Parent-of-origin tumourigenesis is mediated by an essential imprinted modifier in SDHD-linked paragangliomas: SLC22A18 and CDKN1C are candidate tumour modifiers.

Mutations in SDHD and SDHAF2 (both located on chromosome 11) give rise to hereditary paraganglioma almost exclusively after paternal transmission of the mutation, and tumours often show loss of the entire maternal copy of chromosome 11. The 'Hensen' model postulates that a tumour modifier gene located on chromosome 11p15, a region known to harbour a cluster of imprinted genes, is essential to tumour formation. We observed decreased protein expression of the 11p15 candidate genes CDKN1C, SLC22A18 and ZNF215 evaluated in 60 SDHD-mutated tumours compared to normal carotid body tissue and non-SDH mutant tumours.We then created stable knockdown in vitro models, reasoning that the simultaneous knockdown of SDHD and a maternally expressed 11p15 modifier gene would enhance paraganglioma-related cellular characteristics compared to SDHD knockdown alone. Knockdown of SDHD in SNB19 and SHSY5Y cells resulted in the accumulation of succinate, the stabilization of HIF1 protein and a reduction in cell proliferation.Compared to single knockdown of SDHD, knockdown of SDHD together with SLC22A18 or with CDKN1C led to small but significant increases in cell proliferation and resistance to apoptosis, and to a gene expression profile closely related to the known transcriptional profile of SDH-deficient tumours. Of the 60 SDHD tumours investigated, four tumours showing retention of chromosome 11 showed SLC22A18 and CDKN1C expression levels comparable to levels in tumours showing loss of chromosome 11, suggesting loss of protein expression despite chromosomal retention.Our data strongly suggest that SLC22A18 and/or CDKN1C are tumour modifier genes involved in the tumourigenesis of SDHD-linked paraganglioma.

Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population.

Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity.

Fluorescent CXCR4 targeting peptide as alternative for antibody staining in Ewing sarcoma.

BACKGROUND: Ewing sarcoma is an aggressive, highly metastatic primary bone and soft tissue tumor most frequently occurring in the bone of young adolescents. Patients, especially those diagnosed with a metastatic disease, have a poor overall survival. Chemokine receptor CXCR4 has a key pro-tumorigenic role in the tumor microenvironment of Ewing sarcoma and has been suggested to be involved in the increased metastatic propensity. Earlier studies on CXCR4 protein expression in Ewing sarcoma yielded contradictory results when compared to CXCR4 RNA expression studies. Previously, we demonstrated that CXCR4 expression could be detected in vivo using the fluorescently tagged CXCR4-specific peptide MSAP-Ac-TZ14011. Therefore, we studied the membranous CXCR4 expression in Ewing sarcoma cell lines using MSAP-Ac-TZ14011. METHODS: The CXCR4 membrane expression levels were studied in EWS cell lines by flow cytometry using the hybrid peptide MSAP-Ac-TZ14011 and were correlated to CXCR4 RNA expression levels. The measurements were compared to levels detected using the CXCR4 antibody ab2074 under various cell preparation conditions. In addition, the staining patterns were analyzed by confocal fluorescence microscopy over time. RESULTS: The hybrid peptide MSAP-Ac-TZ14011 levels showed a strong and better correlation of CXCR4 membrane expression with the CXCR4 RNA expression levels than observed with the anti-CXCR4 antibody ab2074. With the hybrid peptide MSAP-Ac-TZ14011 using live cell confocal microscopy CXCR4 membrane staining and internalization was detected and the signal intensity correlated well with CXCR4 mRNA expression levels. CONCLUSIONS: The fluorescently labeled CXCR4 targeting peptide-based method provides a reliable alternative to antibody staining to study the CXCR4 membrane expression in live cells using either flow cytometry or live cell fluorescence microscopy. The fluorescently tagged CXCR4 targeting peptide could enable in vivo detection of CXCR4 expression in Ewing sarcoma which may help to stratify cases for anti-CXCR4 therapy.

