Morphological and molecular heterogeneity of epithelial ovarian cancer: Therapeutic implications.

Ovarian epithelial cancer (OEC) is the most lethal gynecologic malignancy. Despite current chemotherapeutic and surgical options, this high lethality can be attributed to multiple factors, including late-stage presentation. In order to optimize OEC treatment, it is important to highlight that it is composed of five main subtypes: high-grade serous ovarian carcinoma (HGSOC), low-grade serous ovarian carcinoma (LGSOC), endometrioid ovarian carcinoma (EOC), ovarian clear cell carcinoma (CCOC), and mucinous ovarian carcinoma (MOC). These subtypes differ in their precursor lesions, as well as in epidemiological, morphological, molecular and clinical features. OEC is one of the tumours in which most pathogenic germline mutations have been identified. Accordingly, up to 20% OC show alterations in BRCA1/2 genes, and also, although with a lower frequency, in other low penetrance genes associated with homologous recombination deficiency (HRD), mismatch repair genes (Lynch syndrome) and TP53. The most important prognostic factor is the 2014 FIGO staging, while older age is also associated with worse survival. HGSOC in all stages and CCC and MOC in advanced stages have the worse prognosis among histological types. Molecular markers have emerged as prognostic factors, particularly mutations in BRCA1/2, which are associated with a better outcome. Regarding treatment, whereas a proportion of HGSOC is sensible to platinum-based treatment and PARP inhibitors due to HRD, the rest of the histological types are relatively chemoresistant. New treatments based in specific molecular alterations are being tested in different histological types. In addition, immunotherapy could be an option, especially for EOC carrying mismatch repair deficiency or POLE mutations.

Molecular genetic heterogeneity in undifferentiated endometrial carcinomas.

Undifferentiated and dedifferentiated endometrial carcinomas are rare and highly aggressive subtypes of uterine cancer, not well characterized at a molecular level. To investigate whether dedifferentiated carcinomas carry molecular genetic alterations similar to those of pure undifferentiated carcinomas, and to gain insight into the pathogenesis of these tumors, we selected a cohort of 18 undifferentiated endometrial carcinomas, 8 of them with a well-differentiated endometrioid carcinoma component (dedifferentiated endometrioid carcinomas), and studied them by immunohistochemistry and massive parallel and Sanger sequencing. Whole-exome sequencing of the endometrioid and undifferentiated components, as well as normal myometrium, was also carried out in one case. According to The Cancer Genome Atlas classification, we distributed 95% of the undifferentiated carcinomas in this series as follows: (a) hypermutated tumors with loss of any mismatch repair protein expression and microsatellite instability (eight cases, 45%); (b) ultramutated carcinomas carrying mutations in the exonuclease domain of POLE (two cases, 11%); (c) high copy number alterations (copy-number high) tumors group exhibiting only TP53 mutations and high number of alterations detected by FISH (two cases, 11%); and (d) low copy number alterations (copy-number low) tumors with molecular alterations typical of endometrioid endometrial carcinomas (five cases, 28%). Two of the latter cases, however, also had TP53 mutations and higher number of alterations detected by FISH and could have progressed to a copy-number high phenotype. Most dedifferentiated carcinomas belonged to the hypermutated group, whereas pure undifferentiated carcinomas shared molecular genetic alterations with copy-number low or copy-number high tumors. These results indicate that undifferentiated and dedifferentiated endometrial carcinomas are molecularly heterogeneous tumors, which may have prognostic value.

A role for the transducer of the Hippo pathway, TAZ, in the development of aggressive types of endometrial cancer.

