Search

Summary of Patients and Clinical Data.

Supplementary Figure 1. Unsupervised Clustering on CCA samples. Supplementary Figure 2. Alterations Found in CCA Clusters. Supplementary Figure 3. MAP2K4 Homozygous Deletions and ERBB2 Amplifications. Supplementary Figure 4. Alterations Related to Structural Variations Found in CCAs. Supplementary Figure 5. FIREFLY Analysis of Pathways Systematically Dysregulated by Somatic Promoter Mutations that Alter Transcription Factor Binding. Supplementary Figure 6. Epigenetic Clusters and Mutation Signatures.

Structural Variations across 71 WGS CCAs.

Supplementary Table S2: Main characteristics of the 36 CTAs profiled in the study according to CTDatabase (http://www.cta.lncc.br/).

Summary of CCA Alterations.

Further details of methods, in addition to the supplementary figure and table legends.

Alterations in CCA Clusters.

Coverage Statistics and List of Genes for Targeted Sequencing.

The content of tumor-derived extracellular vesicles (EVs) can regulate the tumor microenvironment and functionally acts in favor of cancer aggressiveness. To better elucidate the role of EVs in the interplay between immune system and tumor microenvironment, the purpose of this study was to analyze the effect of head and neck squamous cells carcinoma (HNSCC)-derived EVs on the modulation of inflammasomes - mediators of pyroptosis and secretion of inflammatory factors by macrophages. Our results showed that macrophages treated with the Vesicular Secretome Fraction (VSF) isolated from patient-derived HNSCC presented a reduction in the secretion of mature IL-1β and caspase-1 without affecting cell viability. An analysis of the protein content of HNSCC-derived VSF by antibody array revealed that some of the most expressed proteins share a correlation with Transforming Growth Factor-beta (TGF-β) activity. Since TGF-β is related to the inhibition of the NF-kB-related pathways, including those required for the priming phase of the inflammasomes, we sought to evalute the interference of the VSF in the induction of inflammasome components. In fact, HNSCC-derived VSF inhibited the induction of pro-IL-1β and pro-caspase-1 proteins and NLRP3 gene expression during the priming phase of inflammasome activation. Thus, our findings contribute to a better understanding of how tumor-derived EVs modulate inflammatory response by demonstrating their role in inhibiting NLRP3 inflammasomes.

Background The incidence of oral tongue squamous cell carcinoma (OTSCC) is increasing among younger birth cohorts. The etiology of early-onset OTSCC (diagnosed before the age of 50 years) and cancer driver genes remain largely unknown. Methods The Sequencing Consortium of Oral Tongue Cancer was established through the pooling of somatic mutation data of oral tongue cancer specimens (n = 227 [107 early-onset cases]) from 7 studies and The Cancer Genome Atlas. Somatic mutations at microsatellite loci and Catalog of Somatic Mutations in Cancer mutation signatures were identified. Cancer driver genes were identified with the MutSigCV and WITER algorithms. Mutation comparisons between early- and typical-onset OTSCC were evaluated via linear regression with adjustments for patient-related factors. Results Two novel driver genes (ATXN1 and CDC42EP1) and 5 previously reported driver genes (TP53, CDKN2A, CASP8, NOTCH1, and FAT1) were identified. Six recurrent mutations were identified, with 4 occurring in TP53. Early-onset OTSCC had significantly fewer nonsilent mutations even after adjustments for tobacco use. No associations of microsatellite locus mutations and mutation signatures with the age of OTSCC onset were observed. Conclusions This international, multicenter consortium is the largest study to characterize the somatic mutational landscape of OTSCC and the first to suggest differences by age of onset. This study validates multiple previously identified OTSCC driver genes and proposes 2 novel cancer driver genes. In analyses by age, early-onset OTSCC had a significantly smaller somatic mutational burden that was not explained by differences in tobacco use. Lay summary This study identifies 7 specific areas in the human genetic code that could be responsible for promoting the development of tongue cancer. Tongue cancer in young patients (under the age of 50 years) has fewer overall changes to the genetic code in comparison with tongue cancer in older patients, but the authors do not think that this is due to differences in smoking rates between the 2 groups. The cause of increasing cases of tongue cancer in young patients remains unclear.

Evaluate the biological action of valproic acid in the acetylation of histones and in the methylation of tumor suppressor genes via oral rinse in patients with a previous history of head and neck squamous cell carcinoma (HNSCC). Forty-two active or former smokers were included in this randomized, double-blind, placebo-controlled trial. Oral rinse samples were collected prior to treatment with valproic acid or placebo and after 90 days of treatment. The methylation status of five tumor suppressor genes and histone acetylation were evaluated by pyrosequencing and ELISA techniques, respectively. Differences between the 90-day and baseline oral rinse acetylation and methylation results were analyzed by comparing groups. Thirty-four patients were considered for analysis. The mean percentage adherence in the valproic and placebo groups was 93.4 and 93.0, respectively (p = 0.718). There was no statistically significant difference between groups when comparing the medians of the histone acetylation ratio and the methylation ratio for most of the studied genes. A significant reduction in the DCC methylation pattern was observed in the valproic group (p = 0.023). The use of valproic acid was safe and accompanied by good therapeutic adherence. DCC methylation was lower in the valproic acid group than in the placebo group.