Imprinted survival genes preclude loss of heterozygosity of chromosome 7 in cancer cells.

The genomes of a wide range of cancers, including colon, breast, and thyroid cancers, frequently show copy number gains of chromosome 7 and rarely show loss of heterozygosity. The molecular basis for this phenomenon is unknown. Strikingly, oncocytic follicular thyroid carcinomas can display an extreme genomic profile, with homozygosity of all chromosomes except for chromosome 7. The observation that homozygosity of chromosome 7 is never observed suggests that retention of heterozygosity is essential for cells. We hypothesized that cell survival genes are genetically imprinted on either of two copies of chromosome 7, which thwarts loss of heterozygosity at this chromosome in cancer cells. By employing a DNA methylation screen and gene expression analysis, we identified six imprinted genes that force retention of heterozygosity on chromosome 7. Subsequent knockdown of gene expression showed that CALCR, COPG2, GRB10, KLF14, MEST, and PEG10 were essential for cancer cell survival, resulting in reduced cell proliferation, G1 -phase arrest, and increased apoptosis. We propose that imprinted cell survival genes provide a genetic basis for retention of chromosome 7 heterozygosity in cancer cells. The monoallelically expressed cell survival genes identified in this study, and the cellular pathways that they are involved in, offer new therapeutic targets for the treatment of tumours showing retention of heterozygosity on chromosome 7. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chemosensitivity of BRCA1-Mutated Ovarian Cancer Cells and Established Cytotoxic Agents.

OBJECTIVE: Serous adenocarcinomas that arise in patients with inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 are initially well treatable with platinum/paclitaxel. For recurrent disease in patients with BRCA1 or BRCA2 mutations, olaparib treatment is available. To study additional therapeutic regimens, a better understanding of the cellular and molecular mechanisms of the tumors in in vitro models is important. METHODS/MATERIALS: From a high-grade serous ovarian tumor of a BRCA1 mutation carrier, we established 3 distinct cell line subclones, OVCA-TR3.1, -2, and -3. Immunohistochemical characterization, flow cytometric analyses, chemosensitivity, and somatic mutation profiling were performed. RESULTS: The cell lines expressed AE1/AE3, Pax8, WT-1, OC125, estrogen receptor (ER), and p53, comparable to the primary tumor. Synergism could be shown in the combination treatment eremophila-1-(10)-11(13)-dien-12,8ß-olide (EPD), with cisplatin, whereas combination with olaparib did not show synergism. Eremophila-1-(10)-11(13)-dien-12,8ß-olide, a sesquiterpene lactone, is a novel chemotherapeutic agent. The inherited BRCA1 c.2989_2990dupAA mutation was confirmed in the cell lines. Loss of heterozygosity of BRCA1 was detected in each cell line, as well as a homozygous TP53 c.722C>A mutation. Flow cytometry showed that all cell lines had a distinct DNA index. CONCLUSIONS: Three new isogenic ovarian cancer cell lines were developed from a patient with a germ line BRCA1 mutation. Chemosensitivity profiling of the cell lines showed high tolerance for olaparib. Treatment with EPD proved synergistic with cisplatin. The effects of EPD will be further investigated for future clinical efficacy.

Osteosarcoma Stem Cells Have Active Wnt/ß-catenin and Overexpress SOX2 and KLF4.

Osteosarcoma is a bone tumor, displaying significant cellular and histological heterogeneity and a complex genetic phenotype. Although multiple studies strongly suggest the presence of cancer stem cells in osteosarcoma, a consensus on their characterization is still missing. We used a combination of functional assays (sphere-forming, Aldefluor, and side-population) for identification of cancer stem cell populations in osteosarcoma cell lines. Expression of stemness-related transcription factors, quiescent nature, in vivo tumorigenicity, and Wnt/ß-catenin activation were evaluated. We show that different cancer stem cell populations may co-exist in osteosarcoma cell lines exhibiting distinct functional properties. Osteosarcoma spheres are slowly-proliferating populations, overexpress SOX2, and KLF4 stemness-related genes and have enhanced tumorigenic potential. Additionally, spheres show specific activation of Wnt/ß-catenin signaling as evidenced by increased nuclear ß-catenin, TCF/LEF activity, and AXIN2 expression, in a subset of the cell lines. Aldefluor-positive populations were detected in all osteosarcoma cell lines and overexpress SOX2, but not KLF4. The side-population phenotype is correlated with ABCG2 drug-efflux transporter expression. Distinct functional methods seem to identify cancer stem cells with dissimilar characteristics. Intrinsic heterogeneity may exist within osteosarcoma cancer stem cells and can have implications on the design of targeted therapies aiming to eradicate these cells within tumors. J. Cell. Physiol. 231: 876-886, 2016. © 2015 Wiley Periodicals, Inc.