Although TAZ, the final effector of the Hippo pathway that modulates epithelial to mesenchymal transition and stemness, has been implicated in the development of different types of cancer, its role in endometrial cancer has not yet been studied. Thus, we evaluated the expression of TAZ in different types of endometrial cancer by immunohistochemistry. TAZ expression was detected in 76% of undifferentiated endometrial carcinomas, 54% of endometrial carcinosarcomas, 46% of endometrial serous carcinomas, 36% of grade 3 endometrioid carcinomas, and 18% of grade 1-2 endometrioid carcinomas, with statistically significant differences. We analyzed the WWTR1 gene that encodes TAZ by FISH and MassARRAY spectrometry, ruling out gene amplification and differential promoter methylation as the main mechanisms that modulate TAZ expression in endometrial tumors. However, we did detect a significant association between Scribble hypoexpression and delocalization with TAZ expression. Moreover, we demonstrated that TAZ promoted invasiveness, and it favored cell motility and tumor growth, in endometrial cancer cell lines. In addition, TAZ expression was associated with the transition from an epithelial to mesenchymal phenotype, both in vitro and in human tumors. Together, these data reveal a previously unknown role for TAZ and the Hippo pathway in the progression of aggressive subtypes of endometrial cancer.

Genome-wide association study identifies ephrin type A receptors implicated in paclitaxel induced peripheral sensory neuropathy.

BACKGROUND: Peripheral neuropathy is the dose limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat solid tumours. This toxicity exhibits great inter-individual variability of unknown origin. The present study aimed to identify genetic variants associated with paclitaxel induced neuropathy via a whole genome approach. METHODS: A genome-wide association study (GWAS) was performed in 144 white European patients uniformly treated with paclitaxel/carboplatin and for whom detailed data on neuropathy was available. Per allele single nucleotide polymorphism (SNP) associations were assessed by Cox regression, modelling the cumulative dose of paclitaxel up to the development of grade 2 sensory neuropathy. RESULTS: The strongest evidence of association was observed for the ephrin type A receptor 4 (EPHA4) locus (rs17348202, p=1.0×10(-6)), and EPHA6 and EPHA5 were among the top 25 and 50 hits (rs301927, p=3.4×10(-5) and rs1159057, p=6.8×10(-5)), respectively. A meta-analysis of EPHA5-rs7349683, the top marker for paclitaxel induced neuropathy in a previous GWAS (r(2)=0.79 with rs1159057), gave a hazard ratio (HR) estimate of 1.68 (p=1.4×10(-9)). Meta-analysis of the second hit of this GWAS, XKR4-rs4737264, gave a HR of 1.71 (p=3.1×10(-8)). Imputed SNPs at LIMK2 locus were also strongly associated with this toxicity (HR=2.78, p=2.0×10(-7)). CONCLUSIONS: This study provides independent support of EPHA5-rs7349683 and XKR4-rs4737264 as the first markers of risk of paclitaxel induced neuropathy. In addition, it suggests that other EPHA genes also involved in axonal guidance and repair following neural injury, as well as LIMK2 locus, may play an important role in the development of this toxicity. The identified SNPs could form the basis for individualised paclitaxel chemotherapy.

The variant rs1867277 in FOXE1 gene confers thyroid cancer susceptibility through the recruitment of USF1/USF2 transcription factors.

In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.

Molecular Heterogeneity of High Grade Colorectal Adenocarcinoma.

High grade colorectal carcinomas (HG-CRCs), which comprise 15% of colorectal carcinomas, are underrepresented in reported molecular studies. Clinicopathological, immunohistochemical, and molecular features of 40 HG-CRCs are described. Moreover, glandular and solid areas of 25 tumors were separately analyzed. The expression of MLH1, PMS2, MSH2, MSH6, p53, E-cadherin, CDX2, CK20, CD8, PDL1, PAN-TRK, c-MET, SMARCB1, ARID1A, SMARCA2, and SMARCA4 was analyzed by immunohistochemistry. Promoter MLH1 methylation was analyzed in tumors with MLH1/PMS2 loss. Next-generation sequencing was used to screen 161 genes for hotspot mutations, copy number variations and gene fusions. In this series, 72.5% of HG-CRCs showed mismatch repair deficiency (MMRd). MMR deficient tumor and MMR proficient (MMRp) tumors showed striking molecular differences. Thus, whereas BRAF mutations were only observed in MMRd tumors, mutations in KRAS and TP53 were more frequent in MMR proficient tumors. Moreover, gene fusions (NTRK1 and MET) were detected only in MMRd tumors, whereas gene amplification (MYC, CCND1 and EGFR) predominated in MMRp/TP53-mutated tumors. Loss of expression of proteins involved in chromatin remodeling, such as ARID1A, was observed only in MMRd HG-CRCs, which also showed more frequently PD-L1 expression and a higher number of tumor infiltrating lymphocytes. The separate analysis of glandular and solid areas indicated that the clonal or subclonal nature of the molecular alterations also depended on MMR status. Mutations in genes such as TP53 and KRAS were always clonal in MMRp-CRCs but occurred as subclonal events in MMRd-CRCs. Gene amplification was implicated in the progression of MMRp tumors, but not in MMRd tumors, in which clonal diversity was due to accumulation of mutations in genes of different pathways such as NOTCH, MMR, or PIK3CA. In summary, intertumor and intratumor molecular heterogeneity in HG-CRCs is mainly due to MMR status.

