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Background Because more than one neoadjuvant treatment is available for advanced rectal cancer, the aim of this study was to compare the differential clinical and pathologic effects of different combinations of chemoradiation regimens, treatment sequencing, and timing to surgery on patient outcomes. Patients and Methods Between January 2015 and October 2018, 126 newly diagnosed patients with rectal cancer with magnetic resonance imaging-based cT3-4 or N+ rectal disease for curative-intent treatment received 1 of 4 neoadjuvant regimens, followed by immediate surgery or delayed surgery. Whole post-neoadjuvant surgical specimens were assessed by 3-dimensional digital whole-tumor microarray imaging and immunostaining in pathology to analyze the global tumor pathologic regression grades, residual tumor distribution patterns, the extent of lymphovascular permeation, lymph node positivity, and the overall density of lymphocyte infiltration in the tumor microenvironment. These factors were further examined to identify possible correlations with clinical outcomes. Results Among the 4 neoadjuvant treatment groups, including 2 conventional regimens, we found a significant increase of stromal CD3+ and CD8+ immune infiltrates in the postneoadjuvant tumor microenvironment in the 3 groups with delayed surgery after different chemoradiation regimens compared with the group with immediate surgery after a short course of RT alone. Independent of neoadjuvant chemoradiation regimens, the post-induction high-intermediate-low stromal-infiltrating CD8+ T-cell densities corresponded to tumor regression grades, distant metastasis rates, and disease-free survival and were prognostic factors for the further stratification of patients with American Joint Committee on Cancer stage III rectal cancer into different risk groups after surgery. Conclusion The effectiveness of induction strategies on tumor remission and disease recurrence in advanced rectal cancer was significantly correlated with an enhanced cytotoxic immune response in the tumor microenvironment.

Purpose Temporal lobe necrosis (TLN), a late complication of nasopharyngeal carcinoma (NPC) after concurrent chemoradiotherapy (CCRT), causes permanent neurologic deficits. We aimed to investigate the risk factors for the development of CCRT-induced TLN in locally advanced NPC patients. Methods and Materials The incidence of CCRT-induced TLN was assessed in consecutive patients with NPC initially staged with T3-4N0-3M0 receiving curative intensity modulated radiation therapy (IMRT) and cisplatin-based chemotherapy with long-term follow-up. The TLN risk was evaluated with radiation dose-volume histograms (a dosimetric risk indicator of organ injury) and the dynamics of blood circulating neutrophil-to-lymphocyte ratios (a clinical indicator of systemic inflammation) by linear and logistic regression models. High-throughput unbiased T cell receptor-beta (TCRbeta) sequencing was performed to correlate the different TCRbeta repertoires of NPC-infiltrating lymphocytes (a biological factor of the immune microenvironment) with TLN incidence. Results In the era of modern IMRT-based CCRT, radiation doses of up to 74 Gy achieved local control rates of more than 90% in both T3 and T4 diseases but still induced a remarkably higher incidence of TLN in the T4 patients (30.14%) compared with the rare incidence of TLN observed in the T3 patients (2.78%) (P Conclusions The associations of tumor-infiltrating lymphocyte repertoires and blood circulating neutrophil-to-lymphocyte ratios with TLN occurrence in T4 NPC patients suggest that the immune and inflammatory milieus play roles in the late brain damage caused by CCRT. Modulated or provoked by CCRT locally and systemically, the reciprocal interactions of neutrophils and lymphocytes in the intracranial NPC-associated immune microenvironment could be a key driver of chronic TLN pathogenesis.

The nasopharyngeal epithelium is highly susceptible to pathogenic infection. More than 95% of nasopharyngeal carcinomas (NPCs) are Epstein–Barr virus (EBV)-associated epithelial cancers densely infiltrated with EBV-free lymphocytes. It remains unknown whether the immune modulating effects of concurrent chemoradiotherapy (CCRT) on the tumor-infiltrating T-cell priming against EBV, tumor-associated antigens, and/or neoantigens can elicit systemic anti-tumor immunity and decrease recurrence or distant metastasis. Using matched EBV-associated NPCs, nasopharyngeal mucosal tissues, and longitudinal serial peripheral blood samples, we explored the spatiotemporal and quantitative changes in expansion and contraction of intratumoral T-cell clonotypes (ITCs) in peripheral blood samples from before, during, and after CCRT. The pre-treatment nasopharyngeal ITC repertoire contained unique mucosa-resident and commonly system-shared T-cell receptors (TCRs), portraying an individualized tumor-associated and/or metagenomic landscape. We found that the long-term disease-free patients had significantly more robust unique mucosa-resident ITCs that migrated into and expanded in the peripheral blood after CCRT than in the patients with recurrence or distant metastasis (Mann–Whitney U test, p = .0110). However, the system-shared productive ITC TCRs specific to the common viruses, such as EBV, cytomegalovirus, and influenzaA, in all the patients with and without recurrence demonstrated almost no expansion after CCRT. Thus, these findings underline the importance of determining the impact of unique intratumoral immune responses, reflected in the peripheral blood, on disease prognosis after treatment and challenge of mechanistically understanding the common systemic immune evasion of EBV in NPC patients.