Spontaneous bone metastases in a preclinical orthotopic model of invasive lobular carcinoma; the effect of pharmacological targeting TGFß receptor I kinase.

Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the most frequently occurring histological subtypes of breast cancer, accounting for 80-90% and 10-15% of the total cases, respectively. At the time of diagnosis and surgical resection of the primary tumour, most patients do not have clinical signs of metastases, but bone micrometastases may already be present. Our aim was to develop a novel preclinical ILC model of spontaneous bone micrometastasis. We used murine invasive lobular breast carcinoma cells (KEP) that were generated by targeted deletion of E-cadherin and p53 in a conditional K14cre;Cdh1((F/F));Trp53((F/F)) mouse model of de novo mammary tumour formation. After surgical resection of the growing orthotopically implanted KEP cells, distant metastases were formed. In contrast to other orthotopic breast cancer models, KEP cells readily formed skeletal metastases with minimal lung involvement. Continuous treatment with SD-208 (60 mg/kg per day), an orally available TGFß receptor I kinase inhibitor, increased the tumour growth at the primary site and increased the number of distant metastases. Furthermore, when SD-208 treatment was started after surgical resection of the orthotopic tumour, increased bone colonisation was also observed (versus vehicle). Both our in vitro and in vivo data show that SD-208 treatment reduced TGFß signalling, inhibited apoptosis, and increased proliferation. In conclusion, we have demonstrated that orthotopic implantation of murine ILC cells represent a new breast cancer model of minimal residual disease in vivo, which comprises key steps of the metastatic cascade. The cancer cells are sensitive to the anti-tumour effects of TGFß. Our in vivo model is ideally suited for functional studies and evaluation of new pharmacological intervention strategies that may target one or more steps along the metastatic cascade of events.

Towards imaging metabolic pathways in tissues.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging using 9-aminoacridine as the matrix leads to the detection of low mass metabolites and lipids directly from cancer tissues. These included lactate and pyruvate for studying the Warburg effect, as well as succinate and fumarate, metabolites whose accumulation is associated with specific syndromes. By using the pathway information present in the human metabolome database, it was possible to identify regions within tumor tissue samples with distinct metabolic signatures that were consistent with known tumor biology. We present a data analysis workflow for assessing metabolic pathways in their histopathological context.

Near-haploidization significantly associates with oncocytic adrenocortical, thyroid, and parathyroid tumors but not with mitochondrial DNA mutations.

Mitochondrial-rich oncocytic thyroid tumors frequently show near-haploidization and endoreduplication (masked haploidization), which manifests as a near-homozygous genome (NHG). We now extend this investigation to include adrenocortical cancer and parathyroid carcinoma (PaTC), which we studied for a NHG in association with mitochondrial DNA mutations. Sixty endocrine tumors from 59 patients were studied, including 46 thyroid tumor samples of varying histology, 11 adrenocortical cancers, and 3 PaTCs. Genome-wide SNP array analysis and DNA content analysis were combined to determine the chromosomal dosage (allelic state). The entire mitochondrial genome was also studied for mutations. In addition, tumors were characterized for somatic mutations in a subset of genes that are directly or indirectly implicated in cellular metabolism. In addition to a subset of thyroid cancers (n = 5), a NHG was also observed in 1 of 3 PaTCs and 6 of 11 adrenocortical cancers. All but one of the tumors with a NHG (n = 12) showed oncocytic metaplasia (P = 0.0001, two-tailed Fisher's exact). One or more damaging or disrupting mtDNA mutations were found in 68% (41/60) of tumor samples. No correlation was found between mtDNA mutations and the oncocytic phenotype or a NHG, and none of the mutations in nuclear encoded genes correlated with the oncocytic phenotype or a NHG. A subset of oncocytic tumors of the thyroid, parathyroid, and adrenocortical carcinomas carries a NHG. Although damaging/disrupting mtDNA mutations are frequently found in oncocytic and nononcocytic endocrine tumors, neither correlates with a NHG phenotype nor with an oncocytic phenotype.