The Frequency and Prognostic Significance of the Histologic Type in Early-stage Ovarian Carcinoma: A Reclassification Study by the Spanish Group for Ovarian Cancer Research (GEICO).

The frequency and prognostic significance of the histologic type in early-stage ovarian cancer (OC) is not as well established as in advanced stages. In addition, histologic typing based only on morphologic features may be difficult, especially in high-grade tumors. In this study, we have analyzed a prospective cohort of 502 early-stage OCs to investigate their frequency, immunohistochemical characteristics, and survival of the 5 main histologic types. Histotype was assigned according to not only the morphologic features but also according to the expression pattern of WT1, p53, Napsin A, and progesterone receptors. In addition, an extended panel including p16, ß-catenin, HER2, Arid1A, HINF1B, CK7, CDX2, and CK20 was used to refine the diagnosis in difficult cases. In this series, the frequency of the 5 major histologic types was as follows: endometrioid carcinoma, 32.7%; clear cell carcinoma, 25.1%; high-grade serous carcinoma (HGSC), 24.7%; mucinous carcinoma, 10.2%; low-grade serous carcinoma, 4.6%; and others, 2.8%. The combination of morphology and immunohistochemistry allowed the reclassification of 23% of OCs. The lowest concordance was found between samples initially diagnosed as endometrioid, but finally classified as high-grade serous tumors (22% error rate). Endometrioid carcinoma was the most favorable histologic type, whereas HGSC and low-grade serous carcinoma had the worst prognosis. Clear cell carcinoma with abnormal p53 immunostaining pattern also had poor prognosis. Although histologic grade was not a prognostic factor among early-stage endometrioid OCs, distinction between grade 3 endometrioid OC and HGSC is recommended, taking into account differences in prognosis and molecular alterations that can guide different treatments.

Molecular Heterogeneity of Endometrioid Ovarian Carcinoma: An Analysis of 166 Cases Using the Endometrial Cancer Subrogate Molecular Classification.

Endometrioid ovarian carcinoma (EOC) has clinical and biological differences compared with other histologic types of ovarian carcinomas, but it shares morphologic and molecular features with endometrioid endometrial carcinoma. To analyze the molecular heterogeneity of EOC according to the new molecular classification of endometrial cancer and to evaluate the prognostic significance of this molecular classification, we have analyzed 166 early-stage EOC by immunohistochemistry for mismatch repair proteins and p53 expression, and by Sanger sequencing for the exonuclease domain of polymerase epsilon (POLE EDM). In addition, we have carried out next-generation sequencing analysis of tumors with POLE EDM mutations to confirm the ultramutated profile. Eight tumors carried POLE EDM mutations and were classified as ultramutated (5%), 29 showed mismatch repair deficiency and were classified as hypermutated (18%), 16 tumors had a mutated pattern of p53 expression and were classified as p53 abnormal (11%), and 114 tumors did not have any of the previous alterations and were classified as no specific type (66%). Five tumors showed >1 classification criteria. The frequencies of ultramutated and hypermutated tumors were lower in EOC compared with the frequency reported in endometrial cancer. Subrogate molecular groups differed in both morphologic features (histologic grade, squamous and morular metaplasia, and necrosis) and immunohistochemical expression of several biomarkers (ARID1A, nuclear ß-catenin, estrogen receptors, Napsin A, and HINF1B). In addition, the number of CD8 tumor-infiltrating lymphocytes was higher in ultramutated and hypermutated tumors. The most commonly mutated genes in the ultramutated group were ARID1A (100%), PIK3R1, PTEN, BCOR, and TP53 (67% each), whereas no mutations were detected in KRAS. Although the prognosis did not differ among subgroups in the multivariate analysis, a trend toward a better prognosis in POLE-mutated and a worse prognosis in p53 abnormal tumors was observed. In addition, this classification could have important therapeutic implications for the use of immunotherapy in tumors classified as ultramutated and hypermutated.