The persistence of hepatitis B surface antigen (HBsAg) is a risk factor for the development of steatosis-associated tumors in chronic hepatitis B virus (HBV) infection, yet little is known about the metabolic link with this factor. We correlated HBV-related pathogenesis in genetically engineered mice and human carriers with metabolic proteomics and lipogenic gene expression profiles. The immunohistochemistry showed that the promyelocytic leukemia protein (PML, a tumor suppressor involved in genome maintenance and fatty acid oxidation), being inversely influenced by the dynamic HBsAg levels from acute phase to seroclearance, appeared as a lipo-metabolic switch linking HBsAg-induced steatosis (lipogenesis) to HBsAg-lost fat-burning hepatocarcinogenesis (lipolysis). Knockdown of PML in HBsAg-transgenic mice predisposed to obesity and drove early steatosis-specific liver tumorigenesis. Proteome analysis revealed that the signaling pathways corresponding to energy metabolism and its regulators were frequently altered by suppression or depletion of PML in the HBsAg-transgenic mice, mainly including oxidative phosphorylation and fatty acid metabolism. Expression profiling further identified upregulation of stearoyl-CoA desaturase 1 (Scd1) and epigenetic methylation of NDUFA13 in the mitochondrial respiratory chain and the cell cycle inhibitor CDKN1c in concordance to the increased severity of lipodystrophy and neoplasia in the livers of HBsAg-transgenic mice with PML insufficiency. The defect in lipolysis in PML-deficient HBsAg-transgenic mice made the HBsAg-induced adipose-like liver tumors vulnerable to synthetic lethality from toxic saturated fat accumulation with a Scd1 inhibitor. Our findings provide mechanistic insights into the evolution of steatosis-associated hepatic tumors driven by reciprocal interactions of HBsAg and PML, and a potential utility of lipid metabolic reprogramming as a treatment target.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated lymphoepithelioma. The aim of this study was to characterize the homogeneity and distinctness of the T-cell repertoires within and between primary and metastatic NPCs. We used ultra-deep sequencing of the hypervariably rearranged antigen-binding CDR3 regions of T-cell receptor beta (TCRbeta ) to comprehensively profile the T-cell repertoires in NPC patients receiving definitive chemoradiotherapy with long-term follow-up. We observed not only various spatially heterogeneous patient-specific TCRbeta clone compositions that changed with time but also several commonly enriched TCRbeta subclones that were constantly shared between primary NPCs in the head and neck regions, locally recurrent tumors after treatment and later-developed distant metastatic tumors in the liver, lung and bone. Comparison of the overlap frequency of the T-cell clonality between TCRbeta repertoires enabled us to calculate the pairwise genetic distance between primary NPCs of different patients and different sites of metastatic or recurrent NPCs. The constructed NPC phylogeny clearly differentiated the low-risk patients without relapse from the high-risk patients with distant metastasis after chemoradiotherapy. In contrast to the rather low frequency of nonsilent somatic mutations in NPC cells, the degrees of similarity and divergence of NPC-infiltrating lymphocyte TCRbeta repertoires among different patients showed prognostication. Moreover, the persistent presence of commonly NPC-shared in-frame TCRbeta CDR3 gene sequences spatiotemporally identified in the NPC-infiltrating lymphocytes within varied EBV-positive NPCs and their metastases suggest the existence of frequently shared epitopes of neoantigens virally or nonvirally displayed on cancer cells, thereby providing opportunities for the development of precisely tumor-targeted immunotherapy for distant metastasis.

// Yih-Lin Chung 1 , and Mei-Ling Wu 2 1 Department of Radiation Oncology, Koo Foundation Sun-Yat-Sen Cancer Center, Taipei, Taiwan 2 Department of Pathology and Laboratory Medicine, Koo Foundation Sun-Yat-Sen Cancer Center, Taipei, Taiwan Correspondence to: Yih-Lin Chung, email: ylchung@kfsyscc.org Keywords: hepatitis B virus, hepatocarcinogenesis, PML, tumor suppressor, oncogene Received: November 05, 2015 Accepted: March 18, 2016 Published: April 6, 2016 ABSTRACT Proteasome-mediated degradation of promyelocytic leukemia tumor suppressor (PML) is upregulated in many viral infections and cancers. We previously showed that PML knockdown promotes early-onset hepatocellular carcinoma (HCC) in hepatitis B virus surface antigen ( HBsAg) -transgenic mice. Here we report the effects of PML restoration on late-onset HBsAg-induced HCC. We compared protein expression patterns, genetic mutations and the effects of pharmacologically targeting PML in wild-type , PML -/- , PML +/+ HBsAg tg/o and PML -/- HBsAg tg/o mice. PML -/- mice exhibited somatic mutations in DNA repair genes and developed severe steatosis and proliferative disorders, but not HCC. PML -/- HBsAg tg/o mice exhibited early mutations in cancer driver genes and developed hyperplasia, fatty livers and indolent adipose-like HCC. In PML +/+ HBsAg -transgenic mice, HBsAg expression declined over time, and HBsAg-associated PML suppression was concomitantly relieved. Nevertheless, these mice accumulated mutations in genes contributing to oxidative stress pathways and developed aggressive late-onset angiogenic trabecular HCC. PML inhibition using non-toxic doses of arsenic trioxide selectively killed long-term HBsAg-affected liver cells in PML +/+ HBsAg tg/o mice with falling HBsAg and rising PML levels, but not normal liver cells or early-onset HCC cells in PML -/- HBsAg tg/0 mice. These findings suggest dual roles for PML as a tumor-suppressor lost in early-onset HBsAg-induced hepatocarcinogenesis and as an oncogenic promoter in late-onset HBsAg-related HCC progression.

Background In the last 5 decades, there has been significant advancement of breast reconstruction and postmastectomy radiotherapy for breast cancer care. There has been concern that breast reconstruction may adversely affect the efficacy of postmastectomy radiotherapy. This, however, has not been proven by clear clinical evidence. Methods By comparing the locoregional recurrence rates and overall survival after postmastectomy radiotherapy between those with and without prosthesis-based breast reconstruction, a retrospective cohort study of the breast cancer patients was done. Multivariable Cox proportional hazards analysis was used to control the confounding factors. Results From January 1, 1998 to December 31, 2011, 1015 patients receiving postmastectomy radiotherapy were identified. Among them, 111 patients had prosthesis-based breast reconstruction, and the other 904 did not have breast reconstruction. Thirty-four of 904 (3.8%) patients in the nonreconstructed group and 4 of 111 (3.6%) patients in the reconstructed group developed locoregional recurrence. Multivariable survival analysis found no significant difference both in locoregional recurrence-free survival (hazard ratio, 0.852; P = 0.771) and in overall survival (hazard ratio = 1.317; P = 0.246) between the nonreconstructed group and reconstructed group. Conclusions Although postmastectomy radiotherapy has been shown to affect the surgical and cosmetic outcomes of breast reconstruction, prosthesis-based breast reconstruction does not seem to have significant adverse impacts on the locoregional recurrence-free survival and overall survival of postmastectomy radiotherapy.