High-Resolution Multiparameter DNA Flow Cytometry for Accurate Ploidy Assessment and the Detection and Sorting of Tumor and Stromal Subpopulations from Paraffin-Embedded Tissues.

This article contains detailed protocols for the simultaneous flow cytometric identification of tumor cells and stromal cells and measurement of DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for accurate DNA content assessments of FFPE carcinoma tissues. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 (near-haploidy) and of keratin-positive tumor cells with a DNA index close to 1.0 in overall DNA aneuploid samples, thus improving DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin-positive tumor cell fractions can be sorted for further molecular genetic analysis, while DNA from the sorted vimentin-positive stromal cells can serve as a reference when normal tissue of the patient is not available. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Multiparameter DNA content analysis of FFPE carcinomas Alternate Protocol 1: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue and red excitation Alternate Protocol 2: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue excitation Support Protocol: Sorting cell population from FFPE carcinomas.

Differentiating Benign from Malignant Thyroid Tumors by Kinase Activity Profiling and Dabrafenib BRAF V600E Targeting.

Differentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss of iodide transport, a phenotype frequently associated with BRAF V600E mutations. In theory, this should enable the use of existing targeted therapies specifically designed for BRAF V600E mutations. However, in practice, generic or specific drugs aimed at molecular targets identified by next generation sequencing (NGS) are not always beneficial. Detailed kinase profiling may provide additional information to help improve therapy success rates. In this study, we therefore investigated whether serine/threonine kinase (STK) activity profiling can accurately classify benign thyroid lesions and NMTC. We also determined whether dabrafenib (BRAF V600E-specific inhibitor), as well as sorafenib and regorafenib (RAF inhibitors), can differentiate BRAF V600E from non-BRAF V600E thyroid tumors. Using 21 benign and 34 malignant frozen thyroid tumor samples, we analyzed serine/threonine kinase activity using PamChip®peptide microarrays. An STK kinase activity classifier successfully differentiated malignant (26/34; 76%) from benign tumors (16/21; 76%). Of the kinases analyzed, PKC (theta) and PKD1 in particular, showed differential activity in benign and malignant tumors, while oncocytic neoplasia or Graves' disease contributed to erroneous classifications. Ex vivo BRAF V600E-specific dabrafenib kinase inhibition identified 6/92 analyzed peptides, capable of differentiating BRAF V600E-mutant from non-BRAF V600E papillary thyroid cancers (PTCs), an effect not seen with the generic inhibitors sorafenib and regorafenib. In conclusion, STK activity profiling differentiates benign from malignant thyroid tumors and generates unbiased hypotheses regarding differentially active kinases. This approach can serve as a model to select novel kinase inhibitors based on tissue analysis of recurrent thyroid and other cancers.

A clinically applicable molecular classification of oncocytic cell thyroid nodules.