Mismatch Repair Deficiency in Ovarian Carcinoma: Frequency, Causes, and Consequences.

Mismatch repair deficiency (MMRD) is involved in the initiation of both hereditary and sporadic tumors. MMRD has been extensively studied in colorectal cancer and endometrial cancer, but not so in other tumors, such as ovarian carcinoma. We have determined the expression of mismatch repair proteins in a large cohort of 502 early-stage epithelial ovarian carcinoma entailing all the 5 main subtypes: high-grade serous carcinoma, endometrioid ovarian carcinoma (EOC), clear cell carcinoma (CCC), mucinous carcinoma, and low-grade serous carcinoma. We studied the association of MMRD with clinicopathologic and immunohistochemical features, including tumor-infiltrating lymphocytes in EOC, the histologic type in which MMRD is most frequent. In addition, MLH1 promoter methylation status and massive parallel sequencing were used to evaluate the proportion of sporadic and Lynch syndrome-associated tumors, and the most frequently mutated genes in MMRD EOCs. MMRD occurred only in endometriosis-associated histologic types, and it was much more frequent in EOC (18%) than in CCC (2%). The most frequent immunohistochemical pattern was loss of MLH1/PMS2, and in this group, 80% of the cases were sporadic and secondary to MLH1 promoter hypermethylation. The presence of somatic mutations in mismatch repair genes was the other mechanism of MMRD in sporadic tumors. In this series, the minimum estimated frequency of Lynch syndrome was 35% and it was due to germline mutations in MLH1, MSH2, and MSH6. ARID1A, PTEN, KTM2B, and PIK3CA were the most common mutated genes in this series. Interestingly, possible actionable mutations in ERRB2 were found in 5 tumors, but no TP53 mutations were detected. MMRD was associated with younger age and increased tumor-infiltrating lymphocytes. Universal screening in EOC and mixed EOC/CCC is recommended for the high frequency of MMRD detected; however, for CCC, additional clinical and pathologic criteria should be evaluated to help select cases for analysis.

Determination of CYP2D6 gene copy number by multiplex polymerase chain reaction analysis.

Cytochrome P450 2D6 (CYP2D6) copy number variation (CNV) influences the metabolism of 15-25% of clinical drugs. Here we describe a novel multiplex polymerase chain reaction (PCR) analysis method that accurately detects CYP2D6 CNV and CYP2D6*9 allele. It includes the amplification of 2 CYP2D6 and 7 control (AQP1, CYP3A4, MDR1, and SDHB) fluorescent PCR products that are separated on a capillary sequencer and normalized using reference samples. The technique was validated using 27 PCR-restriction fragment length polymorphism (RFLP) pregenotyped samples and further tested in 75 Caucasian samples. The method assigns the correct CYP2D6 copy number, independent of already characterized CYP2D6 single nucleotide polymorphisms (SNPs), and could easily be applied to clinical samples.

High Frequency of ERBB2 Activating Mutations in Invasive Lobular Breast Carcinoma with Pleomorphic Features.