We hypothesized the histone deacetylase inhibitor phenylbutyrate (PB) has beneficial effects on radiation-induced injury by modulating the expression of DNA repair and wound healing genes. Hamsters received a radiosurgical dose of radiation (40 Gy) to the cheek and were treated with varying PB dosing regimens. Gross alteration of the irradiated cheeks, eating function, histological changes, and gene expression during the course of wound healing were compared between treatment groups. Pathological analysis showed decreased radiation-induced mucositis, facilitated epithelial cell growth, and preventing ulcerative wound formation, after short-term PB treatment, but not after vehicle or sustained PB. The radiation-induced wound healing gene expression profile exhibited a sequential transition from the inflammatory and DNA repair phases to the tissue remodeling phase in the vehicle group. Sustained PB treatment resulted in a prolonged wound healing gene expression profile and delayed the wound healing process. Short-term PB shortened the duration of inflammatory cytokine expression, triggered repeated pulsed expression of cell cycle and DNA repair-regulating genes, and promoted earlier oscillatory expression of tissue remodeling genes. Distinct gene expression patterns between sustained and short-term treatment suggest dynamic profiling of wound healing gene expression can be an important part of a biological therapeutic strategy to mitigate radiation-related tissue injury.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated lymphoepithelioma. The aim of this study was to characterize the homogeneity and distinctness of the T-cell repertoires within and between primary and metastatic NPCs. We used ultra-deep sequencing of the hypervariably rearranged antigen-binding CDR3 regions of T-cell receptor beta (TCR

DNA damage response and repair pathways are important barriers to carcinogenesis. Here, we show that promyelocytic leukaemia (PML, also known as TRIM19), involved in sensing DNA damage and executing homologous recombination repair, is down-regulated in non-tumour liver cells surrounding hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). No PML mutation or deletion was found in HBV-infected liver or HCC cells. Immunohistochemical analysis of liver biopsies from patients with breast or liver cancer and HBV reactivation after chemotherapy revealed PML up-regulation and HBV exacerbation in normal liver tissue in response to DNA damage (functional PML), PML down-regulation in HCC peritumour cells associated with high HBsAg accumulation and low HBV replication activity (suppressive PML), and heterogeneous nuclear PML expression in HCC cells that lost HBV DNA and HBsAg and were non-reactive to DNA damage (dysregulated PML). Loss of PML in HBsAg-transgenic mice promoted chromosome breaks in liver cells and accelerated the accumulation of body and liver fat and the development of a liver steatosis-dysplasia-adenoma-carcinoma sequence in an inflammation-independent and male-predominant manner, compared to PML knock-out or HBsAg-transgenic mice during the same time period. These results indicate that PML deficiency facilitates genomic instability and promotes HBsAg-related hepatocarcinogenesis, which also involves androgen and lipid metabolism. These findings uncover a novel PML link between HBV-related tumourigenesis, DNA repair, and metabolism.

The aim of this study was to identify patient-centered, mucositis-associated adverse impact factors and events that might confound physician-assessed oral mucositis (OM) in head and neck cancer (HNC) patients receiving chemoradiotherapy. This was a post hoc analysis of a previously conducted randomized trial to determine the efficacy of 5% phenylbutyrate mouthwash in preventing chemoradiotherapy-induced OM. This analysis identified patient-centered symptomatic, observable, and measurable factors that may confound physician scoring of the severity of OM during chemoradiotherapy. Confounding factors were then combined with physician-rated OM scores according to World Health Organization (WHO) and OM Assessment Scale (OMAS) criteria to investigate the therapeutic implications of OM treatment. The original analysis found no significant differences between experimental and placebo groups with respect to the cumulative incidence of physician-recorded severe OM (WHO ≥3 or OMAS ≥2), patient-reported adverse events, and opioid use. However, patients in the experimental arm had relatively lower rates of OM-associated adverse clinical issues including unplanned short radiation breaks, skipping of chemotherapy, nausea/vomiting, late loss of body weight, and early opioid use, all of which could potentially interfere with physician-assessed OM scoring. When WHO OM grade (functional impact and pain), OMAS ulceration size (organic impact), and prolonged radiation treatment time (cancer treatment impact) were combined, there were significantly fewer interruptions of chemoradiotherapy treatment in symptomatic OM patients in the experimental compared to the placebo group. The benefits conferred by reducing the amount of chemoradiotherapy-related, OM-associated adverse impacts in the experimental group were reflected by better 5-year locoregional recurrence-free survival. This exploratory study raises questions as to whether the severity reflected by physician-rated OM scores is in concordance with OM-induced adverse impacts on HNC patients. Further investigations are warranted to identify patient-related and cancer-associated symptom burdens that may affect tolerance, compliance, and outcome of chemoradiotherapy and confound the evaluation of therapeutic effects on chemoradiotherapy-induced OM.

Background. Environmental exposures to tobacco, alcohol, human papillomavirus (HPV) and/or Epstein-Barr virus (EBV), all of which can perturb multiple cell cycle proteins or tumor suppressors, have been implicated in the pathogenesis of different subsets of head and neck cancers. The aim of this study was to investigate to which extent the virus infection by itself, and/or the altered cell cycle proteins, contributes to prognosis in locally advanced tonsillar squamous cell carcinomas (TSCCs) treated with concurrent chemoradiotherapy (CCRT) alone. Methods. Serial tumor tissue arrays from archival samples were tested for the presence of HPV genome integration or EBV episome by means of DNA sequencing, real-time polymerase chain reaction (PCR), and in situ hybridization. Alterations of cell cycle proteins (p53, pRb, and p21) were evaluated by immunohistochemical staining. The association of viral presence with altered cell cycle proteins was correlated to clinical outcomes. Results. Of the 46 patients with the same T2N2bM0 stage IVA among consecutive patients with TSCC, 23 (50%) had integrated HPV DNA and only 1 (2%) had EBV episome. The HPV types detected were almost all HPV-16. A reduced expression pattern of p53, pRb, and p21 was noted in HPV-positive tumors, and the incremental number of alterations in the 3 proteins was significantly associated with HPV-negative tumors. The presence or absence of HPV together with the number of altered expression of the 3 cell cycle markers resulted in further identification of 4 biologically and clinically distinct subgroups with different outcomes after CCRT. Conclusions. Use of combined biomarkers of oncogenic HPV and tumor suppressors of p53, pRb, and p21 in advanced TSCC provides prognostic molecular classification superior to the TNM stage system and identifies low-risk patients for organ preservation by CCRT alone and high-risk patients who might benefit from planned tonsillectomy and neck dissection before or after CCRT. © 2008 Wiley Periodicals, Inc. Head Neck, 2009