Whole chromosome instability with near-whole genome haploidization (GH) and subsequent endoreduplication is considered a main genomic driver in the tumorigenesis of oncocytic cell thyroid neoplasms (OCN). These copy number alterations (CNA) occur less frequently in oncocytic thyroid adenoma (OA) than in oncocytic carcinoma (OCA), suggesting a continuous process. The current study described the CNA patterns in a cohort of 30 benign and malignant OCN, observed using a next-generation sequencing (NGS) panel that assesses genome-wide loss of heterozygosity (LOH) and chromosomal imbalances using 1500 single-nucleotide polymorphisms (SNPs) across all autosomes and the X chromosome in DNA derived from cytological and histological samples. Observed CNA patterns were verified using multiparameter DNA flow cytometry with or without whole-genome SNP array analysis and lesser-allele intensity-ratio (LAIR) analysis. On CNA-LOH analysis using the NGS panel, GH-type CNA were observed in 4 of 11 (36%) OA and in 14 of 16 OCA (88%). Endoreduplication was suspected in 8 of 16 (50%) OCA, all with more extensive GH-type CNA (P < 0.001). Reciprocal chromosomal imbalance type CNA, characterized by (imbalanced) chromosomal copy number gains and associated with benign disease, were observed in 6 of 11 (55%) OA and one equivocal case of OCA. CNA patterns were different between the histopathological subgroups (P < 0.001). By applying the structured interpretation and considerations provided by the current study, CNA-LOH analysis using an NGS panel that is feasible for daily practice may be of great added value to the widespread application of molecular diagnostics in the diagnosis and risk stratification of OCN.

Conditional immortalization of human atrial myocytes for the generation of in vitro models of atrial fibrillation.

The lack of a scalable and robust source of well-differentiated human atrial myocytes constrains the development of in vitro models of atrial fibrillation (AF). Here we show that fully functional atrial myocytes can be generated and expanded one-quadrillion-fold via a conditional cell-immortalization method relying on lentiviral vectors and the doxycycline-controlled expression of a recombinant viral oncogene in human foetal atrial myocytes, and that the immortalized cells can be used to generate in vitro models of AF. The method generated 15 monoclonal cell lines with molecular, cellular and electrophysiological properties resembling those of primary atrial myocytes. Multicellular in vitro models of AF generated using the immortalized atrial myocytes displayed fibrillatory activity (with activation frequencies of 6-8 Hz, consistent with the clinical manifestation of AF), which could be terminated by the administration of clinically approved antiarrhythmic drugs. The conditional cell-immortalization method could be used to generate functional cell lines from other human parenchymal cells, for the development of in vitro models of human disease.

Targeting EML4-ALK gene fusion variant 3 in thyroid cancer.

Finding targetable gene fusions can expand the limited treatment options in radioactive iodine-refractory (RAI-r) thyroid cancer. To that end, we established a novel cell line 'JVE404' derived from an advanced RAI-r papillary thyroid cancer (PTC) patient, harboring an EML4-ALK gene fusion variant 3 (v3). Different EML4-ALK gene fusions can have different clinical repercussions. JVE404 cells were evaluated for cell viability and cell signaling in response to ALK inhibitors crizotinib, ceritinib and lorlatinib, in parallel to the patient's treatment. He received, after first-line lenvatinib, crizotinib (Drug Rediscovery Protocol (DRUP) trial), and lorlatinib (compassionate use). In vitro treatment with crizotinib or ceritinib decreased viability in JVE404, but most potently and significantly only with lorlatinib. Western blot analysis showed a near total decrease of 99% and 89%, respectively, in pALK and pERK expression levels in JVE404 cells with lorlatinib, in contrast to remaining signal intensities of a half and a third of control, respectively, with crizotinib. The patient had a 6-month lasting stable disease on crizotinib, but progressive disease occurred, including the finding of cerebral metastases, at 8 months. With lorlatinib, partial response, including clinical cerebral activity, was already achieved at 11 weeks' use and ongoing partial response at 7 months. To our best knowledge, this is the first reported case describing a patient-specific targeted treatment with lorlatinib based on an EML4-ALK gene fusion v3 in a thyroid cancer patient, and own cancer cell line. Tumor-agnostic targeted therapy may provide valuable treatment options in personalized medicine.

IGF2 is a potential factor in RAI-refractory differentiated thyroid cancer.

