[Resistance to BRAF inhibitors: A lesson from clinical observations]. / La résistance aux inhibiteurs de BRAF - Les leçons de la clinique.

The MAPK/ERK pathway is an essential intracellular signaling pathway. Its deregulation is involved in tumor transformation and progression. The discovery of activating mutations of BRAF in various cancers has opened new therapeutic avenues with BRAF protein kinase inhibitors. Depending on the type of cancers, these inhibitors have shown either insufficient efficacy due to primary resistance of tumor cells or transient efficacy due to the development of acquired resistance. In this review, we revisit the discoveries that led to the development of BRAF inhibitors and detail the molecular and cellular mechanisms of resistance in cancers treated with these inhibitors. Understanding these mechanisms is crucial for developing more efficient therapeutic strategies.

Title: La résistance aux inhibiteurs de BRAF - Les leçons de la clinique. Abstract: La voie de signalisation MAPK/ERK est une voie centrale de la signalisation intracellulaire. Sa dérégulation participe à la transformation et la progression tumorales. Dans plusieurs cancers, la découverte de mutations activatrices de BRAF, à l'origine de l'activation de cette voie, a ouvert de nouvelles perspectives thérapeutiques avec le développement d'inhibiteurs spécifiques de la protéine. Selon les cancers, ces inhibiteurs ont cependant montré soit une efficacité insuffisante, due à la résistance primaire des cellules tumorales, soit une efficacité transitoire, due à l'apparition d'une résistance acquise. Dans cette revue, nous revenons sur les découvertes qui ont conduit au développement de ces inhibiteurs de BRAF. Nous détaillons également les mécanismes moléculaires et cellulaires de la résistance à ces inhibiteurs observée dans différents types de cancers. Comprendre ces mécanismes est en effet primordial pour développer des stratégies thérapeutiques qui soient plus efficaces.

Proximal Protein Interaction Landscape of RAS Paralogs.

RAS proteins (KRAS, NRAS and HRAS) are frequently activated in different cancer types (e.g., non-small cell lung cancer, colorectal cancer, melanoma and bladder cancer). For many years, their activities were considered redundant due to their high degree of sequence homology (80% identity) and their shared upstream and downstream protein partners. However, the high conservation of the Hyper-Variable-Region across mammalian species, the preferential activation of different RAS proteins in specific tumor types and the specific post-translational modifications and plasma membrane-localization of each paralog suggest they could ensure discrete functions. To gain insights into RAS proteins specificities, we explored their proximal protein-protein interaction landscapes using the proximity-dependent biotin identification technology (BioID) in Flp-In T-REx 293 cell lines stably transfected and inducibly expressing wild type KRAS4B, NRAS or HRAS. We identified more than 800 high-confidence proximal interactors, allowing us to propose an unprecedented comparative analysis of wild type RAS paralogs protein networks. These data bring novel information on poorly characterized RAS functions, e.g., its putative involvement in metabolic pathways, and on shared as well as paralog-specific protein networks that could partially explain the complexity of RAS functions. These networks of protein interactions open numerous avenues to better understand RAS paralogs biological activities.

HP1s modulate the S-Adenosyl Methionine synthesis pathway in liver cancer cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer in adults. Among the altered pathways leading to HCC, an increasing role is attributed to abnormal epigenetic regulation. Members of the Heterochromatin Protein (HP1) 1 family are key players in chromatin organisation, acting as docking sites for chromatin modifiers. Here, we inactivated HP1α in HepG2 human liver carcinoma cells and showed that HP1α participated in cell proliferation. HP1α-depleted cells have a global decrease in DNA methylation and consequently a perturbed chromatin organisation, as exemplified by the reactivation of transcription at centromeric and pericentromeric regions, eventhough the protein levels of chromatin writers depositing methylation marks, such as EZH2, SETDB1, SUV39H1, G9A and DNMT3A remained unaltered. This decrease was attributed mainly to a low S-Adenosyl Methionine (SAM) level, a cofactor involved in methylation processes. Furthermore, we showed that this decrease was due to a modification in the Methionine adenosyl transferase 2A RNA (MAT2A) level, which modifies the ratio of MAT1A/MAT2A, two enzymes that generate SAM. Importantly, HP1α reintroduction into HP1α-depleted cells restored the MAT2A protein to its initial level. Finally, we demonstrated that this transcriptional deregulation of MAT2A in HP1α-depleted cells relied on a lack of recruitment of HP1ß and HP1γ to MAT2A promoter where an improper non-CpG methylation site was promoted in the vicinity of the transcription start site where HP1ß and HP1γ bound. Altogether, these results highlight an unanticipated link between HP1 and the SAM synthesis pathway, and emphasise emerging functions of HP1s as sensors of some aspects of liver cell metabolism.

