Theodoros Foukakis

Molecular Markers for Discrimination of Benign and Malignant Follicular Thyroid Tumors

Objective: To identify molecular markers useful for the diagnostic discrimination of benign and malignant follicular thyroid tumors. Methods: A panel of thyroid tumors was characterized with expression profiling using cDNA microarrays. A robust algorithm for gene selection was developed to identify molecular markers useful for the classification of heterogeneous tumor classes. The study included tumor tissue specimens from 10 patients with benign follicular adenomas and from 10 with malignant tumors. The malignant tumors mainly consisted of clinically relevant minimally invasive follicular carcinomas. The mRNA expression level of a candidate gene, FHL1, was evaluated in an independent series of 61 tumors. Results: 22 gene expression markers were identified as differentially expressed. Several of the identified genes, for example DIO1, CITED1, CA12 and FN1, have previously been observed as differentially expressed in various thyroid tumors. FHL1 was significantly underexpressed in carcinomas compared to adenomas in the independent panel of tumors. The results indicate that a small number of genes can be useful to distinguish follicular adenomas from follicular carcinomas. Conclusions: Our findings clearly corroborate previous studies and identify novel candidate molecular markers. These genes have the potential for molecular classification of follicular thyroid tumors and for providing improved understanding of the molecular mechanisms involved in thyroid malignancies.

Advances in the treatment of patients with gastric adenocarcinoma

Despite a decline in its incidence in the Western world, gastric cancer (GC) remains the fourth most frequent cancer diagnosis worldwide and is, after lung cancer, the second leading cause of death from a malignant disease globally. Based on the published literature, treatment guidelines and reports from international meetings, we here review the current treatment options for GC and discuss insights and perspectives from the latest clinical studies. The management of GC in the early stages of the disease is based on an optimal surgical resection of the primary tumor and the regional lymph nodes. However, less than one third of patients have a resectable disease at diagnosis and among those operated, more than half are not cured by surgery alone, due to a high rate of relapse. Thus, for the majority of patients, systemic cytotoxic therapy, and sometimes radiotherapy, is a treatment option both as an adjunct to surgery and in the palliative setting. Adjuvant chemotherapy offers only a marginal benefit and has not become a standard of care in the West. In North America, adjuvant chemoradiation is broadly used, shown to significantly improve overall survival, albeit with the cost of high toxicity. Furthermore, a recently reported study from the United Kingdom demonstrated a significant disease-free and survival benefit by the use of perioperative combination chemotherapy. Several chemotherapeutic agents have been tested as a palliative therapy in advanced GC including 5- fluorouracil (5-FU), oral pyrimidines, platinum derivatives, anthracyclines, taxanes and camptothecans. It is now accepted that chemotherapy is better than best supportive care only and that 5-FU based combinations are more effective than monotherapy. However, the response rates have generally been moderate and there is no consensus on the optimal combination of cytotoxic agents and the potential role of more recently developed “targeted therapies”.

Array-CGH identifies cyclin D1 and UBCH10 amplicons in anaplastic thyroid carcinoma.

Anaplastic thyroid cancer (ATC) is a rare but highly aggressive disease with largely unexplained etiology and molecular pathogenesis. In this study, we analyzed genome-wide copy number changes, BRAF (V-raf sarcoma viral oncogene homolog B1) mutations, and p16 and cyclin D1 expressions in a panel of ATC primary tumors. Three ATCs harbored the common BRAF mutation V600E. Using array-comparative genomic hybridisation (array-CGH), several distinct recurrent copy number alterations were revealed including gains in 16p11.2, 20q11.2, and 20q13.12. Subsequent fluorescence in situ hybridization revealed recurrent locus gain of UBCH10 in 20q13.12 and Cyclin D1 (CCND1) in 11q13. The detection of a homozygous loss encompassing the CDKN2A locus in 9p21.3 motivated the examination of p16 protein expression, which was undetectable in 24/27 ATCs (89%). Based on the frequent gain in 11q13 (41%; n=11), the role of CCND1 was further investigated. Expression of cyclin D1 protein was observed at varying levels in 18/27 ATCs (67%). The effect of CCND1 on thyroid cell proliferation was assessed in vitro in ATC cells by means of siRNA and in thyroid cells after CCND1 transfection. In summary, the recurrent chromosomal copy number changes and molecular alterations identified in this study may provide an insight into the pathogenesis and development of ATC.

Proteomic profiling of follicular and papillary thyroid tumors.