Background: Characterisation of molecular alterations of pleomorphic lobular carcinoma (PLC), an aggressive subtype of invasive lobular carcinoma (ILC), have not been yet completely accomplished. Methods: To investigate the molecular alterations of invasive lobular carcinoma with pleomorphic features, a total of 39 tumour samples (in situ and invasive lesions and lymph node metastases) from 27 patients with nuclear grade 3 invasive lobular carcinomas were subjected to morphological, immunohistochemical and massive parallel sequencing analyses. Results: Our observations indicated that invasive lobular carcinomas with pleomorphic features were morphologically and molecularly heterogeneous. All cases showed absence or aberrant expression of E-cadherin and abnormal expression of ß-catenin and p120. CDH1 (89%), PIK3CA (33%) and ERRB2 (26%) were the most common mutated genes. ERBB2 mutations preferentially affected the tyrosine-kinase activity domain, being the most frequent the targetable mutation p.L755S (57%). We also observed higher frequency of mutations in ARID1B, KMT2C, MAP3K1, TP53 and ARID1A in PLC than previously reported in classic ILC. Alterations related to progression from in situ to invasive carcinoma and/or to lymph node metastases included TP53 mutation, amplification of PIK3CA and CCND1 and loss of ARID1A expression. Conclusions: The high frequency of ERBB2 mutations observed suggests that ERBB2 mutation testing should be considered in all invasive lobular carcinomas with nuclear grade 3.

Molecular Basis of Tumor Heterogeneity in Endometrial Carcinosarcoma.

Endometrial carcinosarcoma (ECS) represents one of the most extreme examples of tumor heterogeneity among human cancers. ECS is a clinically aggressive, high-grade, metaplastic carcinoma. At the morphological level, intratumor heterogeneity in ECS is due to an admixture of epithelial (carcinoma) and mesenchymal (sarcoma) components that can include heterologous tissues, such as skeletal muscle, cartilage, or bone. Most ECSs belong to the copy-number high serous-like molecular subtype of endometrial carcinoma, characterized by the TP53 mutation and the frequently accompanied by a large number of gene copy-number alterations, including the amplification of important oncogenes, such as CCNE1 and c-MYC. However, a proportion of cases (20%) probably represent the progression of tumors initially belonging to the copy-number low endometrioid-like molecular subtype (characterized by mutations in genes such as PTEN, PI3KCA, or ARID1A), after the acquisition of the TP53 mutations. Only a few ECS belong to the microsatellite-unstable hypermutated molecular type and the POLE-mutated, ultramutated molecular type. A common characteristic of all ECSs is the modulation of genes involved in the epithelial to mesenchymal process. Thus, the acquisition of a mesenchymal phenotype is associated with a switch from E- to N-cadherin, the up-regulation of transcriptional repressors of E-cadherin, such as Snail Family Transcriptional Repressor 1 and 2 (SNAI1 and SNAI2), Zinc Finger E-Box Binding Homeobox 1 and 2 (ZEB1 and ZEB2), and the down-regulation, among others, of members of the miR-200 family involved in the maintenance of an epithelial phenotype. Subsequent differentiation to different types of mesenchymal tissues increases tumor heterogeneity and probably modulates clinical behavior and therapy response.

SDHC mutation in an elderly patient without familial antecedents.

Head and neck paragangliomas are usually asymptomatic and benign tumours arising mainly from the carotid body and the vagal, tympanic or jugular glomus. The majority of patients develop sporadic masses, and around 30% of cases harbour germline mutations in one of the succinate dehydrogenase genes: SDHB, SDHC or SDHD. In these hereditary cases, the presence of familial antecedents of the disease, multiplicity/bilaterality, young age at onset, and more recently, presence of gastrointestinal stromal tumours, are main factors to be considered. Here we describe a new mutation (c.256-257insTTT) affecting the SDHC gene in a 60-year-old-patient with a single head and neck paraganglioma, and without familial antecedents of the disease. In silico splice site analysis showed that this variant created a cryptic splice acceptor site and loss of heterozygosity (LOH) supported the pathogenic role of the mutation. Control population analyses did not detect this variant but revealed a novel SDHC polymorphism that exhibited a frequency of 0.3% (3/1020). This latter finding highlights the importance of assessing the clinical relevance of variants of unknown significance by means of analysing sufficient controls. Despite having found a germline mutation in an older, apparently sporadic patient, we consider that the high costs of analysing all susceptibility genes related to the disease support the recommendation of screening for mutations only in patients fulfilling the above criteria.

Graves' disease is associated with a defective expression of the immune regulatory molecule galectin-9 in antigen-presenting dendritic cells.