Dzip1 and Fam92 form a ciliary transition zone complex with cell type specific roles in Drosophila.

Cilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility. Cilia dysfunctions cause life-threatening ciliopathies, many of which are due to defects in the transition zone (TZ), a complex structure of the ciliary base. Therefore, understanding TZ assembly, which relies on ordered interactions of multiprotein modules, is of critical importance. Here, we show that Drosophila Dzip1 and Fam92 form a functional module which constrains the conserved core TZ protein, Cep290, to the ciliary base. We identify cell type specific roles of this functional module in two different tissues. While it is required for TZ assembly in all Drosophila ciliated cells, it also regulates basal-body growth and docking to the plasma membrane during spermatogenesis. We therefore demonstrate a novel regulatory role for Dzip1 and Fam92 in mediating membrane/basal-body interactions and show that these interactions exhibit cell type specific functions in basal-body maturation and TZ organization.

Synovial-Fluid miRNA Signature for Diagnosis of Juvenile Idiopathic Arthritis.

Juvenile idiopathic arthritis (JIA) is the most common chronic inflammatory rheumatism in childhood; microRNAs (miRNAs) have been proposed as diagnostic biomarkers. Although joints are the primary targets for JIA, a synovial fluid-based miRNA signature has never been studied. We aim to identify miRNA biomarkers in JIA by comparing synovial fluid and serum samples from children with JIA and K.kingae septic arthritis (SA). With next-generation high-throughput sequencing, we measured the absolute levels of 2083 miRNAs in synovial fluid and serum from an exploratory cohort of children and validated differentially expressed miRNAs in a replication study by using RT-qPCR. We identified a 19-miRNA signature only in synovial fluid samples that was significantly deregulated, with at least 2-fold change in expression, in JIA versus SA (p < 0.01). The combination of miR-6764-5p, miR-155, and miR-146a-5p expression in synovial fluid yielded an area under the receiver operating characteristic curve of 1 (95% CI 0.978 to 1), thereby perfectly differentiating JIA from SA in children. We propose, for the first time, a synovial fluid-specific miRNA signature for JIA and associated signaling pathways that may indicate potential biomarkers to assist in the classification and differential diagnosis of JIA and help in understanding JIA pathogenesis.

FKBP4 connects mTORC2 and PI3K to activate the PDK1/Akt-dependent cell proliferation signaling in breast cancer.

Purpose: Among the FKBP family members, FKBP4 has been described to have a potential role in tumorigenesis, and as a putative tissue marker. We previously showed that FKBP4, an HSP90-associated co-chaperone, can elicit immune response as a tumor-specific antigen, and are overexpressed in breast cancer. Experimental design: In this study, we examined how loss of FKBP4 affect breast cancer progression and exploited protein interactomics to gain mechanistic insight into this process. Results: We found that FKBP4 expression is associated with breast cancer progression and prognosis, especially of ER-negative breast cancer. Furthermore, FKBP4 depletion specifically reduces cell growth and proliferation of triple negative breast cancer cell model and xenograft tumor model. Using specific protein interactome strategy by BirA proximity-dependent biotin identification, we demonstrated that FKBP4 is a novel PI3K-Akt-mTOR proximal interacting protein. Conclusion: Our results suggest that FKBP4 interacts with PI3K and can enhance Akt activation through PDK1 and mTORC2.

Quantitative proteomic analysis reveals AK2 as potential biomarker for late normal tissue radiotoxicity.

