Micro-RNAs (miRNAs) are small non-coding RNA molecules, which can act as either oncogenes or tumor suppressors. Dysregulated expression of miRNA genes have been implicated in the development of many different cancers. We hypothesize that genetic variations in miRNA biogenesis genes may be associated with the prognosis of bladder cancer. We genotyped 76 single nucleotide polymorphisms (SNPs) in eight miRNA biogenesis genes in 421 patients with non-muscle-invasive bladder cancer (NMIBC). We analyzed the associations of SNPs with recurrence and progression in all patients as well as stratified by treatment: transurethral resection (TUR) alone or TUR plus intravesical bacillus Calmette–Guérin (BCG) instillation. Two SNPs were significantly associated with tumor recurrence in TUR only subgroup after adjustment for multiple comparisons (Q < 0.1). The most significant SNP was rs197412 in DDX20: the variant allele conferred a decreased risk of recurrence [hazard ratio (HR) = 0.58, 95% confidence interval (95% CI) = 0.40–0.82]. This SNP was validated in a separate group of 586 NMIBC patients and the pooled HR was 0.62 (95% CI = 0.48–0.81, P < 0.001). Two linked SNPs (rs2073778 and rs720012) in DGCR8 showed significant association with tumor progression (HR = 4.00, 95% CI = 1.53–10.46, P = 0.005). A strong gene-dosage effect was observed with higher risk for tumor recurrence and progression with increasing number of unfavorable genotypes. Haplotype and survival tree analyses further characterized the association of miRNA-related SNPs with tumor recurrence and progression. Taken together, our results indicate that genetic variants in miRNA biogenesis pathway may influence bladder cancer clinical outcome in NMIBC patients.
Phenotypic biomarkers of DNA damage repair may enhance cancer risk prediction. The γ-H2AX formed at the sites of double strands break (DSB) after ionizing radiation (IR) is a specific marker of DNA damage.
In an ongoing case-control study, the baseline and IR-induced γ-H2AX levels in peripheral blood lymphocytes (PBLs) from frequency-matched 306 untreated lung cancer patients and 306 controls were measured by a laser scanning cytometer-based immunocytochemical method. The ratio of IR-induced γ-H2AX level to the baseline was used to evaluate inter-individual variation of DSB damage response and to assess the risk of lung cancer by using unconditional multivariable logistic regression with adjustment of age, sex, ethnicity, smoking status, family history of lung cancer, dust exposure and emphysema.
The mean γ-H2AX ratio was significantly higher in cases than controls (1.46±0.14 vs. 1.41±0.12, P < 0.001). Dichotomized at the median in controls, high γ-H2AX ratio was significantly associated with increased risk of lung cancer (OR = 2.43, 95% CI: 1.66–3.56). There was also a significant dose-response relationship between γ-H2AX ratio and lung cancer risk in quartile analysis. Analysis of joint effects with other epidemiological risk factors revealed elevated risk with increasing number of risk factors.
γ-H2AX activity as shown by measuring DSB damage in IR-irradiated PBLs may be a novel phenotypic marker of lung cancer risk.
γ-H2AX assay is a robust and quantifiable image-based cytometer method that measures mutagen-induced DSB response in PBLs as a potential biomarker in lung cancer risk assessment.
Double strands break; γ-H2AX; mutagen sensitivity; lung cancer risk
Barrett’s esophagus (BE) is the precursor lesion of esophageal adenocarcinoma (EA), whose progression follows sequential stages. However, the low progression rate and the inadequacy and subjective interpretation of histological grading in predicting BE progression call for more objective biomarkers that can improve risk prediction. We performed a genome-wide profiling of 754 human microRNAs (miRNAs) in 35 normal epithelium (NE), 34 BE, and 36 EA tissues using Taqman real-time PCR-based profiling. Unsupervised hierarchical clustering using 294 modestly to highly expressed miRNAs showed clear clustering of two groups: NE versus BE/EA tissues. Moreover, there was an excellent clustering of Barrett’s metaplasia (BM, without dysplasia) tissues from NE tissues. However, BE tissues of different stages and EA tissues were interspersed. There were differentially expressed miRNAs at different stages. The majority of miRNA aberrations involved upregulation of expression in BE and EA tissues, with the most dramatic alterations occurring at the BM stage. Known oncomirs, such as miR-21, miR-25 and miR-223, and tumor suppressor miRNAs, including miR-205, miR-203, let-7c, and miR-133a, showed progressively altered expression from BE to EA. We also identified a number of novel miRNAs that showed progressively altered expression, including miR-301b, miR-618, and miR-23b. The significant miRNA alterations that were exclusive to EA but not BE included miR-375 downregulation and upregulation of five members of the miR-17-92 and its homologue clusters, which may become promising biomarkers for EA development.
microRNA expression; Barrett’s esophagus; esophageal cancer
Lung cancer in lifetime never smokers is distinct from that in smokers, but the role of separate or overlapping carcinogenic pathways has not been explored. We therefore evaluated a comprehensive panel of 11,737 SNPs in inflammatory-pathway genes in a discovery phase (451 lung cancer cases, 508 controls from Texas). SNPs that were significant were evaluated in a second external population (303 cases, 311 controls from the Mayo Clinic). An intronic SNP in the ACVR1B gene, rs12809597, was replicated with significance and restricted to those reporting adult exposure to environmental tobacco smoke Another promising candidate was a SNP in NR4A1, although the replication OR did not achieve statistical significance. ACVR1B belongs to the TGFR-β superfamily, contributing to resolution of inflammation and initiation of airway remodeling. An inflammatory microenvironment, (second hand smoking, asthma, or hay fever) is necessary for risk from these gene variants to be expressed. These findings require further replication, followed by targeted resequencing, and functional validation.
