous TSC1 variant, that was absent in the primary tumor. In the absence of RAS variants, panitumumab showed no real-life efficacy. This might be explained by high constitutive AKT signaling in the two thyroid cancer cell lines with NHG, with panitumumab only being a potent inhibitor of pEGFR in all cancer cell lines tested. Conclusions: In progressive HCC, several treatment options outside or inside clinical trials are available. WGS of metastatic tumors might direct the timing of therapy. Unlike other cancers, the absence of RAS variants seems to provide insufficient justification of single-agent panitumumab administration in HCC cases harboring a near-homozygous genome. Show less
Aydemirli, M.D.; Corver, W.; Beuk, R.; Roepman, P.; Solleveld-Westerink, N.; Wezel, T. van; ... ; Morreau, H. 2019
Objective: To evaluate the efficacy and treatment rationale of Hurthle cell carcinoma (HCC) following a patient with progressive and metastatic HCC. HCC was recently shown to harbor a distinct... Show moreObjective: To evaluate the efficacy and treatment rationale of Hurthle cell carcinoma (HCC) following a patient with progressive and metastatic HCC. HCC was recently shown to harbor a distinct genetic make-up and the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kiase (PI3K)/AKT signaling pathways are potential targets for anti-cancer agents in the management of recurrent HCC. The presence or absence of gene variants can give a rationale for targeted therapies that could be made available in the context of drug repurposing trials. Methods: Treatment included everolimus, sorafenib, nintedanib, lenvatinib, and panitumumab. Whole genome sequencing (WGS) of metastatic tumor material obtained before administration of the last drug, was performed. We subsequently evaluated the rationale and efficacy of panitumumab in thyroid cancer and control cell lines after epidermal growth factor (EGF) stimulation and treatment with panitumumab using immunofluorescent Western blot analysis. EGF receptor (EGFR) quantification was performed using flow cytometry. Results: WGS revealed a near-homozygous genome (NHG) and a somatic homozygous TSC1 variant, that was absent in the primary tumor. In the absence of RAS variants, panitumumab showed no real-life efficacy. This might be explained by high constitutive AKT signaling in the two thyroid cancer cell lines with NHG, with panitumumab only being a potent inhibitor of pEGFR in all cancer cell lines tested. Conclusions: In progressive HCC, several treatment options outside or inside clinical trials are available. WGS of metastatic tumors might direct the timing of therapy. Unlike other cancers, the absence of RAS variants seems to provide insufficient justification of single-agent panitumumab administration in HCC cases harboring a near-homozygous genome. Show less
Sghaier, R. ben; Jansen, A.M.L.; Bdioui, A.; Wezel, T. van; Ksiaa, M.; Elgolli, L.; ... ; Morreau, H. 2019
A high colorectal cancer (CRC) incidence is observed in Tunisia, with a relatively high proportion of patients developing CRC before the age of 40. While this suggests a genetic susceptibility,... Show moreA high colorectal cancer (CRC) incidence is observed in Tunisia, with a relatively high proportion of patients developing CRC before the age of 40. While this suggests a genetic susceptibility, only a few Tunisian Lynch Syndrome families have been described. In this study we aimed to identify the underlying genetic cause in 32 patients with early onset CRC and/or a positive family history. Of twenty-four patients' tumor or biopsies could be analyzed with immunohistochemical staining to detect loss of expression of one of the MMR proteins. Ten tumors showed loss of expression, of which one tumor was from a patient where a germline pathogenic MSH2 variant was detected previously with Sanger sequencing. Next generation sequencing of the MMR, POLE and POLD1 genes was performed in leukocyte and tumor DNA of the remaining nine patients, as well as in two patients with MMR-proficient tumors, but with severe family history. In six of 11 patients a germline variant was detected in MLH1 (n=5) or MSH2 (n=1). Two of six patients were from the same family and both were found to carry a novel in-frame MLH1 deletion, predicted to affect MLH1 function. All MLH1 variant carriers had loss of heterozygosity with retention of the variant in the tumors, while a somatic pathogenic variant was detected in the patient with the germline MSH2 variant. Show less
Tuin, K. van der; Garcia, M.V.; Corver, W.E.; Khalifa, M.N.; Neto, D.R.; Corssmit, E.P.M.; ... ; Morreau, H. 2019
In colorectal cancer (CRC), T-cell checkpoint blockade is only effective in patients diagnosed with mismatch repair-deficient (MMRd) cancers. However, defects in Human Leukocyte Antigen (HLA) class... Show moreIn colorectal cancer (CRC), T-cell checkpoint blockade is only effective in patients diagnosed with mismatch repair-deficient (MMRd) cancers. However, defects in Human Leukocyte Antigen (HLA) class I expression were reported to occur in most MMR-d CRCs, which would preclude antigen presentation in these tumours, considered essential for the clinical activity of this immunotherapeutic modality. We revisited this paradox by characterising HLA class I expression in two independent cohorts of CRC. We determined that loss of HLA class I expression occurred in the majority (73-78%) of MMR-d cases. This phenotype was rare in CRC liver metastases, irrespective of MMR status, whereas weak, inducible expression of HLA class I molecules was frequent in liver lesions. We propose that HLA class I is an important determinant of metastatic homing in CRCs. This observation is paramount to understand CRC carcinogenesis and for the application of immunotherapies in the metastatic setting. Show less