BACKGROUND: Biomarkers for predicting late normal tissue toxicity to radiotherapy are necessary to personalize treatments and to optimize clinical benefit. Many radiogenomic studies have been published on this topic. Conversely, proteomics approaches are not much developed, despite their advantages. METHODS: We used the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic approach to analyze differences in protein expression levels in ex-vivo irradiated (8 Gy) T lymphocytes from patients with grade ≥ 2 radiation-induced breast fibrosis (grade ≥ 2 bf+) and patients with grade < 2 bf + after curative intent radiotherapy. Patients were selected from two prospective clinical trials (COHORT and PHRC 2005) and were used as discovery and confirmation cohorts. RESULTS: Among the 1979 quantified proteins, 23 fulfilled our stringent biological criteria. Immunoblotting analysis of four of these candidate proteins (adenylate kinase 2, AK2; annexin A1; heat shock cognate 71 kDa protein; and isocitrate dehydrogenase 2) confirmed AK2 overexpression in 8 Gy-irradiated T lymphocytes from patients with grade ≥ 2 bf + compared with patients with grade < 2 bf+. As these candidate proteins are involved in oxidative stress regulation, we also evaluated radiation-induced reactive oxygen species (ROS) production in peripheral blood mononuclear cells from patients with grade ≥ 2 bf + and grade < 2 bf+. Total ROS level, and especially superoxide anion level, increased upon ex-vivo 8 Gy-irradiation in all patients. Analysis of NADPH oxidases (NOXs), a major source of superoxide ion in the cell, showed a significant increase of NOX4 mRNA and protein levels after irradiation in both patient groups. Conversely, only NOX4 mRNA level was significantly different between groups (grade ≥ 2 bf + and grade < 2 bf+). CONCLUSION: These findings identify AK2 as a potential radiosensitivity candidate biomarker. Overall, our proteomic approach highlights the important role of oxidative stress in late radiation-induced toxicity, and paves the way for additional studies on NOXs and superoxide ion metabolism.

Protein interactions study through proximity-labeling.

Introduction: The proteome is a dynamic system in which protein-protein interactions play a crucial part in shaping the cell phenotype. However, given the current limitations of available technologies to describe the dynamic nature of these interactions, the identification of protein-protein interactions has long been a major challenge in proteomics. In recent years, the development of BioID and APEX, two proximity-tagging technologies, have opened-up new perspectives and have already started to change our conception of protein-protein interactions, and more generally, of the proteome. With a broad range of application encompassing health, these new technologies are currently setting milestones crucial to understand fine cellular mechanisms. Area covered: In this article, we describe both the recent and the more conventional available tools to study protein-protein interactions, compare the advantages and the limitations of these techniques, and discuss the recent advancements led by the proximity tagging techniques to refine our conception of the proteome. Expert opinion: The recent development of proximity labeling techniques emphasizes the growing importance of such technologies to decipher cellular mechanism. Although several challenges still need to be addressed, many fields can benefit from these tools and notably the detection of new therapeutic targets for patient care.

[New approaches for protein-protein interaction study]. / Approches nouvelles pour l'étude des interactions protéine-protéine.

The proteome is a dynamic system in which protein-protein interactions play a crucial role to model together the cellular phenotype. However, given the inherent limitation of the available technologies to depict the dynamic nature of these interactions, identify protein-protein interaction has for a long time represented an important challenge in proteomic. The recent development of BioID and APEX, two proximity-dependent labeling technologies, opens today new perspectives and yet start changing our vision of protein-protein interaction, and more globally our vision of the proteome. In this review, we describe the recent and conventional tools available to study protein-protein interactions, compare the advantages and limitations of these technics, and discuss the recent progress brought by the proximity-dependent labelling to complete our vision of the proteome, and thus better understand cellular mechanisms.

An integrated cell line-based discovery strategy identified follistatin and kallikrein 6 as serum biomarker candidates of breast carcinoma.