Dorival Mendes Rodrigues-Junior,1,2 Maria Fernanda de Andrade Pelarin,1 Helena Bonciani Nader,1 André Luiz Vettore,3 Maria Aparecida Silva Pinhal1,4 1Department of Biochemistry, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil; 2Institute of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden; 3Department of Biological Science, Universidade Federal de São Paulo (UNIFESP), Diadema, Brazil; 4Department of Biochemistry, Faculdade de Medicina do ABC, Santo André, BrazilCorrespondence: Dorival Mendes Rodrigues-JuniorLaboratório de Biologia Molecular do Câncer, UNIFESP, Rua Pedro de Toledo, 669 – 11° Andar, São Paulo, SP 04039-032, BrazilEmail dorivalmrjr@gmail.comMaria Aparecida Silva PinhalDepartment of Biochemistry, Universidade Federal de São Paulo (UNIFESP), Rua Três de Maio, 100, Vila Clementino, São Paulo, SP 04044-020, BrazilEmail maspinhal@yahoo.com.brIntroduction: Multiple myeloma (MM) remains an incurable disease, and patient survival requires a better understanding of this malignancy’s molecular aspects. Heparanase (HPSE) is highly expressed in aggressive MM cells and related to tumor growth, metastasis, and bortezomib (BTZ) resistance. Thus, targeting HPSE seems to be a promising approach for MM treatment, and because microRNAs (miRNAs) have emerged as potential regulators of HPSE expression, the use of extracellular vesicles (EVs) can allow the efficient delivery of therapeutic miRNAs.Methods: We used prediction algorithms to identify potential miRNAs that regulate negatively HPSE expression. RT-qPCR was performed to assess miRNAs and HPSE expression in MM lines (U266 and RPMI-8226). Synthetic miRNA mimics were electroporated in MM cells to understand the miRNA contribution in HPSE expression, glycosaminoglycans (GAGs) profile, cell proliferation, and cell death induced by BTZ. EVs derived from HEK293T cells were engineered with miRNAs to evaluate their therapeutic potential combined with BTZ.Results: It revealed a direct association between BTZ sensitivity, HPSE, and miR-1252-5p expressions. Moreover, overexpression of miR-1252-5p significantly reduced HPSE expression and HPSE enzymatic activity in MM cells. The higher level of miR-1252-5p was correlated with a reduction of cell viability and higher sensitivity to BTZ. Further, EVs carrying miR-1252-5p increased MM cells’ sensitivity to BTZ treatment.Conclusion: These results showed that miR-1252-5p could negatively regulate HPSE in MM, indicating the use of EVs carrying miR-1252-5p as a potential novel BTZ sensitization approach in MM cells.Keywords: bortezomib, cancer, extracellular vesicles, exosomes, heparanase, multiple myeloma, microRNA

Background In the last decades, survival rates in head and neck squamous cell carcinoma (HNSCC) have not changed, with a five-year survival of only 50%. Thus, there is a great need for the identification of new molecular targets and development of novel therapeutic strategies. Cancer-testis antigens (CTAs) are expressed in various types of tumor but rarely in healthy normal tissues. Therefore, they appear as ideal targets for immunotherapy approaches, as well as, unique markers for cancer diagnosis/prognosis. Objective This study evaluated the expression pattern of cancer/testis antigens (CTA) in HNSCC samples and correlated the expression data with the clinicopathological prognostic variables. Methods An in silico screening was performed using all CTA genes cataloged on the CTDatabase and the expression of the eight CTA genes (ARMC3, DDX53, FTHL17, GAGE1, MAGEA11, SYCE1, TCP11, and XAGE1) was examined in 89 HNSCC and 20 normal mucosa samples using RT-PCR analysis. Results GAGE1 (48.3%), XAGE1 (40.4%) and MAGEA11 (19.1%) were frequently and specifically expressed in HNSCC samples and 68.5% of the cases expressed at least one of these antigens. Moreover, GAGE1 and XAGE1 mRNA positivity was significantly associated with the presence of metastasis in the lymph nodes (p=0.038 and p=0.023, respectively) and, by multivariate analysis, male gender (p=0.032), advanced clinical stage (p=0.018) and mRNA positivity for GAGE1 (p=0.010) were independent prognostic factors for overall survival. Conclusion These findings suggest GAGE1 and XAGE1 expressions to be useful as prognostic markers for HNSCC.

Management of locally advanced head and neck squamous cell carcinoma (HNSCC) requires a multi-prong approach comprising surgery, radiation and/or chemotherapy, yet outcomes are limited. This is largely due to a paucity of biomarkers that can predict response to specific treatment modalities. Here, we evaluated TGFβ3 protein levels in extracellular vesicles (EVs) released by HNSCC cells as a predictor for response to chemoradiation therapy (CRT). To this end, specific EV-fractions were isolated from cell lines or HNSCC patient plasma, and TGFβ3 protein was quantified. In patients treated with CRT, TGFβ3 levels were found to be significantly higher in plasma EV-fractions or non-responders compared with responders. High levels of TGFβ3 levels in Annexin V-EVs were associated with the worst progression-free survival. In vitro experiments demonstrated that TGFβ3 silencing sensitized HNSCC cells to cytotoxic therapies, and this phenotype could be rescued by treatment with exogenous. In addition, specific EV-fractions shed by cisplatin-resistant cells were sufficient to transfer the resistant phenotype to sensitive cells through activation of TGFβ-signaling pathway. Therefore, our data show that TGFβ3 transmitted through EV plays a significant role in response to cytotoxic therapy, which can be exploited as a potential biomarker for CRT response in HNSCC patients treated with curative intent.

Multiple myeloma (MM) is an incurable disease; a better understanding of the molecular aspects of this hematological malignancy could contribute to the development of new treatment strategies and help to improve the survival rates of patients with MM. Previously, the methylation status of the deleted in colorectal cancer (DCC) gene was correlated with the survival rate of patients with MM, thus the main goal of this study was to understand DCC contribution to MM tumorigenesis, and to assess the impact of DCC inhibition in the MM response to treatment with bortezomib. Our results demonstrated that hypermethylation of the DCC promoter inhibits gene expression, and DCC silencing is significantly correlated with a reduction in cell viability and an increase in cell death induced by bortezomib. In conclusion, our results suggested that hypermethylation is an important mechanism of DCC expression regulation in MM and that the absence of DCC contributes to the enhanced sensitivity to treatment with bortezomib.