Elsayed, F.A.; Tops, C.M.J.; Nielsen, M.; Ruano, I.; Vasen, H.F.A.; Morreau, H.; ... ; Wezel, T. van 2019
Background Germline mutations affecting the exonuclease domains of POLE and POLD1 predispose to colorectal adenomas and carcinoma. Here, we aimed to screen the exonuclease domains to find the... Show moreBackground Germline mutations affecting the exonuclease domains of POLE and POLD1 predispose to colorectal adenomas and carcinoma. Here, we aimed to screen the exonuclease domains to find the genetic causes of multiple colorectal polyps in unexplained cases. Methods Using a custom next-generation sequencing panel, we sequenced the exonuclease domains of POLE and POLD1 in 332 index patients diagnosed with multiple colorectal polyps without germline alteration in colorectal polyposis predisposing genes. Results We identified two variants of unknown significance. One germline POLD1 c.961G>A, p.(Gly321Ser) variant was found in two cases. The first patient was diagnosed with multiple polyps at age 35 and colorectal cancer (CRC) at age 37, with no known family history of CRC. The second patient was diagnosed with CRC at age 44 and cumulatively developed multiple polyps; this patient had two sisters with endometrial cancer who did not carry the variant. Furthermore, we identified a novel POLD1 c.955 T>G, p.(Cys319Gly) variant in a patient diagnosed with multiple colorectal adenomas at age 40. Co-segregation analysis showed that one sister who cumulatively developed multiple adenomas from age 34, and another sister who developed CRC at age 38 did not carry the variant. We did not identify pathogenic variants in POLE and POLD1. Conclusion This study confirms the low frequency of causal variants in these genes in the predisposition for multiple colorectal polyps, and also establishes that these genes are a rare cause of the disease. Show less
Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic... Show moreBiallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome. Show less
Tuin, K. van der; Kock, L. de; Kamping, E.J.; Hannema, S.E.; Pouwels, M.J.M.; Niedziela, M.; ... ; Morreau, H. 2019
Context: DICER1 syndrome is a rare autosomal-dominantly inherited disorder that predisposes to a variety of cancerous and noncancerous tumors of mostly pediatric and adolescent onset, including... Show moreContext: DICER1 syndrome is a rare autosomal-dominantly inherited disorder that predisposes to a variety of cancerous and noncancerous tumors of mostly pediatric and adolescent onset, including differentiated thyroid carcinoma (DTC). DTC has been hypothesized to arise secondarily to the increased prevalence of thyroid hyperplastic nodules in syndromic patients.Objective: To determine somatic alterations in DICER1-associated DTC and to study patient outcomes.Design: Retrospective series.Setting: Tertiary referral centers.Patients: Ten patients with germline pathogenic DICER1 variants and early-onset DTC.Methods: Somatic DICER1 mutation analysis, extensive somatic DNA variant and gene fusion analyses were performed on all tumors.Results: Median age at DTC diagnosis was 13.5 years and there was no recurrent or metastatic disease (median follow-up, 8 years). All thyroid specimens showed diffuse nodular hyperplasia with at least one focus suspicious of DTC but without infiltrative growth, extrathyroidal extension, vascular invasion, or lymph node metastasis. Most of the individual nodules (benign and malignant) sampled from the 10 tumors harbored distinct DICER1 RNase IIIb hotspot mutations, indicating a polyclonal composition of each tumor. Furthermore, nine of 10 DICER1-related DTCs lacked well known oncogenic driver DNA variants and gene rearrangements.Conclusion: On the basis of our clinical, histological, and molecular data, we consider that most DICER1-related DTCs form a low-risk subgroup. These tumors may arise within one of multiple benign monoclonal nodules; thus, hemi-thyroidectomy or, more likely, total thyroidectomy may often be required. However, radioiodine treatment may be unnecessary given the patients' ages and the tumors' low propensity for metastases. Show less
Boot, A.; Oosting, J.; Doorn, S.; Ouahoud, S.; Garcia, M.V.; Ruano, D.; ... ; Wezel, T. van 2019
Allele-specific expression (ASE) is found in approximately 20-30% of human genes. During tumorigenesis, ASE changes due to somatic alterations that change the regulatory landscape. In colorectal... Show moreAllele-specific expression (ASE) is found in approximately 20-30% of human genes. During tumorigenesis, ASE changes due to somatic alterations that change the regulatory landscape. In colorectal cancer (CRC), many chromosomes show frequent gains or losses while homozygosity of chromosome 7 is rare. We hypothesized that genes essential to survival show allele-specific expression (ASE) on both alleles of chromosome 7. Using a panel of 21 recently established low-passage CRC cell lines, we performed ASE analysis by hybridizing DNA and cDNA to Infinium HumanExome-12 v1 BeadChips containing cSNPs in 392 chromosome 7 genes. The results of this initial analysis were extended and validated in a set of 89 paired normal mucosa and CRC samples. We found that 14% of genes showed ASE in one or more cell lines and identified allelic switching of the potential cell survival genes DLX5, GRB10, and SVOPL on chromosome 7, whereby the most abundantly expressed allele in the normal tissue is the lowest expressed allele in the tumor and vice versa. We established that this allelic switch does not result from loss of imprinting. The allelic switching of SVOPL may be a result of transcriptional downregulation, while the exact mechanisms resulting in the allelic switching of DLX5 and GRB10 remain to be elucidated. In conclusion, our results show that profound changes take place in allelic transcriptional regulation during the tumorigenesis of CRC. Show less