Differentiated thyroid cancer (DTC) is the most frequent endocrine tumor with a good prognosis after primary treatment in most cases. By contrast, 30-40% of patients with metastatic DTC are unresponsive to 131I radioactive iodide (RAI) treatment due to tumor dedifferentiation. Currently, underlying molecular mechanisms of dedifferentiation remain elusive and predictive biomarkers are lacking. Therefore, the present study aimed to identify molecular biomarkers in primary tumors associated with RAI refractoriness. A retrospective cohort was gathered consisting of RAI-sensitive patients with DTC and RAI-refractory patients with poorly DTC. In all patients, extensive intratumoral mutation profiling, gene fusions analysis, telomerase reverse transcriptase (TERT) promoter mutation analysis and formalin-fixed paraffin-embedded-compatible RNA sequencing were performed. Genetic analyses revealed an increased mutational load in RAI-refractory DTC, including mutations in AKT1, PTEN, TP53 and TERT promoter. Transcriptomic analyses revealed profound differential expression of insulin-like growth factor 2 (IGF2), with up to 100-fold higher expression in RAI-refractory DTC compared with in RAI-sensitive DTC cases. ELISA revealed significant lower IGF2 plasma concentrations after surgery and subsequent 131I RAI therapy in patients with DTC compared with pretreatment baseline. Overall, the current findings suggested that the tumor-promoting growth factor IGF2 may have a potential role in acquiring RAI refractoriness.

Digoxin treatment reactivates in vivo radioactive iodide uptake and correlates with favorable clinical outcome in non-medullary thyroid cancer.

PURPOSE: Non-medullary thyroid cancer (NMTC) treatment is based on the ability of thyroid follicular cells to accumulate radioactive iodide (RAI). However, in a subset of NMTC patients tumor dedifferentiation occurs, leading to RAI resistance. Digoxin has been demonstrated to restore iodide uptake capacity in vitro in poorly differentiated and anaplastic NMTC cells, termed redifferentiation. The aim of the present study was to investigate the in vivo effects of digoxin in TPO-Cre/LSL-BrafV600E mice and digoxin-treated NMTC patients. METHODS: Mice with thyroid cancer were subjected to 3D ultrasound for monitoring tumor growth and 124I PET/CT for measurement of intratumoral iodide uptake. Post-mortem analyses on tumor tissues comprised gene expression profiling and measurement of intratumoral autophagy activity. Through PALGA (Dutch Pathology Registry), archived tumor material was obtained from 11 non-anaplastic NMTC patients who were using digoxin. Clinical characteristics and tumor material of these patients were compared to 11 matched control NMTC patients never treated with digoxin. RESULTS: We found that in mice, tumor growth was inhibited and 124I accumulation was sustainably increased after short-course digoxin treatment. Post-mortem analyses revealed that digoxin treatment increased autophagy activity and enhanced expression of thyroid-specific genes in mouse tumors compared to vehicle-treated mice. Digoxin-treated NMTC patients exhibited significantly higher autophagy activity and a higher differentiation status as compared to matched control NMTC patients, and were associated with favourable clinical outcome. CONCLUSIONS: These in vivo data support the hypothesis that digoxin may represent a repositioned adjunctive treatment modality that suppresses tumor growth and improves RAI sensitivity in patients with RAI-refractory NMTC.

Single nucleotide polymorphisms in antigen processing machinery component ERAP1 significantly associate with clinical outcome in cervical carcinoma.