Background The study is to evaluate the patterns of failure, toxicities and long-term outcomes of aggressive treatment using 18F-FDG PET/CT-guided chemoradiation plannings for advanced cervical cancer with extensive nodal extent that has been regarded as a systemic disease. Methods We retrospectively reviewed 72 consecutive patients with 18F-FDG PET/CT-detected widespread pelvic, para-aortic and/or supraclavicular lymph nodes treated with curative-intent PET-guided cisplatin-based extended-field dose-escalating intensity-modulated radiotherapy (IMRT) and adaptive high-dose-rate intracavitary 3D-brachytherapy between 2002 and 2010. The failure sites were specifically localized by comparing recurrences on fusion of post-therapy recurrent 18F-FDG PET/CT scans to the initial PET-guided radiation plannings for IMRT and brachytherapy. Results The median follow-up time for the 72 patients was 66 months (range, 3–142 months). The 5-year disease-free survival rate calculated by the Kaplan-Meier method for the patients with extensive N1 disease with the uppermost PET-positive pelvic-only nodes (26 patients), and the patients with M1 disease with the uppermost PET-positive para-aortic (31 patients) or supraclavicular (15 patients) nodes was 78.5 %, and 41.8–50 %, respectively (N1 vs. M1, p = 0.0465). Eight (11.1 %), 18 (25.0 %), and 3 (4.2 %) of the patients developed in-field recurrence, out-of-field and/or distant metastasis, and combined failure, respectively. The 6 (8.3 %) local failures around the uterine cervix were all at the junction between IMRT and brachytherapy in the parametrium. The rate of late grade 3/4 bladder and bowel toxicities was 4.2 and 9.7 %, respectively. When compared to conventional pelvic chemoradiation/2D-brachytherapy during 1990–2001, the adoption of 18F-FDG PET-guided extended-field dose-escalating chemoradiation plannings in IMRT and 3D-brachytherapy after 2002 appeared to provide higher disease-free and overall survival rates with acceptable toxicities in advanced cervical cancer patients. Conclusions For AJCC stage M1 cervical cancer with supraclavicular lymph node metastases, curability can be achieved in the era of PET and chemo-IMRT. However, the main pattern of failure is still out-of-field and/or distant metastasis. In addition to improving systemic treatment, how to optimize and integrate the junctional doses between IMRT and 3D-brachytherapy in PET-guided plannings to further decrease local recurrence warrants investigation. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2226-0) contains supplementary material, which is available to authorized users.

Clinical characteristics and distribution of treatment modalities of 564 consecutive cervical cancer patients without visceral metastasis diagnosed in1990-2010. (DOC 38Â kb)

The integrated 18Â F-FDG PET/CT staging, modern multi-modalities of radiotherapy (RT) planning and concurrent chemotherapy for treatment of an advanced cervical cancer patient with extensive pelvic, para-aortic and supraclavicular nodal diseases but no visceral metastasis at diagnosis. (A) An integrated concurrent chemoradiotherapy (CCRT) showing a combination of PET-guided cisplatin-based extended-field dose-escalating IMRT/IGRT and adaptive high-dose-rate (HDR) image-based intracavitary 3D brachytherapy. External beam RT (EBRT) and brachytherapy doses are transformed to EQD2 (equivalent dose of 2-Gy fraction) for combination. HR-CTV: SUVs of 4.5 greater at the delayed phase of PET; IR-CTV: SUVs of 2.5-4.5 at the delayed phase of PET; R, rectum; B, bladder; Si, sigmoid. (B) The hematologic toxicity profile of the patient during the extended-field CCRT. (DOC 258Â kb)

Two Parallel Staging Systems for Advanced Cervical Cancer. (DOC 4920Â kb)

PET-guided RT planning for a locally bulky FIGO IB2 cervical cancer with multiple FDG-avid pelvic-only nodes. The relatively different intensity of standardized uptake values (SUVs) of 18 F-FDG PET were used to delineate HR- and IR-clinical target volumes (CTVs) ((high risk defined as SUVs of 4.5 greater at the delayed phase and intermediate risk defined as SUVs of 2.5–4.5 at the delayed phase). A representative of fusion between pre-therapy PET and post-implantated CT scans at time of the 1st brachytherapy after concurrent chemoradiation of 4500 cGy with one tandem and two ovoid applicators inserted into the uterus and vaginal fornix, respectively. The 3D RT, IMRT and brachytherapy doses are transformed to EQD2 (equivalent dose of 2-Gy fraction) for combination. Adding to external beam radiation, the brachytherapy planning aimed to deliver a minimum of total EQD2 85 Gy to 90 % of the HR/IR-CTVs in 5 fractions (Frs). HR-CTV: FDG-avid dark color; IR-CTV: FDG-avid light color; B, FDG-avid bladder. Note that the green, red and yellow arrows indicate the area only receiving external beam radiation, the junction of IMRT and brachytherapy, and the overlapping of IMRT and brachytherapy, respectively. There is yet no optimization method to match the dose junction between IMRT and brachytherapy. (DOC 399 kb)

Clinical impacts of concurrent chemotherapy, IMRT and PET on survival of cervical cancer patients. The year 2002 represents a point at which our institution started to use 18 F-FDG PET/CT for staging, planning and/or follow-up in cancer patients, and commonly adopted the IMRT technique for cancer patients who needed RT. (A) Proportions of patients alive and free of disease at 8 years after treatment with a specific or combined treatment modality during the periods 1990–2001 and 2002–2010.The disease-free survival of patients treated by conventional RT in 1990–2001 was compared to that of patients treated by IMRT in 2002–2010 (conventional RT vs. IMRT). The disease-free survival of patients treated with concurrent chemoradiotherapy (CCRT) was compared to that of patients treated with RT alone (CCRT vs. RT alone). * represents p