Objective Thyroid proteomics is a new direction in thyroid cancer research aiming at etiological understanding and biomarker identification for improved diagnosis. Methods Two-dimensional electrophoresis was applied to cytosolic protein extracts from frozen thyroid samples (ten follicular adenomas, nine follicular carcinomas, ten papillary carcinomas, and ten reference thyroids). Spots with differential expression were revealed by image and multivariate statistical analyses, and identified by mass spectrometry. Results A set of 25 protein spots significant for discriminating between the sample groups was identified. Proteins identified for nine of these spots were studied further including 14-3-3 protein beta/alpha, epsilon, and zeta/delta, peroxiredoxin 6, selenium-binding protein 1, protein disulfide-isomerase precursor, annexin A5 (ANXA5), tubulin alpha-1B chain, and α1-antitrypsin precursor. This subset of protein spots carried the same predictive power in differentiating between follicular carcinoma and adenoma or between follicular and papillary carcinoma, as compared with the larger set of 25 spots. Protein expression in the sample groups was demonstrated by western blot analyses. For ANXA5 and the 14-3-3 proteins, expression in tumor cell cytoplasm was demonstrated by immunohistochemistry both in the sample groups and an independent series of papillary thyroid carcinomas. Conclusion The proteins identified confirm previous findings in thyroid proteomics, and suggest additional proteins as dysregulated in thyroid tumors.

The Ras effectors NORE1A and RASSF1A are frequently inactivated in pheochromocytoma and abdominal paraganglioma

NORE1A (RASSF5) and RASSF1A are newly described Ras effectors with tumour suppressor functions. Both molecules are frequently inactivated in various cancers. In this study, we aimed to explore the potential involvement of NORE1A and RASSF1A in pheochromocytoma and abdominal paraganglioma tumorigenesis. A panel of 54 primary tumours was analysed for NORE1A and RASSF1A mRNA expression by TaqMan quantitative RT-PCR. Furthermore, NORE1A and RASSF1A promoter methylation was assessed by combined bisulphite restriction endonuclease assay and methylation-sensitive Pyrosequencing respectively. The anti-tumorigenic role of NORE1A was functionally investigated in Nore1A-transfected PC12 rat pheochromocytoma cells by fluorescent inhibition of caspase activity and soft agar assays. Significantly suppressed NORE1A and RASSF1A mRNA levels were detected in primary tumours compared with normal adrenal medulla (P<0.001). Methylation of the NORE1A promoter was not observed in primary tumours. On the other hand, 9% (5/54) of the primary tumours examined showed RASSF1A promoter methylation greater than 20% as detected by Pyrosequencing. Methylation of the RASSF1A promoter was significantly associated with malignant behaviour (P<0.05). Transient expression of Nore1a resulted in enhanced apoptosis and impaired colony formation in soft agar. Our study provides evidence that NORE1A and RASSF1A are frequently suppressed in pheochromocytoma and abdominal paraganglioma. Silencing of NORE1A contributes to the transformed phenotype in these tumours.

Anaplastic carcinoma of the thyroid gland: Treatment and outcome over 13 years at one institution

Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy of the thyroid gland. Patients at our institution are treated with external radiotherapy up to 46 Gray (Gy) and low‐dose doxorubicin prior to surgery. We retrospectively evaluated the outcome of ATC patients over a 13‐year period.

Proteomic study of thyroid tumors reveals frequent up-regulation of the Ca2+ -binding protein S100A6 in papillary thyroid carcinoma.

BACKGROUND The accurate diagnosis of thyroid tumors is challenging. Proteomics has emerged as a promising approach for the discovery of molecular diagnostic markers as a potential complement to routine diagnostics. METHODS Protein fractions from 29 frozen thyroid tumor tissue samples (10 papillary carcinomas, 9 follicular carcinomas, and 10 follicular adenomas) as well as from normal thyroid tissue were analyzed by surface enhanced laser desorption/ionization time-of-flight mass spectrometry followed by validation by Western blotting and immunohistochemistry. RESULTS A Ca2+ binding protein belonging to the S100 family, S100A6, was differentially expressed between papillary and follicular thyroid tumors. Moreover, two posttranslationally modified forms of S100A6 were observed and verified by liquid chromatography-coupled tandem mass spectrometry. Validation by Western blotting displayed a significantly higher expression of S100A6 in papillary thyroid carcinoma (PTC) in comparison with the other tumor groups or normal tissue (p < 0.05). Immunohistochemical analysis on 98 tumors showed that PTC cases had a significantly stronger cytosolic staining and a larger proportion of stained nuclei than follicular tumors. BRAF gene mutation was not significantly associated with S100A6 protein levels. CONCLUSION This study supports a role of S100A6 in thyroid tumorigenesis and as a potential aid in the discrimination between follicular thyroid tumors and PTC.