Background There is a paucity of plasma-based biomarkers that prospectively segregate the outcome of patients with head and neck squamous-cell carcinoma (HNSCC) treated with chemoradiation therapy (CRT). Plasma extracellular vesicles (EVs) might be an alternative source for discovery of new specific markers present in patients with HNSCC, which could help to re-direct patients to appropriate curative therapies without delay. Methods In order to identify new markers in plasma compartments, Cholerae toxin B chain (CTB) and Annexin V (AV) were used to isolate EVs from pooled plasma samples from patients with locally advanced HNSCC who responded (CR, n = 6) or presented incomplete response (NR, n = 6) to CRT. The crude plasma and EVs cargo were screened by antibody array. Results Of the 370 polypeptides detected, 119 proteins were specific to NR patients while 38 were exclusive of the CR subjects. The Gene Set Enrichment Analysis (GSEA) and Search Tool for the Retrieval of Interacting Genes (STRING) database analysis indicated that the content of circulating plasma EVs might have a relevant function for the tumor intercellular communication in the HNSCC patients. Conclusion This study provides a list of potential markers present in plasma compartments that might contribute to the development of tools for prediction and assessment of CRT response and potentially guide therapeutic decisions in this context. Electronic supplementary material The online version of this article (10.1186/s12885-019-5565-9) contains supplementary material, which is available to authorized users.

Purpose Among alkaloids, abundant secondary metabolites in plants, aporphines constitute a class of compounds with interesting biological activities, including anticancer effects. The present study evaluated the anticancer activities of 14 substances, including four aporphine derivatives acquired through the biomonitoring of (±)-apomorphine hydrochloride total synthesis from 2-phenethylamine and 3,4-dimethoxybenzaldehyde against head and neck squamous cell carcinoma (HNSCC). Methods The cytotoxic effects of compounds against a panel of HNSCC cell lines were determined by PrestoBlue cell viability assay, while the genotoxicity of substances was evaluated by micronucleus test. Cell death was detected by flow cytometry (Annexin V/7AAD) and western blot analysis was used to detect the presence of cleaved Caspase-3 molecules. Results The aporphine and isoquinoline derivatives APO, C1, and A5 significantly reduced HNSCC cell viability and promoted DNA damages in these cells. Further, by activating the Caspase-3 pathway, these substances were able to induce apoptosis. Conclusion Our results revealed that APO, C1, and A5 exhibit cytotoxic effects in HNSCC cells. The mechanisms of action appear to be partly via the generation of DNA damages and apoptosis induction through Caspase-3 pathway activation. This study provides preclinical data that suggest a potential therapeutic role for APO, C1, and A5 against head and neck cancer cells.

Background: Aberrant methylation is a frequent event in oral cancer. Methods: In order to better characterize these alterations, a search for genes downregulated by aberrant methylation in oral squamous cell carcinoma (OSCC) was conducted through the mining of ORESTES dataset. Findings were further validated in OSCC cell lines and patients? samples and confirmed using TCGA data. Differentially expressed genes were identified in ORESTES libraries and validated in vitro using RT-PCR in HNSCC cell-lines and OSCC tumor samples. Further confirmation of these results was performed using mRNA expression and methylation data from The Cancer Genome Atlas (TCGA) data. Results: From the set of genes selected for validation, CA3 and FHL1 were downregulated in 60% (12/20) and 75% (15/20) of OSCC samples, respectively, and in HNSCC cell lines. The treatment of cell lines JHU-13 and FaDu with the demethylating agent 5'-aza-dC was efficient in restoring CA3 and FHL1 expression. TCGA expression and methylation data on OSCC confirms the downregulation of these genes in OSCC samples and also suggests that expression of CA3 and FHL1 is probably regulated by methylation. The downregulation of CA3 and FHL1 observed in silico was validated in HNSCC cell lines and OSCC samples, showing the feasibility of integrating different datasets to select differentially expressed genes in silico. Conclusions: These results showed that the downregulation of CA3 and FHL1 data observed in the ORESTES libraries was validated in HNSCC cell lines and OSCC samples and in a large cohort of samples from the TCGA database. Moreover, it suggests that expression of CA3 and FHL1 could probably be regulated by methylation having an important role the oral carcinogenesis. Made available in DSpace on 2020-01-07T18:10:32Z (GMT). No. of bitstreams: 1 APInVitroFelipe.pdf: 1315888 bytes, checksum: ea33d6217f7d184bc536baa3eee21e68 (MD5) Previous issue date: 2018 Article number: 193.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

Despite significant advances in the treatment of head and neck squamous cell carcinoma (HNSCC), the survival rate has not changed in the last decades. Therefore, the development of novel therapeutic strategies is pursued. Cancer–testis antigens (CTA) are strong immunogenic proteins with a tumor-restricted expression pattern, and are considered ideal targets for tumor-specific immunotherapeutic approaches. In this study, using an in silico approach, we selected, among 139 previously described CTA, candidates to be evaluated in 89 HNSCC and 20 normal mucosa samples. SPANX-CD (71.9%), MAGEB2 (44.9%), MAGEA1 (44.9%), MAGEB6 (32.6%), and CXORF48 (27.0%) were found frequently expressed in HNSCC, and over 85% of the tumors expressed at least one of these five CTAs. The mRNA positivity of CXORF48, MAGEB6, and CRISP2 presented significant associations with recognized clinical features for poor outcome. Furthermore, MAGEA3/6 positivity was associated with significantly better disease-free survival (DFS, P = 0.014), and the expression of this antigen was shown to be an independent prognostic factor for tumor recurrence. In conclusion, one of five selected CTAs is expressed in at least 85% of the HNSCCs, suggesting a possible usage as target for immunotherapeutic approaches, and the mRNA-positivity for MAGEA3/6 is shown to be an independent marker for DFS. Mol Cancer Ther; 14(3); 828–34. ©2015 AACR.