Flow-chart of curative-intent treatment for advanced cervical cancer patients with extensive nodal disease but no known visceral metastasis in the era of PET and IMRT at our institution. (DOC 71Â kb)

Neoadjuvant chemoradiation therapy has improved the local control rate and overall survival in locally advanced rectal cancers. The purpose of this retrospective study is to evaluate the correlation between the final pathologic stage and survival in these patients.Patients with biopsy-proven rectal carcinoma, pretreatment staging by magnetic resonance imaging such as T3 or T4 tumors, or node-positive disease were treated with preoperative concomitant 5-fluorouracil-based chemotherapy and radiation, followed by radical surgical resection. Clinical outcome with survival, disease-free survival, recurrence rate, and local recurrence rate were compared with each T and N findings using the American Joint Committee on Cancer Tumor-Node-Metastasis (TNM) staging system.A total of 248 patients were enrolled in this study. Overall survival and disease-free survival at 1, 3, and 5 years were 97.1, 92, and 89.9% and 87.5, 71.1, and 69.5%, respectively. Thirty-six patients (14.5%) had a pathologic complete response after neoadjuvant therapy. The recurrence rate was significantly different between the pathologic complete response group and residual group (5.6 vs 31.1%; P = .002). Five-year disease-free survival was significantly better in the complete response group than the residual tumor group (93 vs 66%; P = .0045). There was no statistical difference in survival or locoregional recurrence rate between these two groups.Posttreatment pathologic TNM stage is correlated to disease-free survival and tumor recurrence rate in locally advanced rectal cancer after preoperative chemoradiation. Also, pathologic complete response to neoadjuvant treatment has its oncologic benefit in both overall recurrence and disease-free survival.

Purpose: This study established a prognostic scoring system for nasopharyngeal carcinoma (NPC), which estimates the probability of locoregional (LR) control following definitive conformal radiotherapy. Methods and Materials: Patients with nondisseminated NPC at initial presentation ( n = 630) were enrolled in this study. All patients had magnetic resonance imaging of the head and neck and were treated with conformal radiotherapy. Among them, 93% had concurrent chemotherapy, and 76% had postradiation chemotherapy. The extent of the primary tumor, age at diagnosis, primary tumor size, tumor and nodal classification, histology, and serum lactate dehydrogenase (LDH) level before treatment were included in the analysis for building a prognostic scoring system. The end point for this study was LR control. Results: The prognostic score was defined as the number of adverse prognostic factors present at diagnosis. Four factors had similarly independent prognostic effects (hazard ratio, 2.0–2.6): age >40 years, histologic WHO type I-II, serum LDH level ≥410 U/L, and involvement of two or more sites of the following anatomic structures, i.e., sphenoid floor, clivus marrow, clivus cortex, prevertebral muscles, and petrous bone. The score predicted the 5-year probability of LR control as follows: 0 (15% of the patients), 100%; 1 (42% of the patients), 93%; 2 (29% of the patients), 83%; 3 or higher (13% of the patients), 71%. Conclusion: This scoring system is useful in the decision-making for individual patients and the design of clinical trials to improve LR control for advanced-stage NPC.

Purpose: To investigate the clinical benefit of additional radiotherapy to patients with unresectable hepatocellular carcinoma treated with transcatheter arterial chemoembolization (TACE) and the molecular effects of radiation on gene expression in hepatoma cells. Experimental Design: Between August 1996 and August 2003, 276 and 64 patients with American Joint Committee on Cancer stage T3N0M0 hepatocellular carcinoma receiving TACE alone and TACE followed by three-dimensional conformal radiotherapy, respectively, at our institution were studied. Clinical outcome and pattern of failure were analyzed for the association of survival benefit with radiotherapy. The molecular effects of radiotherapy were studied in vitro and in vivo using human hepatoma cells with different p53 mutation and hepatitis B virus infection status. Results: Median follow-up and survival time in the TACE alone and TACE + radiotherapy groups were 39 and 19 months, and 51 and 17 months, respectively. Additional radiotherapy to TACE did not improve overall survival (P = 0.65). However, different failure patterns were noted after TACE and after radiotherapy. Although all irradiated tumors regressed substantially, radiotherapy rapidly enhanced both intrahepatic and extrahepatic tumor progression outside the radiotherapy treatment field in a significant portion of patients, which offset the benefit of radiotherapy on overall survival. In molecular analysis of the radiation effects on human hepatoma cells, radiotherapy rapidly induced p53-independent transcriptional up-regulation of vascular endothelial growth factor (VEGF), increased VEGF secretion in a dose-, time-, and cell type–dependent manner, and promoted hepatoma cell growth in vivo with enhanced intratumor angiogenesis, which correlated well with elevated levels of serum VEGF. Conclusions: Radiotherapy to eradicate a primary hepatocellular carcinoma might result in the outgrowth of previously dormant microtumors not included in the radiotherapy treatment field. Radiotherapy-induced VEGF could be a paracrine proliferative stimulus. Therapeutic implications of the study justify the combination of three-dimensional conformal radiotherapy with anti-VEGF angiogenic modalities for the treatment of unresectable hepatocellular carcinoma to reduce relapses.