UNLABELLED: Secreted proteins constitute a relevant source of putative cancer biomarkers. Here, we compared the secretome of a series of four genetically-related breast cancer cell lines as a model of aggressiveness using quantitative mass spectrometry. 537 proteins (59.5% of the total identified proteins) predicted to be released or shed from cells were identified. Using a scoring system based on i) iTRAQ value, ii) breast cancer tissue mRNA expression levels, and iii) immunohistochemical staining (public database), a short list of 10 candidate proteins was selected. Using specific ELISA assays, the expression level of the top five proteins was measured in a verification set of 56 patients. The four significantly differentially expressed proteins were then validated in a second independent set of 353 patients. Finally, follistatin (FST) and kallikrein 6 (KLK6) in serum were significantly higher (p-value < 0.0001) in invasive breast cancer patients compared with non-cancerous controls. Using specific cut-off values, FST distinguished breast cancer samples from healthy controls with a sensitivity of 65% and an accuracy of 68%, whereas KLK6 achieved a sensitivity of 55% and an accuracy of 61%. Therefore, we concluded that FST and KLK6 may have significance in breast cancer detection. BIOLOGICAL SIGNIFICANCE: Discovery of new serum biomarkers that exhibit increased sensitivity and specificity compared to current biomarkers appears to be an essential field of research in cancer. Most biological markers show insufficient diagnostic sensitivity for early breast cancer detection and, for the majority of them, their concentrations are elevated only in metastatic forms of the disease. It is therefore essential to identify clinically reliable biomarkers and develop effective approaches for cancer diagnosis. One promising approach in this field is the study of secreted proteins through proteomic analysis of in vitro progression breast cancer models. Here we have shown that FST and KLK6 are elevated in breast cancer patient serum compared to healthy controls. We expect that our discovery strategy will help to identify cancer-specific and body-fluid-accessible biomarkers.

D25V apolipoprotein C-III variant causes dominant hereditary systemic amyloidosis and confers cardiovascular protective lipoprotein profile.

Apolipoprotein C-III deficiency provides cardiovascular protection, but apolipoprotein C-III is not known to be associated with human amyloidosis. Here we report a form of amyloidosis characterized by renal insufficiency caused by a new apolipoprotein C-III variant, D25V. Despite their uremic state, the D25V-carriers exhibit low triglyceride (TG) and apolipoprotein C-III levels, and low very-low-density lipoprotein (VLDL)/high high-density lipoprotein (HDL) profile. Amyloid fibrils comprise the D25V-variant only, showing that wild-type apolipoprotein C-III does not contribute to amyloid deposition in vivo. The mutation profoundly impacts helical structure stability of D25V-variant, which is remarkably fibrillogenic under physiological conditions in vitro producing typical amyloid fibrils in its lipid-free form. D25V apolipoprotein C-III is a new human amyloidogenic protein and the first conferring cardioprotection even in the unfavourable context of renal failure, extending the evidence for an important cardiovascular protective role of apolipoprotein C-III deficiency. Thus, fibrate therapy, which reduces hepatic APOC3 transcription, may delay amyloid deposition in affected patients.

Anti-heat shock protein autoantibody profiling in breast cancer using customized protein microarray.

Heat shock proteins (HSPs) are over-expressed in a wide range of human cancers. It results in the stimulation of the immune system and consequently in elevated concentration of anti-HSP autoantibodies. Elevated anti-HSP autoantibodies were found in breast cancer patients, and they are associated with tumor metastasis. Therefore, screening these autoantibodies could be of diagnostic and prognostic values. Protein microarrays have already demonstrated their great potential as a diagnostic tool. However, protein diversity requires optimization of the microarray fabrication to achieve high sensitivity and specificity. In this study, seven HSPs were immobilized on six different surface chemistries. After evaluation and optimization with purified antibodies of the six surface chemistries, two surfaces were selected to detect anti-HSP autoantibodies in breast cancer sera. Multiplex detection of anti-HSP autoantibodies allowed discrimination of breast cancer patients (50) from healthy controls (26) with a sensitivity of 86% and a specificity of 100%.

Late side-effects after curative intent radiotherapy: Identification of hypersensitive patients for personalized strategy.

Radiation therapy undeniably enhances local control and thus improves overall survival in cancer patients. However, some long-term cancer survivors (less than 10%) develop severe late radio-induced toxicities altering their quality of life. Therefore, there is a need to identify patients who are sensitive to those toxicities and who could benefit from adapted care. In this review, we address all available techniques aiming to detect patients' hyper-radiosensitivity and present the scientific rationales these techniques are based on.

Use of autoantibodies to detect the onset of breast cancer.