Genetic variation of the antigen processing machinery (APM) components TAP2, LMP7, and ERAP1 is related to cervical carcinoma risk, although the relation with expression and clinical outcome remains unknown. We have investigated the occurrence of APM component single nucleotide polymorphisms (SNPs) in cervical carcinoma. Twelve nonsynonymous, coding SNPs in the TAP1, TAP2, LMP2, LMP7, and ERAP1 genes were genotyped in 75 cervical carcinoma patients with known APM component and HLA class I expression levels. Individual genotype distributions were assessed for association with APM component expression, various histopathological parameters and survival. Genotype distributions at the ERAP1-56 and ERAP1-127 loci were significantly associated with overall survival (OS); haplotype construction spanning these two SNPs revealed that the combination of a major allele at ERAP1-56 and a minor allele at ERAP1-127 was significantly associated with survival, homozygosity for this haplotype being associated with decreased OS (5-year survival 50% vs. 70 and 81% for complete absence or heterozygosity for this haplotype, respectively; P = 0.021). Heterozygosity for this haplotype was an independent predictor for better OS in multivariate analysis (HR = 0.219; P = 0.014). These data indicate that genetic variation in APM component genes, particularly ERAP1, is an important contributing factor in cervical carcinogenesis, progressive tumor growth and survival. The location of the ERAP1-127 SNP in the peptidase M1 domain of the ERAP1 aminopeptidase suggests the possibility of direct functional consequences of variation at this locus.

Metabolic reprogramming related to whole-chromosome instability in models for Hürthle cell carcinoma.

Hürthle 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 α-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.

Lack of TNFalpha mRNA expression in cervical cancer is not associated with loss of heterozygosity at 6p21.3, inactivating mutations or promoter methylation.

Infection with oncogenic human papillomavirus (HPV) is considered to be the major etiologic event for cervical cancer. Tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine, may be involved in orchestrating an antitumor immune response against human papillomavirus expressing cervical cancer cells. Hence, loss of TNFalpha could be advantageous for tumor cells to escape immune clearance. The aim of our study was to investigate TNFalpha gene expression and epigenetic characteristics associated with the loss of TNFalpha expression in cervical cancer. To this end, we examined TNFalpha expression, loss of heterozygosity (LOH) at 6p21.3, the locus of TNFalpha, mutational status of the TNFalpha locus, loss of the TNFalpha promoter variant 2 allele and CpG hypermethylation of the TNFalpha promoter. RNA in situ hybridization showed absence of TNFalpha expression in 45% of 63 tumors. LOH occurred in 57% of the tumors and was not concordant with absence of TNFalpha mRNA. No mutations in the TNFalpha gene were identified in 15 cases deficient in TNFalpha expression exhibiting LOH. Furthermore, lack of TNFalpha expression did not correlate with promoter methylation. In conclusion, TNFalpha mRNA expression is absent in nearly half of the cervical tumors analyzed. Neither promoter methylation nor genetic causes for lack of expression were evident.

Expression of Smad2 and Smad4 in cervical cancer: absent nuclear Smad4 expression correlates with poor survival.

Alterations in transforming growth factor-beta signaling, due to a decrease in Smad2 and especially Smad4 expression, has primarily been reported in pancreatic and colorectal cancers, although loss of the chromosomal region 18q21.1, containing the loci of Smad2 and Smad4, is among the most frequent molecular alterations in cervical cancer. The aim of our study was to investigate whether decreased Smad2 and Smad4 protein expression in primary cervical cancers is associated with molecular alterations at 18q21.1, mutations in the functional domains of Smad2 and Smad4 or hypermethylation, and to assess the biological relevance of decreased Smad2 and Smad4 expression. Subsequently, Smad2, Smad4 and p21 protein expression was determined by immunohistochemistry in 117 primary cervical carcinomas, assembled in a tissue array. Smad signaling was shown to be associated with p21 mRNA expression. All the tumors expressed Smad2 or Smad4. Weak cytoplasmic Smad2 or weak cytoplasmic Smad4 expression could not be attributed to loss of heterozygosity at 18q21.1. Despite weak/moderate Smad2 expression and absent nuclear Smad4 expression, the coding regions of the functional MH1 and MH2 domains of Smad2 and Smad4 were unchanged, as assessed by sequence analysis. The Smad4 promoter region was unmethylated in tumor samples with weak/moderate cytoplasmic Smad4 expression. Remarkably, both weak cytoplasmic Smad4 expression and absent nuclear Smad4 expression significantly correlated with poor disease-free (P=0.003 and P=0.003, respectively) and overall 5-year survival (P=0.003 and P=0.010, respectively). Our findings support the hypothesis that Smad4 is a target molecule for functional inactivation in cervical cancer.