Objective. The purpose of this prospective study was to investigate the toxicity and efficacy of integrating extended-field para-aortic and pelvic external radiation, high-dose-rate intracavity brachytherapy, and concurrent and adjuvant cisplatin-based chemotherapy for locally advanced cervical cancer. Method. A phase I/II study was performed from 1998 to 2003 including sixty-three patients with both clinical FIGO and MRI/CT-based TNM stage IIB-IVA cervical cancer. Patients were treated with extended-field external radiation to the para-aortic and pelvic regions with 45 Gy in 25 fractions, and an additional boost to the gross nodes to 50.4 Gy and the parametrium to 59.4 Gy. Patients also received a high-dose-rate (HDR) intracavity brachytherapy with doses of 22–31 Gy to point A in 4–6 fractions. Concurrently, two cycles of cisplatin (50–80 mg/m 2 ) were administered in weeks 1 and 5 during radiotherapy, as well as two cycles of cisplatin (60–80 mg/m 2 ) for 1 day and 5-fluorouracil (600–800 mg/m 2 ) for 4 days at 1 and 2 months after completion of radiotherapy. The treatment-related acute and late side effects were evaluated using RTOG criteria, and the disease control and survival rate were calculated using the Kaplan–Meier method. The median follow-up interval was 36 months. Results. All sixty-three patients completed the planned extended-field radiotherapy and high-dose-rate brachytherapy with 2 concurrent cycles of cisplatin. Fifty-eight (92%) patients received 2 cycles of the post-radiation adjuvant chemotherapy of cisplatin and 5-fluorouracil. RTOG grade III acute toxicity was gastrointestinal (2%) and hematological (10%). No patient had grade IV acute toxicity. Late grades III–IV morbidity actuarial risk of 6% at 6.5 years primarily involved the injuries to the bowels requiring surgical intervention for intestinal obstruction or fistula formation. Initial sites of recurrence were locoregional failure alone (pelvic and para-aortic regions within the radiation field), 3%; distant metastases only, 8%; and locoregional failure plus distant metastases, 8%. The observed rates at 3-year and 5-year of locoregional control, freedom from distant metastasis, and overall survival were 86% and 86%, 81% and 81%, and 81% and 77%, respectively. Conclusion. Incorporating HDR brachytherapy into a regimen including concurrent chemotherapy and extended radiation appears safe and effective.

Rheumatoid arthritis (RA) is characterized by progressive destruction of the affected joints. The pathophysiology results from genetic susceptibility and autoimmune phenomena, leading to tissue inflammation and synovial hyperplasia termed pannus, which irreversibly destroys cartilage and bone. The current treatment options, which suppress immune responses or ameliorate inflammation, do not halt the destructive process. We found that the histone deacetylase (HDAC) inhibitors (phenylbutyrate and trichostatin A) causing histone hyperacetylation to modulate multiple gene expression not only induced the expression of p21(Cip1) and p16(INK4) in synovial cells but also inhibited the expression of tumor necrosis factor-alpha in affected tissues in adjuvant arthritis, an animal model of RA. Based on the observations that joint swelling is reduced, subintimal mononuclear cell infiltration is decreased, synovial hyperplasia is inhibited, pannus formation is suppressed, and no cartilage or bone destruction is seen, the HDAC inhibitors may represent a new class of compounds for the treatment of RA by simultaneously, coordinately, synergistically, or epigenetically modulating multiple molecular targets in the pathogenesis of RA.

Treatment of hepatocellular carcinoma (HCC) cells with butyrate can induce apoptosis irrespective of hepatitis B virus integration. No information is available, however, regarding the effect of butyrate on HCC in the presence of hepatitis C virus (HCV) because some HCV proteins can regulate cell survival. By gene transfer, we found that HCV core enhances but HCV NS5A antagonizes sodium phenylbutyrate (NaPB)-induced apoptosis in HCC cells, which is independent of p53. We then chose the p53-negative Hep3B HCC cell to investigate the mechanism of anti-apoptosis mediated by NS5A. In the NaPB-treated Hep3B cells without NS5A expression, induction of apoptosis was associated with Bax redistribution from the cytosol to the nucleus interior and subsequently, to a nuclear membrane-bound form. In the NS5A expressing Hep3B cells, NaPB treatment also triggered relocalization of both Bax and NS5A from the cytosol to the nucleus interior but Bax retained inside the nucleus and did not finally move to the nuclear membrane. Using double immunofluorescence and coimmunoprecipitation, we demonstrated that NS5A co-localizes and interacts with Bax in the nucleus. The HCV NS5A protein was further found to contain Bcl-2 homology domains (BH3, BH1 and BH2). Additional studies using deleted NS5A constructs were carried out to determine whether the BH2 domain or nuclear localization signal (NLS) in NS5A is required for interaction with Bax in the nucleus or inhibition of apoptosis. NS5A with deletion of both BH2 domain and NLS localized in the cytoplasm, dissociated with Bax, and lost anti-apoptosis activity during NaPB treatment. In contrast, NS5A with intact BH domains except NLS still bound directly to Bax in the perinuclear region or the nucleus, but showed less association with Bax in the nucleus and lower effect in apoptosis inhibition than full-length NS5A. These results suggest that HCV NS5A as a Bcl-2 homologue interacts with Bax to protect p53-negative HCC cells from NaPB-induced apoptosis. © 2003 Wiley-Liss, Inc.

We hypothesized the histone deacetylase inhibitor phenylbutyrate (PB) has beneficial effects on radiation-induced injury by modulating the expression of DNA repair and wound healing genes. Hamsters received a radiosurgical dose of radiation (40 Gy) to the cheek and were treated with varying PB dosing regimens. Gross alteration of the irradiated cheeks, eating function, histological changes, and gene expression during the course of wound healing were compared between treatment groups. Pathological analysis showed decreased radiation-induced mucositis, facilitated epithelial cell growth, and preventing ulcerative wound formation, after short-term PB treatment, but not after vehicle or sustained PB. The radiation-induced wound healing gene expression profile exhibited a sequential transition from the inflammatory and DNA repair phases to the tissue remodeling phase in the vehicle group. Sustained PB treatment resulted in a prolonged wound healing gene expression profile and delayed the wound healing process. Short-term PB shortened the duration of inflammatory cytokine expression, triggered repeated pulsed expression of cell cycle and DNA repair-regulating genes, and promoted earlier oscillatory expression of tissue remodeling genes. Distinct gene expression patterns between sustained and short-term treatment suggest dynamic profiling of wound healing gene expression can be an important part of a biological therapeutic strategy to mitigate radiation-related tissue injury.