Background Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. The purposes of this study were to determine the methylation profile of exfoliated tumors cells collected from patients with head and neck squamous cell carcinoma (HNSCC) and to evaluate its prognostic significance. Methods The methylation profile and level of a 20-gene panel were evaluated by quantitative methylation-specific polymerase chain reaction (qMSP) in exfoliated tumor cell samples from 96 patients with HNSCC. Results CCNA1 (60.4%), DCC (54.2%), and TIMP3 (35.4%) were frequently methylated in these samples. Patients with exfoliated tumors cells positive for DCC methylation showed a trend toward a lower local recurrence-free survival. Conclusion These findings indicate that a low invasive method could be used to access the methylation profile of exfoliated cells from patients with HNSCC. Moreover, our data provide evidence that hypermethylation of DCC could be useful as prognostic indicator for this malignancy. © 2013 Wiley Periodicals, Inc. Head Neck 36: 631–637, 2014

Viviane Sonaglio,1 Ana C de Carvalho,2 Silvia R C Toledo,3,4 Carolina Salinas-Souza,3,4 André L Carvalho,5 Antonio S Petrilli,3 Beatriz de Camargo,6 André L Vettore21Pediatrics Department, A C Camargo Hospital, São Paulo, Brazil; 2Biological Science Department, Federal University of São Paulo, Diadema, Brazil; 3Department of Pediatrics, Pediatric Oncology Institute, GRAACC/Federal University of São Paulo, São Paulo, Brazil; 4Department of Morphology and Genetics, Federal University of São Paulo, São Paulo, Brazil; 5Department of Head and Neck Surgery, PIO XII Foundation, Barretos Cancer Hospital, Barretos, São Paulo, Brazil; 6Research Program Pediatric Oncology Program, CPNq, Instituto Nacional do Cancer, Rio de Janeiro, BrazilAbstract: Osteosarcoma (OS) is the eighth most common form of childhood and adolescence cancer. Approximately 10%–20% of patients present metastatic disease at diagnosis and the 5-year overall survival remains around 70% for nonmetastatic patients and around 30% for metastatic patients. Metastatic disease at diagnosis and the necrosis grade induced by preoperative treatment are the only well-established prognostic factors for osteosarcoma. The DNA aberrant methylation is a frequent epigenetic alteration in humans and has been described as a molecular marker in different tumor types. This study evaluated the DNA aberrant methylation status of 18 genes in 34 OS samples without previous chemotherapy treatment and in four normal bone specimens and compared the methylation profile with clinicopathological characteristics of the patients. We were able to define a three-gene panel (AIM1, p14ARF, and ESR1) in which methylation was correlated with OS cases. The hypermethylation of p14ARF showed a significant association with the absence of metastases at diagnoses, while ESR1 hypermethylation was marginally associated with worse overall survival. This study demonstrated that aberrant promoter methylation is a common event in OS and provides evidence that p14ARF and ESR1 hypermethylation could be useful as a prognostic indicator for this disease.Keywords: DNA methylation, ESR1, hypermethylation, osteosarcoma, p14ARF

Mutations in JAK-STAT signaling pathway genes have been associated with the development of various hematological tumors, but have not been investigated in head and neck tumors, and the PIK3CA, BRAF and KRAS genes have been described in a few cases of head and neck squamous cell carcinoma (HNSCC). In the present study, we determined the mutation status in members of the MAPK, PI3K-AKT and JAK-STAT pathways in HNSCC. Mutations in the KRAS, BRAF, PIK3CA, JAK1 and JAK2 genes were evaluated in 94 HNSCCs by direct DNA sequencing analysis using cDNA synthesized from RNA extracted from patient tumor cells. All patients evaluated had wild-type KRAS, BRAF and PIK3CA genes. Furthermore, although some known polymorphisms have been found in JAK1 genes (rs45598436, rs17127063, rs2230587, rs3737139, rs2230588 and rs12129819) and JAK2 (rs10429491, rs2230723, rs2230724 and rs41316003), no mutation could be detected. Our data indicate that mutations in these kinase genes seem to be rare events in HNSCC.

Hypermethylation in the promoter regions of genes is associated with suppression of gene expression and has been considered a potential molecular marker for several tumor types, including head and neck squamous cell carcinomas (HNSCC). Moreover, hypermethylation can be detected in body fluids such as saliva and can be useful for the diagnosis and prognosis of patients suffering from cancer. To evaluate the hypermethylation profile as a tool for early detection of tumor recurrences, this study determines the methylation status of 24 genes in salivary rinses collected from HNSCC patients at diagnosis, just after the last curative treatment and in the patients' follow-up visit at 6 months after treatment. In the analysis of salivary rinse samples taken at diagnosis of HNSCC patients, five genes (CCNA1, DAPK, DCC, MGMT and TIMP3) showed high specificity and sensitivity. Hypermethylation in any of these five genes was correlated with the presence of tumors in the oral cavity. Patients with TIMP3 methylation in samples collected 6 months after the last curative treatment had lower local recurrence-free survival (P = 0.008). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (P = 0.025). This study presents, for the first time, the detection of TIMP3 promoter hypermethylation in post-treatment salivary rinse as an independent prognostic maker for local recurrence-free survival in patients with HNSCC, justifying the use of DNA hypermethylation detection in saliva as a tool for identifying and monitoring HNSCC patients' subgroups with high risk of developing local recurrence.

Dacomitinib, an irreversible pan-HER inhibitor, had shown modest clinical activity in squamous cell carcinoma of head and neck (SCCHN) patients. Therefore, validated predictive biomarkers are required to identify patients most likely to benefit from this therapeutic option. To characterize the genetic landscape of cisplatin-treated SCCHN genomes and identify potential predictive biomarkers for dacomitinib sensitivity, we performed whole exome sequencing on 18 cisplatin-resistant metastatic SCCHN tumors and their matched germline DNA. Platinum-based chemotherapy elevated the mutation rates of SCCHN compared to chemotherapy-naïve SCCHNs. Cisplatin-treated SCCHN genomes uniquely exhibited a novel mutational signature characterized by C:G to A:T transversions at CCR sequence contexts that may have arisen due to error-prone translesional synthesis. Somatic mutations in REV3L, the gene encoding the catalytic subunit of DNA polymerase ζ involved in translesional synthesis, are significantly enriched in a subset of patients who derived extended clinical benefit to dacomitinib (P = 0.04). Functional assays showed that loss-of-function of REV3L dramatically enhanced the sensitivity of SCCHN cells to dacomitinib by the loss of both translesion synthesis and homologous recombination pathways. Our data suggest that the ‘platinum’ mutational signature and inactivation of REV3L may inform treatment options in patients of recurrent SCCHN.