The widespread use of screening mammography has resulted in increased detection of early-stage breast disease, particularly for in situ carcinoma and early-stage breast cancer. However, the majority of women with abnormalities noted on screening mammograms are not diagnosed with cancer because of several factors, including radiologist assessment, patient age, breast density, malpractice concerns, and quality control procedures. Although magnetic resonance imaging is a highly sensitive detection tool that has become standard for women at very high risk of developing breast cancer, it lacks sufficient specificity and costeffectiveness for use as a general screening tool. Therefore, there is an important need to improve screening and diagnosis of early-invasive and noninvasive tumors, that is, in situ carcinoma. The great potential for molecular tools to improve breast cancer outcomes based on early diagnosis has driven the search for diagnostic biomarkers. Identification of tumor-specific markers capable of eliciting an immune response in the early stages of tumor development seems to provide an effective approach for early diagnosis. The aim of this review is to describe several autoantibodies identified during breast cancer diagnosis. We will focus on these molecules highlighted in the past two years and discuss the potential future use of autoantibodies as biomarkers of early-stage breast cancer.

Comparative evaluation of the new FDA approved THxID™-BRAF test with High Resolution Melting and Sanger sequencing.

BACKGROUND: Since patients diagnosed with BRAF V600E and V600K mutated advanced melanoma show response to treatment with MAP kinase inhibitors, several sensitive methods have been developed to determine the V600 allele status of melanoma patients. Vemurafenib (Zelboraf) and dabrafenib (Tafinlar) are specific BRAF V600 inhibitors recently approved by the US FDA as single agent treatments for unresectable or metastatic melanoma in patients with the BRAF V600 mutation. METHODS: We assessed the new CE THxID™-BRAF diagnostic test, which is also FDA-approved as a companion diagnostic test in the US under a specific reference and compared the results of this assay with both High Resolution Melting (HRM) and Sanger sequencing in 113 melanoma FFPE samples. RESULTS: Invalid results were observed in 0/113 specimen with HRM, 5/113 (4.4%) with Sanger sequencing, and 1/113 (0.9%) with the THxID™-BRAF test. Positive percentage agreement (PPA) was 93.5% (95% CI 82.5 - 97.8) for V600E and V600K mutations combined for the THxID™-BRAF test and HRM, and negative percentage agreement (NPA) was 100.0% (95% CI 94.5 - 100.0). For the THxID™-BRAF test and Sanger, PPA was 100.0% (95% CI 92.1 - 100.0) and NPA 100.0% (95% CI 94.2 - 100.0). One V600E sample identified by THxID™-BRAF test was detected as wild-type by HRM and uninterpretable by Sanger. All V600K (n = 3) were detected using the 3 different approaches. Finally, percent agreement values were not significantly different when using punches (n = 77) vs. slides (n = 36) or depending on samples characteristics such as pigmentation, necrosis, and tumor content. CONCLUSIONS: This study demonstrated the high agreement between the FDA approved THxID™-BRAF assay, HRM, and Sanger sequencing. It has also highlighted the potential of THxID™-BRAF to be applied to a broader range of sample types than claimed in the current "instructions for use", an extension that would require the ad hoc validation and approval.

A multiparametric serum marker panel as a complementary test to mammography for the diagnosis of node-negative early-stage breast cancer and DCIS in young women.

BACKGROUND: The sensitivity of mammography for the detection of small lesions, including node-negative early-stage (T1N0) primary breast cancer (PBC) and ductal carcinoma in situ (DCIS), is significantly decreased in young patients. From a clinical standpoint, an inconclusive mammogram reflects the inability of clinicians to confidently decide whether patients should be referred for biopsy or for follow-up with repeat imaging. METHODS: Specific ELISAs were developed for a panel of 13 well-recognized breast autoantigens (HSP60, FKBP52, PRDX2, PPIA, MUC1, GAL3, PAK2, P53, CCNB1, PHB2, RACK1, RUVBL1, and HER2). Circulating autoantibody levels were measured in a cohort of 396 serum samples from histologically confirmed DCIS (n = 87) or T1N0 PBC (n = 153) and healthy controls (n = 156). RESULTS: Individually, antibodies against CCNB1, FKBP52, GAL3, PAK2, PRDX2, PPIA, P53, and MUC1 demonstrated discriminatory power between breast cancer and healthy control groups. At 90% sensitivity, the overall combined specificity of the autoantibody serum screening test was 42%. Adjustment for higher sensitivities of 95% and 99% resulted in 30% and 21% specificities, respectively (33% and 18% in T1N0 PBC and 28% and 21% in DCIS). Finally, in patients with node-negative early-stage breast cancer younger than 50 years, the autoantibody assay exhibited 59% specificity with a fixed sensitivity at 90%. CONCLUSIONS: Our autoantibody panel allows accurate detection of early breast cancer and DCIS, notably in younger patients. IMPACT: Clinical assessment of this autoantibody panel displays a potential to facilitate clinical management of early-stage breast cancer detection in cases of inconclusive mammogram.