Studies of Drosophila photoreceptor development have illustrated the means by which signal transduction events regulate cell fate decisions in a multicellular organization. Development of the R7 photoreceptor is best understood, and its formation is dependent on the seven in absentia (sina) gene. We have characterized two highly conserved human homologs of sina, termed SIAH1 and SIAH2. SIAH1 maps to chromosome 16q12 and encodes a 282-amino-acid protein with 76% amino acid identity to the Drosophila SINA protein. SIAH2 maps to chromosome 3q25 and encodes a 324-amino-acid protein that shares 68% identity with Drosophila SINA and 77% identity with human SIAH1. SIAH1 and SIAH2 were expressed in many normal and neoplastic tissues, and only subtle differences in their expression were noted. However, one of three murine homologs, Siah1B, was strongly induced in fibroblasts undergoing apoptotic cell death. While a previous study suggested that SINA was a nuclear protein, epitope-tagged SINA and SIAH1 proteins were found in the cytoplasm of Drosophila and mammalian cells. Their substantial evolutionary conservation, role in specifying cell fate, and activation in apoptotic cells suggest the SIAH proteins have important roles in vertebrate development. Furthermore, given the role of sina in Drosophila photoreceptor development, SIAH2 is a candidate for the Usher syndrome type 3 gene at chromosome 3q21-q25.

Hepatitis B virus (HBV) is implicated in liver cancer. The aim of this study was to find out whether HBV or its components [HBV surface antigen (HBsAg), HBV core protein (HBc), and HBV X protein (HBx)] could interfere with the host DNA damage response and repair pathway. The full HBV genome or individual HBV open-reading frame (ORF) was introduced into HepG2 cells to examine the effect on host genomic stability, DNA repair efficacy in response to double-strand DNA damage, and DNA damage-induced cell death. Responses to apoptosis induction in the HBV ORF-transfected HepG2 cells were also compared with those in HBV-positive and HBV-negative human hepatocellular carcinoma (HCC) cells. In the absence of HBV replication, accumulation of HBsAg in liver cells without other HBV proteins enhanced DNA repair protein and tumor suppressor promyelocytic leukemia (PML) degradation, which resulted in resistance to apoptosis induction and deficient double-strand DNA repair. However, HBsAg-positive cells exhibited increased cell death with exposure to the poly(ADP-ribose) polymerase inhibitor that blocks single-strand DNA repair. These results indicate that suppression of PML by HBsAg disrupts cellular mechanisms that respond to double-strand DNA damage for DNA repair or apoptosis induction, which may facilitate hepatocarcinogenesis and open up a synthetic lethality strategy for HBsAg-positive HCC treatment.

DNA damage response and repair pathways are important barriers to carcinogenesis. Here, we show that promyelocytic leukaemia (PML, also known as TRIM19), involved in sensing DNA damage and executing homologous recombination repair, is down-regulated in non-tumour liver cells surrounding hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). No PML mutation or deletion was found in HBV-infected liver or HCC cells. Immunohistochemical analysis of liver biopsies from patients with breast or liver cancer and HBV reactivation after chemotherapy revealed PML up-regulation and HBV exacerbation in normal liver tissue in response to DNA damage (functional PML), PML down-regulation in HCC peritumour cells associated with high HBsAg accumulation and low HBV replication activity (suppressive PML), and heterogeneous nuclear PML expression in HCC cells that lost HBV DNA and HBsAg and were non-reactive to DNA damage (dysregulated PML). Loss of PML in HBsAg-transgenic mice promoted chromosome breaks in liver cells and accelerated the accumulation of body and liver fat and the development of a liver steatosis-dysplasia-adenoma-carcinoma sequence in an inflammation-independent and male-predominant manner, compared to PML knock-out or HBsAg-transgenic mice during the same time period. These results indicate that PML deficiency facilitates genomic instability and promotes HBsAg-related hepatocarcinogenesis, which also involves androgen and lipid metabolism. These findings uncover a novel PML link between HBV-related tumourigenesis, DNA repair, and metabolism.

Background Epidermal growth factor receptor inhibitors (EGFRIs) cause skin inflammation, and understanding the factors that mediate this reaction is fundamental for designing therapies for EGFRI-related cutaneous side effects. Objective We characterized EGFRI-enhanced skin reactions and evaluated the therapeutic efficacy of phenylbutyrate, a histone deacetylase inhibitor. Methods PD168393, an EGFRI, was applied topically to the ear skin of mice with or without mast cell deficiency. The skin was then irritated once or pre-sensitized and repeatedly challenged with 2,4-dinitrofluorobenzene (DNFB). The reaction pattern, the type and number of infiltrating cells, changes in protein, cytokine (TNF-α) and chemokine (CCL2) expression, and the immune response were analyzed. Phenylbutyrate, formulated as a gel for topical treatment or dissolved in water for intraperitoneal administration, was tested as a treatment. Results EGFRI rapidly upregulated the mast cell chemotactic factor, stem cell factor (SCF) and augmented DNFB-induced immediate contact dermatitis within hours of treatment in the presence of mast cells. Topical phenylbutyrate treatment suppressed EGFRI-induced SCF expression in the epithelium, inhibited DNFB-induced mast cell recruitment in the dermis, and ameliorated the EGFRI-enhanced acute skin reaction. EGFRI also enhanced the delayed-type DNFB-induced hypersensitive reaction that was mast-cell independent but was associated with T lymphocytes. Systemic phenylbutyrate administration suppressed EGFRI-enhanced delayed-type skin hypersensitivity by increasing the number and function of Foxp3+ T regulatory suppressor cells, which inhibited T helper cell proliferation. Conclusions Our data suggest that phenylbutyrate has dual beneficial therapeutic effects on EGFRI-enhanced acute (local inflammatory) and late (systemic immune) skin reactions.

Purpose Deleterious oral mucositis (OM) develops during radiotherapy (RT) or chemoradiotherapy for head-and-neck cancer (HNC) patients. There are currently no effective cytoprotective treatments for OM without a potential risk of tumor protection. This randomized, double-blind, placebo-controlled pilot study aimed to determine the therapeutic safety and efficacy of phenylbutyrate (an antitumor histone deacetylase inhibitor and chemical chaperone) 5% mouthwash for treating OM caused by cancer therapy. Methods and Materials Between September 2005 and June 2006, 36 HNC patients were randomized to standard oral care plus 5 mL of either phenylbutyrate 5% mouthwash ( n = 17) or placebo (mouthwash vehicle, n = 19) taken four times daily (swish and spit). Treatment began when mild mucositis (Radiation Therapy Oncology Group Grade 1) occurred, and ended 4 weeks after RT completion. Safety and efficacy were based on adverse events, physical examination, laboratory determinations, vital signs, Oral Mucosa Assessment Scale (OMAS) and World Health Organization scores, the ability to eat, body weight change, local control, and survival. Results We found no severe drug-related side effect. At RT doses of 5500–7500 cGy, phenylbutyrate significantly mitigated the severity of mucositis compared with placebo, based on both the WHO score (severity ≥ 3; p = 0.0262) and the OMAS scale (ulceration score ≥ 2; p = 0.0049). The Kaplan-Meier estimates for 2- and 3-year local control, and overall survival were 100% and 80.8%, and 78.6% and 64.3%, respectively, in the phenylbutyrate group and 74.2% and 74.2%, and 57.4% and 50.2%, respectively, in the placebo group. Conclusions This pilot trial suggested that phenylbutyrate mouthwash significantly decreased the impact of OM in HNC patients receiving RT or chemoradiotherapy and did not confront the tumor control. Larger Phase II randomized trials are needed to confirm these results.