SummaryThe development of locoregional recurrence is the main reason for treatment failure in head and neck squamous cell carcinomas (HNSCC) and the remaining of tumor cells in surgical margins is associated with recurrence. Surgical margins are considered negative based on histologic assessment of the pathological specimen. However, this method lacks sensitivity in identifying cells that already started malignant transformation but have not yet developed a pathologic phenotype. We investigated the usefulness of assessing the expression of PTHLH, EPCAM, MMP9, LGALS1 and MET for the detection of molecular alterations in histologically negative surgical margins and determine the correlation of these tumor-related alterations with clinical and prognostic parameters. Differential gene expression was determined by quantitative RT–PCR analyses in normal mucosa, HNSCC and negative margin samples. Thirty-eight percent of the histologically negative surgical margins examined were margin-positive for overexpression of at least one of the genes evaluated. Moreover, MMP9 and PTHLH overexpression in the surgical margins was associated with the development of second primary tumors (p=0.002) and lower rates of local control (log rank test p=0.022; HR=4.186; p=0.035), respectively. These findings demonstrate that the overexpression of tumor-related genes in histologically negative surgical margins is a frequent event. The use of qRT–PCR may be an useful tool in detecting actually negative HNSCC surgical margins and the overexpression of specific genes in these margins could be helpful in the identification of patients with a higher risk of developing second primary tumors and local recurrences, thus aiding the surgeon in the delineation of the HNSCC resection extent and helping in the planning of adjuvant therapy.

Lourenco S V, Coutinho-Camillo C M, Buim M E C, de Carvalho A C, Lessa R C, Pereira C M, Vettore A L, Carvalho A L, Fregnani J H, Kowalski L P & Soares F A (2010) Histopathology57, 689–698 Claudin-7 down-regulation is an important feature in oral squamous cell carcinoma Aims: Claudins, a large family of essential tight junction (TJ) proteins, are abnormally regulated in human carcinomas, especially claudin-7. The aim of this study was to investigate claudin-7 expression and alterations in oral squamous cell carcinoma (OSCC). Methods and results: Expression of claudin-7 was analysed in 132 cases of OSCC organized in a tissue microarray. Claudin-7 mRNA transcript was evaluated using real-time polymerase chain reaction and the methylation status of the promoter was also assessed. Claudin-7 was negative in 58.3% of the cases. Loss of claudin-7 protein expression was associated with recurrence (P = 0.019), tumour size (P = 0.014), clinical stage of OSCC (P = 0.055) and disease-free survival (P = 0.015). Down-regulation of the claudin-7 mRNA transcripts was observed in 78% of the cases, in accordance with immunoexpression. Analysis of the methylation status of the promoter region of claudin-7 revealed that treatment of O28 cells (that did not express claudin-7 mRNA transcripts) with 5-Aza-2′-Deoxycytidine (5-Aza-dC) led to the re-expression of claudin-7 mRNA transcript. Conclusion: Loss of claudin-7 expression is associated with important subcellular processes in OSCC with impact on clinical parameters.

AIM We aimed to evaluate the amount and quality of the RNA obtained from lymph nodes of non-Hodgkin lymphomas (NHLs) patients using fine-needle aspiration cytology (FNAC), and to develop strategies to overcome eventual technical drawbacks. MATERIALS AND METHODS Twenty-six patients with NHL and 10 tonsils from children submitted to tonsillectomy underwent FNAC. The aspirates were performed using both cytoaspirator (sample A) and syringe and needle (sample B). The RNA was extracted using Trizol reagent and transcribed with the Superscript kit (Invitrogen). The quality of RNA was verified through the amplification of a beta-actin 155-bp fragment. RESULTS Fifty-two NHL and 20 tonsil samples were analyzed. The total amount of RNA in the tonsil samples varied from

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Multiple myeloma (MM) is an incurable hematological malignancy. Different studies demonstrated the occurrence of genetic and epigenetic alterations in MM. the aberrant methylation is one of the most frequent epigenetic alterations in human genome. This study evaluated the aberrant methylation status or 20 genes in 51 MM samples by quantitative methylation-specific PCR (QMSP) and compared the methylation profile with clinicopathological characteristics of the patients. the QMSP analyses showed that PTGS2 (100.0%), SFN (100.0%), CDKN2B (90.2%), CDH1 (88.2%), ESR1 (72.5%), HIC1 (70.5%), CCND2 (62.7%), DCC (45.1%) and TGF beta R2 (39.2%) are frequently hypermethylated in MM while aberrant methviation of RAR beta (16.6%), MGMT (12.5%), AIM1 (12.5%), CDKN2A (8.3%), SOCS1 (8.3%), CCNA1 (8.3%) and THBS1 (4.1%) are rare events. There was no methylation of GSTP1, MINT31, p14ARF and RB1 in the samples tested. Hypermethylation of ESR1 was correlated positively with isotype IgA, while aberrant methylation of THBS1 correlated negatively with isotype IgG. Furthermore, hypermethylation of DCC and TGF beta R2 were correlated with poor survival. the multivariate analysis showed ISS and TGF beta R2 hypermethylation strongly correlated with poor outcome. This study represents the first quantitative evaluation of promoter methylation in MM and our data provide evidence that TGF beta R2 hypermethylation, besides ISS, may be useful as prognostic indicator in this disease. (C) 2009 UICC Universidade Federal de São Paulo, Dept Sci Biol, São Paulo, Brazil Universidade Federal de São Paulo, Discipline Hematol & Hemotherapy, EPM, São Paulo, Brazil Barretos Canc Hosp, Res & Training Inst, Barretos, Brazil Universidade Federal de São Paulo, Dept Sci Biol, São Paulo, Brazil Universidade Federal de São Paulo, Discipline Hematol & Hemotherapy, EPM, São Paulo, Brazil FAPESP: 04/13213-3 FAPESP: 06/61572-8 CNPq: 472193/2004-0 CNPq: 306429/2004-7 Web of Science

Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance. We generated SAGE libraries from normal and neoplastic plasma cells to identify genes differentially expressed in multiple myeloma (MM). Normal plasma cells were obtained from palatine tonsils and MM SAGE library was generated from bone marrow plasma cells of MM patients. We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 46 upregulated genes in the MM library. Ten upregulated genes were selected for further investigation. Differential expression was validated by quantitative real-time PCR in purified plasma cells of 31 patients and three controls. P53CSV, DDX5, MAPKAPK2 and RANBP2 were found to be upregulated in at least 50% of the MM cases tested. All of them were also found upregulated in MM when compared to normal plasma cells in a meta-analysis using ONCOMINE microarray database. Antibodies specific to DDX5, RANBP2 and MAPKAPK2 were used in a TMA containing 57 MM cases and confirmed the expression of these proteins in 74%, 96%, and 21% of the MM samples, respectively. Analysis of differential expression using SAGE could identify genes important for myeloma tumorigenesis (P53CSV, DDX5, MAPKPK2 and RANBP2) and that could potentially be useful as therapeutic targets.