Elevated concentrations of milk ß2-microglobulin are associated with increased risk of breastfeeding transmission of HIV-1 (Vertical Transmission Study).

There is increasing evidence to support a relationship between human immunodeficiency virus (HIV-1) transmission through breastfeeding and milk host factors. We analyzed skim milk proteome to further determine the contribution of host factors to the risk of mother-to-child transmission of HIV-1. Quantitative mass spectrometry analysis was performed on nine case-control pairs of HIV+ transmitter/nontransmitter mothers, and specific biochemical assays on two selected proteins were assessed in an independent validation set of 127 samples. 33 identified proteins were differentially expressed between HIV+ transmitter and nontransmitter mothers. Among them, ß2-microglobulin was significantly higher in the maternal transmitter than in the nontransmitter groups (p value = 0.0007), and S100A9 was significantly higher in the early maternal transmitter cases (before 4 months of age) compared with the nontransmitters (p value = 0.004). ß2-Microglobulin correlated with milk and plasma HIV viral load and CD4+ cell count, whereas S100A9 correlated with the estimated timing of infection of the infant through breastfeeding. Finally, ß2-microglobulin concentration in milk could accurately predict the risk of HIV-1 postnatal transmission by breastfeeding (p value < 0.0001, log-rank test). In conclusion, milk ß2-microglobulin and S100A9 are host factors that are found to be associated with mother-to-child transmission of HIV-1.

EGFR Expression and KRAS and BRAF Mutational Status in Intestinal-Type Sinonasal Adenocarcinoma.

Accumulation of molecular alterations, including EGFR overexpression and mutations in KRAS and BRAF, contribute to colorectal carcinogenesis. Since intestinal-type adenocarcinoma (ITAC) of the nasal cavity and paranasal sinus has morphologic and phenotypic features that are usually indistinguishable from colorectal cancer (CRC), it is likely that both tumor types share equivalent genetic alterations. Data from a series of 43 patients treated surgically for ITAC in Montpellier, France between November 1998 and December 2012 were collected. Tumors were characterized for mutations in KRAS and BRAF as well as EGFR overexpression. Kaplan-Meier survival curves were constructed using overall survival as the primary end points. Patient survival was analyzed using the hazards ratio. Twenty seven tumors (63%) showed EGFR positivity and 30% exhibited a high expression level (+2/+3). KRAS mutations were detected in 43% of cases. BRAF mutations were identified in 3.6% of specimens. Patients with age superior to 60 years, metastatic status, and KRAS mutations had significant overall survival values (p = 0.026, p = 0.001 and p = 0.03, respectively). Our results indicate that KRAS mutations and EGFR expression are frequent in ITAC and that KRAS mutations predict good patient prognosis in ITAC. Finally, EGFR directed molecular treatments could be investigated in a subset of patients affected by ITAC.

Proteomic approaches to identify biomarkers predictive of radiotherapy outcomes.

To be highly successful, a radiotherapeutic dose must be sufficiently large to destroy radioresistant tumors, yet avoid injuring the surrounding healthy tissue. However, many patients exhibit high radiosensitivity and may develop radiation-induced early and late side effects. Because the identification of these radiosensitive patients remains largely problematic, general radiotherapy protocols currently limit the dose given, which risks delivering an insufficient dose to a significant number of less sensitive patients. Therefore, one of the main current challenges of radiobiology is to predict a patient's tumor radioresistance and normal tissue radiosensitivity to tailor a personalized treatment to that individual. Although predictive assays exist, none has demonstrated highly significant results that would be useful in a clinical setting. Therefore, proteomics represents a promising approach for identifying new relevant predictive biomarkers. In this review, the authors first explain the main characteristics of tumor radioresistance and normal tissue radiosensitivity. The authors next describe the existing predictive assays. Finally, the proteomics studies performed to date to identify new biomarkers that probably predicts radiotherapy outcomes are discussed.