Effect of tailored dose-dense chemotherapy vs standard 3-weekly adjuvant chemotherapy on recurrence-free survival among women with high-risk early breast cancer: A randomized clinical trial

Importance Standard dosing of chemotherapy based on body surface area results in marked interpatient variation in pharmacokinetics, toxic effects, and efficacy. Whether tailored dosing can improve outcomes is unknown, as is the role of dose-dense adjuvant chemotherapy. Objective To determine whether tailored dose-dense adjuvant chemotherapy improves the outcomes of early breast cancer compared with a standard 3-weekly chemotherapy schedule. Design, Setting, and Participants A randomized, open-label, phase 3 trial of women aged 65 years and younger who had surgery for nonmetastatic node-positive or high-risk node-negative breast cancer at 86 sites in Sweden, Germany, and Austria between February 20, 2007, and September 14, 2011. Interventions Patients were randomized 1:1 either to 4 cycles of leukocyte nadir-based tailored and dose-dense adjuvant epirubicin and cyclophosphamide every 2 weeks followed by 4 cycles of tailored dose-dense docetaxel every 2 weeks, or to standard-interval chemotherapy with 3 cycles of fluorouracil and epirubicin-cyclophosphamide every 3 weeks followed by 3 cycles of docetaxel every 3 weeks. Main Outcomes and Measures The primary end point was breast cancer recurrence-free survival (BCRFS). Secondary end points included 5-year event-free survival (EFS), distant disease-free survival (DDFS), overall survival (OS), and rates of grade 3 or 4 toxic effects. Results Among 2017 randomized patients (1006 in the tailored dose-dense group and 1011 in the control group; median [IQR] age, 51 [45-58] years; 80% with hormone receptor-positive tumors; 97% with node-positive disease), 2000 received study treatment (≥1 cycle of chemotherapy; 1001 in the tailored dose-dense group and 999 in the control group). After a median follow-up of 5.3 years (IQR, 4.5-6.1 years), 269 BCRFS events were reported, 118 in the tailored dose-dense group and 151 in the control group (HR, 0.79; 95% CI, 0.61-1.01; log-rank P = .06; 5-year BCRFS, 88.7% vs 85.0%). The tailored dose-dense group had significantly better EFS than the control group (HR, 0.79; 95% CI, 0.63-0.99; P = .04; 5-year EFS, 86.7% vs 82.1%). The groups did not differ in OS (HR, 0.77; 95% CI, 0.57-1.05; P = .09; 5-year OS, 92.1% vs 90.2%) or DDFS (HR, 0.83; 95% CI, 0.64-1.08; P = .17; 5-year DDFS, 89.4% vs 86.7%). Grade 3 or 4 nonhematologic toxic effects occurred in 527 (52.6%) in the tailored dose-dense group and 366 (36.6%) in the control group. Conclusions and Relevance Among women with high-risk early breast cancer, the use of tailored dose-dense chemotherapy compared with standard adjuvant chemotherapy did not result in a statistically significant improvement in breast cancer recurrence-free survival. Nonhematologic toxic effects were more frequent in the tailored dose-dense group. Trial Registration clinicaltrials.gov Identifier: NCT00798070; isrctn.org Identifier: ISRCTN39017665.

First results of the preoperative accelerated partial breast irradiation (PAPBI) trial

BACKGROUND AND PURPOSE The aim of this study is to assess the toxicity and cosmetic outcome of preoperative accelerated partial breast irradiation (PAPBI) for breast cancer patients with low risk on local recurrence. MATERIAL AND METHODS Women aged ⩾60years with an invasive, unifocal ⩽3cm on MRI, (non-lobular) adenocarcinoma of the breast and a negative sentinel node received PAPBI (40Gray in 10 fractions over 2 weeks). Six weeks after radiotherapy a wide local excision was performed. RESULTS 70 patients with a median follow-up of 23 months (3-44 months) were evaluated. The overall postoperative infection rate was 11%. At 1, 2 and 3 years of follow-up respectively 89%, 98% and 100% of patients had no or mild induration-fibrosis. Fibrosis was only found in a small volume of the breast. The global cosmetic outcome was good to excellent in 77% at 6 months to 100% at 3 years. Two patients developed a local recurrence. CONCLUSION Our first results show limited fibrosis in a small volume and good to excellent cosmetic outcome. In selected patients, preoperative radiotherapy appears to be a good option for breast conserving therapy.

Nanogrid single-nucleus RNA sequencing reveals phenotypic diversity in breast cancer

Single cell RNA sequencing has emerged as a powerful tool for resolving transcriptional diversity in tumors, but is limited by throughput, cost and the ability to process archival frozen tissue samples. Here we develop a high-throughput 3′ single-nucleus RNA sequencing approach that combines nanogrid technology, automated imaging, and cell selection to sequence up to ~1800 single nuclei in parallel. We compare the transcriptomes of 485 single nuclei to 424 single cells in a breast cancer cell line, which shows a high concordance (93.34%) in gene levels and abundance. We also analyze 416 nuclei from a frozen breast tumor sample and 380 nuclei from normal breast tissue. These data reveal heterogeneity in cancer cell phenotypes, including angiogenesis, proliferation, and stemness, and a minor subpopulation (19%) with many overexpressed cancer genes. Our studies demonstrate the utility of nanogrid single-nucleus RNA sequencing for studying the transcriptional programs of tumor nuclei in frozen archival tissue samples.Single cell RNA sequencing is a powerful tool for understanding cellular diversity but is limited by cost, throughput and sample preparation. Here the authors use nanogrid technology with integrated imaging to sequence thousands of cancer nuclei in parallel from fresh or frozen tissue.