The aims of this study were to correlate HGF, VEGF and FGF serum levels and microvessel density (MVD) with cell origin, biological behavior, tumor load and prognosis in NHL. Eighty-seven consecutive previously untreated NHL patients had serum samples collected; 37 (42%) of them also had serum follow-up samples; the control group was composed of 10 healthy blood donors. Cytokine serum levels were determined by ELISA, and MVD was measured by CD34 staining in paraffin blocks. HGF mean serum level was significantly higher in both early and advanced NHL stages when compared with the control group. HGF was also significantly higher in aggressive and indolent NHL when compared with the control group. Also, mean serum level of HGF in aggressive NHL was significantly higher than in indolent NHL. Regarding International Prognostic Index (IPI), HGF mean serum level at diagnosis was significantly higher for patients with IPI >2 when compared to IPI

A TP53 germline mutation, R337H, has been previously described in children from southern Brazil with adrenocortical tumours but no documented familial history of other cancers. Here, we have screened for TP53 mutation 45 Brazilian unrelated individuals with family histories fulfilling the clinical definitions of Li-Fraumeni (LFS) or Li-Fraumeni-like (LFL) syndromes. Mutations were found in 13 patients (28.9%), including six (46.1%) R337H mutations, and four novel germline mutations (V173M, V197M, G244D and IVS6+1G>T). Families with the R337H mutation presented a wide spectrum of tumours, including breast cancers (30.4%), brain cancers (10.7%), soft tissue sarcomas (10.7%) and adrenocortical carcinomas (8.9%). Testing of 53 Brazilian subjects with no cancer history showed that R337H was not a common polymorphism in that population. Moreover, loss of heterozygocity with retention of the R337H allele was observed in a breast adenocarcinoma, supporting a role for this mutation in breast tumorigenesis. These results show that the TP53 R337H germline mutation predisposes to a larger spectrum of tumours, similar to the one reported for other TP53 mutations.

Purpose Definitive radiation therapy is the mainstay of treatment for early stage laryngeal squamous cell carcinoma (LSCC). However, up to 30% of the patients do not respond to radiotherapy. Unfortunately, we are unable to predict which tumors are likely to respond to radiation, and which will be resistant and persist. Therefore, the development of novel markers to predict response to radiotherapy is urgently needed. This study was designed to evaluate the expression pattern of microRNAs (miRNAs) in LSCC in order to identify markers capable of segregating radioresistant and radiosensitive tumors and to investigate the relationship between the expression of these miRNAs and the prognosis of LSCC. Methods The expression profile of 667 miRNAs was determined in an initial screening of nine early-stage LSCC samples (5 radioresistant and 4 radiosensitive) using TaqMan Low-Density Array (TLDA). Real-time polymerase chain reactions were performed to validate the expression of selected miRNAs in an expanded LSCC cohort (20 radioresistant and 14 radiosensitive). The miRNA expression level was scored as high or low based on the median of the expression in the LSCC samples. Results A comprehensive miRNA expression profiling enabled the identification of four miRNAs (miR-296-5p miR-452, miR-183* and miR-200c) differentially expressed in radioresistant LSCC. Moreover, the analysis of additional 34 LSCC samples, confirmed the expression of miR-296-5p as significantly related to radioresistance (p = 0.002) as well as an association of this marker with recurrence (p = 0.025) in early stage laryngeal cancer. Conclusions This study indicates that miR-296-5p expression is associated with resistance to radiotherapy and tumor recurrence in early stage LSCC, showing the feasibility of this marker as a novel prognostic factor for this malignance. Furthermore, miR-296-5p expression could be helpful in the identification of tumors resistant to radiotherapy; thus aiding the clinicians in the choice of the best therapeutic scheme to be used in each case. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0621-y) contains supplementary material, which is available to authorized users.