INTRODUCTION: Patients with autoimmune thyroid disease (AITD) show defects in their immune-regulatory mechanisms. Herein we assessed the expression and function of galectin-1 and galectin-9 (Gal-1, Gal-9) in dendritic cells (DCs) from patients with AITD. MATERIALS AND METHODS: Peripheral blood samples from 25 patients with Graves' disease (GD), 11 Hashimoto's thyroiditis (HT), and 24 healthy subjects were studied. Thyroid tissue samples from 44 patients with AITD and 22 patients with goiter were also analyzed. Expression and function of Gal-1 and Gal-9 was assessed by quantitative RT-PCR, immunofluorescence and flow cytometry. RESULTS: A diminished expression of Gal-9, but not of Gal-1, by peripheral blood DCs was observed in GD patients, mainly in those with Graves´ ophthalmopathy, and a significant negative association between disease severity and Gal-9 expression was detected. In addition, the mRNA levels of Gal-9 and its ligand TIM-3 were increased in thyroid tissue from AITD patients and its expression was associated with the levels of Th1/Th12/Th17 cytokines. Immunofluorescence studies proved that intrathyroidal Gal-9 expression was confined to DCs and macrophages. Finally, in vitro functional assays showed that exogenous Gal-9 had a suppressive effect on the release of Th1/Th2/Th17 cytokines by DC/lymphocyte autologous co-cultures from both AITD patients and healthy controls. CONCLUSIONS: The altered pattern of expression of Gal-9 in peripheral blood DCs from GD patients, its correlation with disease severity as well as its ability to suppress cytokine release suggest that Gal-9 could be involved in the pathogenesis of AITD.

In vitro adhesion of an avian pathogenic Escherichia coli O78 strain to surfaces of the chicken intestinal tract and to ileal mucus.

The role of fimbria in adherence of an avian pathogenic Escherichia coli (APEC) O78 strain 789 to chicken intestine was studied. Bacterial adhesion to tissue sections representing the regions within the chicken intestinal tract was determined by using immunohistochemical methods. E. coli 789 grown to express the type 1 fimbria adhered efficiently to the crop epithelium, to the lamina propria of intestinal villi, and to the apical surfaces of both the mature as well as the crypt-located enterocytes in intestinal villi, whereas no adhesion to mucus-producing goblet cells was detected. The adhesion was inhibited by mannoside and the role of type 1 fimbriae in the observed adhesion was confirmed with a recombinant strain expressing type 1 fimbriae genes cloned from E. coli and Salmonella enterica. E. coli 789 strain grown to favor AC/I fimbriae expression as well as the recombinant E. coli strain expressing the fac genes adhered to goblet cells but only poorly to the other epithelial sites. E. coli strain 789 as well as S. enterica serovar Typhimurium IR715 and S. enterica serovar Enteriditis TN2 strains were able to multiply in ileal mucus medium. The type 1 fimbria expressing bacteria adhered to the ileal mucus, whereas the AC/I fimbriated strains showed poor adherence to the mucus. The adhesion of E. coli 789 onto the crop epithelium and the follicle associated epithelium of the chicken ileum was efficiently inhibited by an adhesive strain ST1 of Lactobacillus crispatus isolated from chicken, whereas poor inhibition of E. coli adherence was observed with the weakly adhesive L. crispatus strain 134mi. The type 1 fimbriae may be important in colonization of the chicken intestine by APEC and Salmonella.

Plasmacytoid dendritic cells in patients with autoimmune thyroid disease.

BACKGROUND: Patients with autoimmune thyroid diseases (AITD) show defects in immunoregulatory mechanisms. Herein we assessed the expression of different regulatory receptors in circulating and thyroid dendritic cells (DCs). DESIGN: Peripheral blood samples from 49 patients with Hashimoto's thyroiditis, 35 with Graves' disease, and 34 healthy subjects were studied. Clinical parameters included grades of goiter and ophthalmopathy, thyroid function, and antibody tests. Thyroid tissue samples from 10 AITD patients were also analyzed. Levels of DCs and their expression of different regulatory molecules (IDO, ILT2, ILT3, PSGL-1, PD-L1) were studied. In vitro interferon-α response by plasmacytoid DCs (pDCs) and tryptophan (Trp) metabolites were determined. RESULTS: Significant low levels of pDCs, but not conventional DCs, were detected in the peripheral blood from AITD patients, mainly in those with severe disease. Furthermore, a diminished expression of ILT3, PSGL-1, and CD69 by peripheral blood pDCs from AITD patients was observed. An increased number of pDCs was found in thyroid tissue, showing a diminished expression of ILT3 and PSGL-1. A lower proportion of IDO+ pDCs, a significant increase in Trp levels, a decrease in the kyneruine/Trp ratio, and an increased in vitro interferon-α response were present in AITD patients. Finally, a significant correlation was found between the in vitro synthesis of IL-10 by stimulated T cells and expression of IDO by pDCs. CONCLUSIONS: The diminished number of pDCs in the peripheral blood from AITD patients as well as their abnormal phenotype could contribute significantly to the pathogenesis.