The mechanism responsible for hepatitis B virus (HBV) exacerbation during chemotherapy and radiotherapy remains unknown. We investigated whether the activation of DNA repair pathways influences HBV replication. The upregulation of the promyelocytic leukemia (PML) protein and its associated PML nuclear body (PML-NB) by chemotherapy and irradiation-induced DNA repair signaling correlated with the upregulation of HBV pregenomic transcription, HBV-core expression, and HBV DNA replication. The HBV-core protein and HBV DNA localized to PML-NBs, where they associated with PML and histone deacetylase 1 (HDAC1). Chemotherapy and radiotherapy affected the interactions between PML, HBV-core, and HDAC1. The enhanced protein-protein interaction between PML and HBV-core inhibited PML-mediated apoptosis and decreased PML-associated HDAC activity. The reversal of HDAC-mediated repression on the HBV covalently closed circular DNA basal core promoter resulted in the amplification of HBV-core and pregenomic expression. These results suggest that PML in PML-NBs links the DNA damage response with HBV replication and may cooperate with HBV-core and HDAC1 on the HBV covalently closed circular DNA basal core promoter to form a positive feedback loop for HBV exacerbation during chemotherapy and radiotherapy. (Mol Cancer Res 2009;7(10):1672–85)

Neoadjuvant concomitant chemoradiotherapy has been used in cases of locally advanced rectal cancer to preserve sphincter function, decrease local recurrence, and improve survival. Preoperative staging is essential for planning and providing optimal therapy. The purpose of this study is to determine the accuracy of staging with magnetic resonance imaging and to define any factors that interfere in interpretation of images obtained after preoperative chemoradiation therapy. Thirty-six patients with biopsy-proven, locally advanced rectal cancer were treated with preoperative concomitant 5-fluorouracil-based chemotherapy and radiation, followed six to eight weeks later by radical surgery. Preoperative magnetic resonance images were reinterpreted by one radiologist and the results compared with histopathologic staging. T-level downstaging occurred in 10 of 36 patients (28 percent), and N-level downstaging occurred in 29 of 36 patients (80 percent) after completion of chemoradiation therapy. Pathologic complete remission after chemoradiotherapy occurred in five patients (12 percent). Of the 36 patients, 17 (47 percent) were overstaged and 2 (6 percent) were understaged in T-level, whereas 10 patients (28 percent) were overstaged and 3 patients (8 percent) were understaged in N-level. The accuracy of magnetic resonance imaging for determining depth of wall invasion was 47 percent, with 64 percent accuracy for nodal staging. Magnetic resonance imaging is commonly used in staging of pelvic malignancies because of its fine resolution, but chemoradiotherapy may decrease its accuracy. Thickening of the rectal wall after radiation by marked fibrosis, and peritumoral infiltration of inflammatory cells and vascular proliferation may contribute to overestimation of stage. By contrast, pathologic residual cancer beneath normal mural structure after chemoradiation therapy may result in understaging of rectal cancer.

Radiotherapy is an effective treatment for head and neck, skin, anogenital, and breast cancers. However, radiation-induced skin morbidity limits the therapeutic benefits. A low-toxicity approach to selectively reduce skin morbidity without compromising tumor killing by radiotherapy is needed. We found that the antitumor agents known as histone deacetylase (HDAC) inhibitors (phenylbutyrate, trichostatin A, and valproic acid) could suppress cutaneous radiation syndrome. The effects of HDAC inhibitors in promoting the healing of wounds caused by radiation and in decreasing later skin fibrosis and tumorigenesis were correlated with suppression of the aberrant expression of radiation-induced transforming growth factor β and tumor necrosis factor α. Our findings implicate that the inhibition of HDAC may provide a novel strategy to increase the therapeutic gain in cancer radiotherapy by not only inhibiting tumor growth but also protecting normal tissues.

Overexpression of Her2/neu is implicated in the development of resistance to the antiestrogen tamoxifen (TAM) that exerts its inhibitory effect through interaction with estrogen receptor (ER). Whereas Her2/neu and ER are believed to be important cell survival/death factors in human breast cancer cells, if and how they interact to confer resistance to hormone therapy is not known. This prompted us to investigate whether modulation of the effect of TAM occurs via the Her2/neu pathway and whether targeting the interaction between the Her2/neu pathway and the ER pathway is beneficial. There are 2 forms of ER that are localized to the cell membrane and to the nucleus. For the first time, we found that Her2/neu directly interacts with ER at the cell membrane. We then investigated the role of Her2/neu overexpression in the regulation of the cell membrane ER pathway in TAM-resistant breast cancer cells and the nature of this interaction in apoptotic signaling. Relief of TAM resistance was associated with Her2/neu downregulation and ER upregulation. TAM-induced apoptosis occurred immediately after dissociation of Her2/neu from cell membrane ER. These results demonstrate a novel mechanism by which Her2/neu regulates the cell membrane ER-coupled apoptosis and the possible involvement of the Her2/neu in TAM resistance of breast cancer cells. Moreover, the antiproliferative activity of TAM should rely on the integration between the signal transduction from the cell membrane ER and the gene regulation by the nuclear ER. Coordinated modulation on the cell membrane ER/Her2/neu pathway and the nuclear ER/RAR pathway may provide a new approach for treatment of ER-positive, Her2/neu overexpressing breast cancer.