Background The presence of metastatic disease in cervical lymph nodes of head and neck squamous cell carcinoma (HNSCC) patients is a very important determinant in therapy choice and prognosis, with great impact in overall survival. Frequently, routine lymph node staging cannot detect occult metastases and the post-surgical histologic evaluation of resected lymph nodes is not sensitive in detecting small metastatic deposits. Molecular markers based on tissue-specific microRNA expression are alternative accurate diagnostic markers. Herein, we evaluated the feasibility of using the expression of microRNAs to detect metastatic cells in formalin-fixed paraffin-embedded (FFPE) lymph nodes and in fine-needle aspiration (FNA) biopsies of HNSCC patients. Methods An initial screening compared the expression of 667 microRNAs in a discovery set comprised by metastatic and non-metastatic lymph nodes from HNSCC patients. The most differentially expressed microRNAs were validated by qRT-PCR in two independent cohorts: i) 48 FFPE lymph node samples, and ii) 113 FNA lymph node biopsies. The accuracy of the markers in identifying metastatic samples was assessed through the analysis of sensitivity, specificity, accuracy, negative predictive value, positive predictive value, and area under the curve values. Results Seven microRNAs highly expressed in metastatic lymph nodes from the discovery set were validated in FFPE lymph node samples. MiR-203 and miR-205 identified all metastatic samples, regardless of the size of the metastatic deposit. Additionally, these markers also showed high accuracy when FNA samples were examined. Conclusions The high accuracy of miR-203 and miR-205 warrant these microRNAs as diagnostic markers of neck metastases in HNSCC. These can be evaluated in entire lymph nodes and in FNA biopsies collected at different time-points such as pre-treatment samples, intraoperative sentinel node biopsy, and during patient follow-up. These markers can be useful in a clinical setting in the management of HNSCC patients from initial disease staging and therapy planning to patient surveillance. Electronic supplementary material The online version of this article (doi:10.1186/s12916-015-0350-3) contains supplementary material, which is available to authorized users.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: the presence of metastatic disease in cervical lymph nodes of head and neck squamous cell carcinoma (HNSCC) patients is a very important determinant in therapy choice and prognosis, with great impact in overall survival. Frequently, routine lymph node staging cannot detect occult metastases and the post-surgical histologic evaluation of resected lymph nodes is not sensitive in detecting small metastatic deposits. Molecular markers based on tissue-specific microRNA expression are alternative accurate diagnostic markers. Herein, we evaluated the feasibility of using the expression of microRNAs to detect metastatic cells in formalin-fixed paraffin-embedded (FFPE) lymph nodes and in fine-needle aspiration (FNA) biopsies of HNSCC patients.Methods: An initial screening compared the expression of 667 microRNAs in a discovery set comprised by metastatic and non-metastatic lymph nodes from HNSCC patients. the most differentially expressed microRNAs were validated by qRT-PCR in two independent cohorts: i) 48 FFPE lymph node samples, and ii) 113 FNA lymph node biopsies. the accuracy of the markers in identifying metastatic samples was assessed through the analysis of sensitivity, specificity, accuracy, negative predictive value, positive predictive value, and area under the curve values.Results: Seven microRNAs highly expressed in metastatic lymph nodes from the discovery set were validated in FFPE lymph node samples. MiR-203 and miR-205 identified all metastatic samples, regardless of the size of the metastatic deposit. Additionally, these markers also showed high accuracy when FNA samples were examined.Conclusions: the high accuracy of miR-203 and miR-205 warrant these microRNAs as diagnostic markers of neck metastases in HNSCC. These can be evaluated in entire lymph nodes and in FNA biopsies collected at different time-points such as pre-treatment samples, intraoperative sentinel node biopsy, and during patient follow-up. These markers can be useful in a clinical setting in the management of HNSCC patients from initial disease staging and therapy planning to patient surveillance. Universidade Federal de São Paulo, Dept Biol Sci, Lab Canc Mol Biol, BR-04039032 São Paulo, SP, Brazil Barretos Canc Hosp, Mol Oncol Res Ctr, BR-14784400 Barretos, SP, Brazil Barretos Canc Hosp, Dept Pathol, BR-14784400 Barretos, SP, Brazil Barretos Canc Hosp, Dept Head & Neck Surg, BR-14784400 Barretos, SP, Brazil Duke NUS Grad Med Sch, Canc Stem Cell Biol Program, Singapore 169857, Singapore Universidade Federal de São Paulo, Dept Biol Sci, Lab Canc Mol Biol, BR-04039032 São Paulo, SP, Brazil FAPESP: 2012/14837-7 Web of Science

The transcriptome-wide endosperm-preferred expression of maize genes was addressed by analyzing a large database of expressed sequence tags (ESTs). We generated 30,531 high quality sequence-reads from the 5'-ends of cDNA libraries from maize endosperm harvested at 10, 15, and 20 days after pollination. A further 196,900 maize sequence-reads retrieved from public databases were added to this endosperm collection to generate MAIZEST, a database with tools for data storage and analysis. MAIZEST contains 227,431 ESTs, one third of which represents developing endosperm and the remaining two-thirds represent transcripts from 49 cDNA libraries constructed from different organs and tissues. Assembling the MAIZEST ESTs generated 29,206 putative transcripts, of which a set of 4032 assembled sequences was composed exclusively of sequences derived from endosperm cDNA libraries. After sequence analysis using overlapping parameters, a sub-set of 2403 assembled sequences was functionally annotated and revealed a wide variety of putative new genes involved in endosperm development and metabolism.

The possible correlation among Epstein-Barr virus (EBV) load, interleukin-6 (IL-6) and interleukin-10 (IL-10) levels has become an attractive issue and can provide a useful tool for diagnosis and monitoring of patients at risk for post-transplant lymphoproliferative disease (PTLD) development. At the time of diagnosis of PTLD, 11 patients were prospectively enrolled and 55 nested controls were selected from a 1800 renal transplant cohort. Real-time polymerase chain reaction (PCR) was used to quantify EBV load in peripheral blood mononuclear cells (PBMC). Serum IL-6 and IL-10 levels were determined using an enzyme-linked immunosorbent assay (ELISA). The median EBV load of PTLD cases was 17400 copies/10(6) PBMC, statistically different from controls (P=0.001). The median IL-6 level of PTLD cases was not different from controls (P=0.079). However, median IL-10 levels showed a significant difference in both groups (Por = 0.001). The receiver-operating characteristic (ROC) curve analysis was applied to estimate the IL-10 cut-off value predictive of PTLD development. We found that 73.5 pg/ml has high sensitivity (1.00) and specificity (0.85). Also, Pearson's analysis showed a strong correlation between EBV load and serum IL-10 concentration (Por = 0.001). This nested case-control study demonstrates that EBV load at diagnosis of PTLD correlates with IL-10 levels, and that monitoring of IL-10 can provide a less expensive and less time-consuming tool for PTLD diagnosis and close follow-up of patients at risk. Furthermore, we were able to define a cut-off value of IL-10 mostly predictive of PTLD development in this cohort. Our data suggest that serial measurements prior to PTLD development must be carried out to validate our hypothesis.

In view of the medical, sanitary and forensic importance of Chrysomya species, a knowledge of their nucleotide sequences would be useful for the molecular characterization of this genus, and would help in designing primers and in improving the molecular identification of Calliphoridae species. In this work, the mitochondrial genome of the blowfly Chrysomya chloropyga (Diptera: Calliphoridae) was completely sequenced. The entire mitochondrial DNA (mtDNA) molecule was 15,837 bp long and was sequenced using the shotgun approach. The overall nucleotide composition was heavily biased towards As and Ts, which accounted for 76.7% of the whole genome. The cox1 gene had a serine as the start codon, while incomplete termination codons mediated by tRNA signals were found for cox2 , nd4 and nd5 . The C. chloropyga genes were in the same order and orientation as the mitochondrial genome of other dipteran species, except for the occurrence of a 123 bp region that included a complete duplication of tRNA Ile and a partial duplication of tRNA Gln genes. C. chloropyga is the first species of Diptera with 23 tRNA genes instead of the usual 22 already described. A phylogenetic analysis showed a split of Brachycera into Calyptratae and Acalyptratae subdivisions. The complete sequence of C. chloropyga mtDNA described here will be a useful source of sequence information for general molecular and evolutionary studies in Diptera.