Regulatory polymorphisms in ß-tubulin IIa are associated with paclitaxel-induced peripheral neuropathy.

PURPOSE: Peripheral neuropathy is the dose-limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat several solid tumors such as breast, lung, and ovary. The cytotoxic effect of paclitaxel is mediated through ß-tubulin binding in the cellular microtubules. In this study, we investigated the association between paclitaxel neurotoxicity risk and regulatory genetic variants in ß-tubulin genes. EXPERIMENTAL DESIGN: We measured variation in gene expression of three ß-tubulin isotypes (I, IVb, and IIa) in lymphocytes from 100 healthy volunteers, sequenced the promoter region to identify polymorphisms putatively influencing gene expression and assessed the transcription rate of the identified variants using luciferase assays. To determine whether the identified regulatory polymorphisms were associated with paclitaxel neurotoxicity, we genotyped them in 214 patients treated with paclitaxel. In addition, paclitaxel-induced cytotoxicity in lymphoblastoid cell lines was compared with ß-tubulin expression as measured by Affymetrix exon array. RESULTS: We found a 63-fold variation in ß-tubulin IIa gene (TUBB2A) mRNA content and three polymorphisms located at -101, -112, and -157 in TUBB2A promoter correlated with increased mRNA levels. The -101 and -112 variants, in total linkage disequilibrium, conferred TUBB2A increased transcription rate. Furthermore, these variants protected from paclitaxel-induced peripheral neuropathy [HR, 0.62; 95% confidence interval (CI), 0.42-0.93; P = 0.021, multivariable analysis]. In addition, an inverse correlation between TUBB2A and paclitaxel-induced apoptosis (P = 0.001) in lymphoblastoid cell lines further supported that higher TUBB2A gene expression conferred lower paclitaxel sensitivity. CONCLUSIONS: This is the first study showing that paclitaxel neuropathy risk is influenced by polymorphisms regulating the expression of a ß-tubulin gene.

Hematologic ß-tubulin VI isoform exhibits genetic variability that influences paclitaxel toxicity.

Cellular microtubules composed of α-ß-tubulin heterodimers that are essential for cell shape, division, and intracellular transport are valid targets for anticancer therapy. However, not all the conserved but differentially expressed members of the ß-tubulin gene superfamily have been investigated for their role in these settings. In this study, we examined roles for the hematologic isoform ß-tubulin VI and functional genetic variants in the gene. ß-tubulin VI was highly expressed in blood cells with a substantial interindividual variability (seven-fold variation in mRNA). We characterized DNA missense variations leading to Q43P, T274M, and R307H, and a rare nonsense variant, Y55X. Because variations in the hematologic target of microtubule-binding drugs might alter their myelosuppressive action, we tested their effect in cell lines stably expressing the different ß-tubulin VI full-length variants, finding that the T274M change significantly decreased sensitivity to paclitaxel-induced tubulin polymerization. Furthermore, patients treated with paclitaxel and carrying ß-tubulin VI T274M exhibited a significantly lower thrombocytopenia than wild-type homozygous patients (P = 0.031). Together, our findings define ß-tubulin VI as a hematologic isotype with significant genetic variation in humans that may affect the myelosuppresive action of microtubule-binding drugs. A polymorphism found in a tubulin isoform expressed only in hemapoietic cells may contribute to the patient variation in myelosuppression that occurs after treatment with microtubule-binding drugs.