Over 40,000 sugarcane (Saccharum officinarum) consensus sequences assembled from 237,954 expressed sequence tags were compared with the protein and DNA sequences from other angiosperms, including the genomes of Arabidopsis and rice (Oryza sativa). Approximately two-thirds of the sugarcane transcriptome have similar sequences in Arabidopsis. These sequences may represent a core set of proteins or protein domains that are conserved among monocots and eudicots and probably encode for essential angiosperm functions. The remaining sequences represent putative monocot-specific genetic material, one-half of which were found only in sugarcane. These monocot-specific cDNAs represent either novelties or, in many cases, fast-evolving sequences that diverged substantially from their eudicot homologs. The wide comparative genome analysis presented here provides information on the evolutionary changes that underlie the divergence of monocots and eudicots. Our comparative analysis also led to the identification of several not yet annotated putative genes and possible gene loss events in Arabidopsis.

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.

Background Hypermethylation in the promoter regions is associated with the suppression of gene expression and has been considered a potential molecular marker for several tumor types, including head and neck squamous cell carcinomas (HNSCC). Methods To evaluate the gene hypermethylation profile as a prognostic marker, this retrospective study used a QMSP approach to determine the methylation status of 19 genes in 70 HNSCC patients. Results The methylation profile analysis of primary HNSCC revealed that genes CCNA1, DAPK, MGMT, TIMP3 and SFRP1 were frequently hypermethylated, with high specificity and sensitivity. TIMP3 and CCNA1 hypermethylation was significantly associated with lower rates of second primary tumor-free survival (p = 0.007 and p = 0.001; log-rank test, respectively). Conclusion This study, for the first time, presents CCNA1 and TIMP3 hypermethylation as a helpful tool to identify HNSCC subjects at risk of developing second primary carcinomas.

Objective: To study the potential chemopreventive effects of polyunsaturated fatty acids omega-3 in Swiss mice submitted to oral and oesophageal carcinogenesis induction by 4-nitroquinoline-1-oxide (4-NQO). Study design: The animals underwent carcinogenesis induction with 50 µg/mL 4-NQO for eight weeks. The animals were divided into groups: Group I—4-NQO induction without chemoprevention, Group II—chemoprevention with the addition of 5% fish oil (FO) in their diet after 4-NQO carcinogenesis induction, and Group III—chemoprevention with 5% FO in their diet during and after 4-NQO carcinogenesis induction. Results: The incidence of invasive oral carcinoma was: Group I (72.9%), Group II (84.2%), and Group III (64.7%); p = 0.34. The difference in the incidence of invasive oesophageal carcinoma was statistically significant: Group I (37.8%), Group II (68.4%), and Group III (29.4%); p = 0.02. Conclusions: 4-NQO induction led to cancer in the majority of animals. Chemoprevention with FO brought no benefit in preventing the carcinogenesis process initiated by 4-NQO for oral cancer. The suggestive pro-tumour action of FO when given after tumour post-initiation seems to demonstrate that this fatty acid can potentialise the action of 4-NQO in the oesophagus carcinogenesis of the Swiss mice.

The complete sequence of the mitochondrial genome of the screwworm Cochliomyia hominivorax was determined. This genome is 16,022 bp in size and corresponds to a typical Brachycera mtDNA. A Serine start codon for COI and incomplete termination codons for COII, NADH 5 and NADH 4 genes were described. The nucleotide composition of C. hominivorax mtDNA is 77% AT-rich, reflected in the predominance of AT-rich codons in protein-coding genes. Non-optimal codon usage was commonly observed in C. hominivorax mitochondrial genes. Phylogenetic analysis distributed the Acalypterate species as a monophyletic group and assembled the C. hominivorax (Calyptratae) and the Acalyptratae in a typical Brachycera cluster. The identification of diagnostic restriction sites on the sequenced mitochondrial genome and the correlation with previous RFLP analysis are discussed.

Instituto Ludwig de Pesquisa sobre o Câncer, Rua Prof. Antonio Prudente, 109-4 andar, 01509-010, Sao Paulo-SP

The maize Opaque2 (O2) protein is a basic leucine zipper transcription factor that controls the expression of distinct classes of endosperm genes through the recognition of different cis -acting elements in their promoters. The O2 target region in the promoter of the α-coixin gene was analyzed in detail and shown to comprise two closely adjacent binding sites, named O2u and O2d , which are related in sequence to the GCN4 binding site. Quantitative DNase footprint analysis indicated that O2 binding to α-coixin target sites is best described by a cooperative model. Transient expression assays showed that the two adjacent sites act synergistically. This synergy is mediated in part by cooperative DNA binding. In tobacco protoplasts, O2 binding at the O2u site is more important for enhancer activity than is binding at the O2d site, suggesting that the architecture of the O2–DNA complex is important for interaction with the transcriptional machinery.

The seed storage proteins of Coix, sorghum and maize are codified by homologous genes which are coordinately expressed in the endosperm in a temporal-specific fashion. Opaque2 (O2), a bZIP protein originally isolated from maize, has been described as a transcription activator of α- and β-prolamin genes. The isolation and characterization of cDNA and genomic clones encoding the Opaque2 homologue from Coix are reported here. The coding region of the Coix O2 gene is interrupted by five introns and codifies a polypeptide of 408 amino acids. Comparison of the deduced amino acid sequence with two different sequences of maize O2 protein showed that the Coix O2 protein is similar to the maize O2 isolated from W22 maize inbred line. The Coix O2 protein has the same binding specificity and expression pattern of the maize O2. The O2 proteins together with OHP1, OsBZIPPA, SPA, CPRF2 and RITA1 were assigned to one of the five bZIP plant families in an updated classification of plant bZIP according to bZIP domain similarity.