lung cancer; never smokers; inflammation genes; sidestream exposure
Urinary bladder cancer is a heterogeneous disease with diverse genetic and environmental risk factors that can influence disease risk or clinical course for recurrence, progression, and survival. Therefore, identification of these factors is paramount for disease prevention and optimal clinical management of bladder cancer patients. Of particular interest is the need to identify molecular biomarkers that can give accurate assessment of tumor biological potential and to predict treatment response. Recent advances in molecular biology, cytogenetic and genomic research have spurred discovery efforts for novel genetic, epigenetic, and proteomic biomarkers that are prognostic for cancer. This review focuses on some of the important germline polymorphisms found to be correlated with clinical outcomes in bladder cancer. So far the majority of the identified candidate loci was based on prior knowledge of pathogenesis and had not been validated for clinical applications. The future challenges are to analyze the wealth of information from whole-genome studies, to understand the underlying biological mechanisms of these associations, the network of gene-gene and gene-environment interactions, and to apply these markers for the identification of high-risk population for targeted, personalized therapy.
Polymorphisms; prognostic markers; clinical outcome; bladder cancer
We adopted a two-stage study design to screen 927 single nucleotide polymorphisms (SNPs) located in 73 apoptotic-pathway genes in a case-control study and then performed a fast-track validation of the significant SNPs in a replication population to identify sequence variations in the apoptotic pathway modulating lung cancer risk. Fifty-five SNPs showed significant associations in the discovery population comprised of 661 lung cancer cases and 959 controls. Six of these SNPs located in three genes (Bcl-2, CASP9 and ANKS1B) were validated in a replication population with 1154 cases and 1373 controls. Additive model was the best-fitting model for five SNPs (rs1462129 and rs255102 of Bcl-2, rs6685648 of CASP9 and rs1549102, rs11110099 of ANKS1B) and recessive model was the best fit for one SNP (rs10745877 of ANKS1B). In the analysis of joint effects with subjects carrying no unfavorable genotypes as the reference group, those carrying one, two, and three or more unfavorable genotypes had an odds ratio (OR) of 2.22 [95% confidence interval (CI) = 1.08–4.57, P = 0.03], 2.70 (95% CI = 1.33–5.49; P = 0.006) and 4.13 (95% CI = 2.00–8.57; P = 0.0001), respectively (P for trend = 6.05E-06). The joint effect of unfavorable genotypes was also validated in the replication population. The SNPs identified are located in or near key genes known to play important roles in apoptosis regulation, supporting the strong biological relevance of our findings. Future studies are needed to identify the causal SNPs and elucidate the underlying molecular mechanisms.
In January of 2011, the Biomedical Engineering Society (BMES) and the Society for Physical Regulation in Biology and Medicine (SPRBM) held its inaugural Cellular and Molecular Bioengineering (CMBE) conference. The CMBE conference assembled worldwide leaders in the field of CMBE and held a very successful Round Table discussion among leaders. One of the action items was to collectively construct a white paper regarding the future of CMBE. Thus, the goal of this report is to emphasize the impact of CMBE as an emerging field, identify critical gaps in research that may be answered by the expertise of CMBE, and provide perspectives on enabling CMBE to address challenges in improving human health. Our goal is to provide constructive guidelines in shaping the future of CMBE.
Lymph node dissection is often performed as a part of surgical treatment for breast cancer and malignant melanoma to prevent malignant cells from traveling via the lymphatic system. Currently little is known about postoperative lymphatic drainage pattern alterations. This knowledge may be useful for management of recurrent cancer and prevention of breast cancer related lymphedema. We mapped the complete superficial lymphatic system of a dog and used this canine model to perform preliminary studies of lymphatic architectural changes in postoperative condition.
Lymphatic territories (lymphosomes) were mapped with 4 female mongrel carcasses using an indocyanine green (ICG) fluorescent lymphography and a radiographic microinjection technique. Two live dogs were then subjected to unilateral lymph node dissection of lymph basins of the forelimb, and ICG lymphography and lymphangiogram were performed 6 months after the surgery to investigate lymphatic changes. Lymphatic patterns in the carcass were then compared with postoperative lymphatic patterns in the live dogs.
Ten lymphosomes were identified, corresponding with ten lymphatic basins. Postoperative fluorescent lymphographic images and lymphangiograms in the live dogs revealed small caliber lymphatic network fulfilling gaps in the surgical area and collateral lymphatic vessels arising from the network connecting to lymph nodes in the contralateral and ipsilateral neck in one dog and the ipsilateral subclavicular vein in another dog.
Our canine lymphosome map allowed us to observe lymphatic collateral formations after lymph node dissection in live dogs. This canine model may help clarify our understanding of postoperative lymphatic changes in humans in future studies.
The free muscle-sparing transverse rectus abdominis myocutaneous (MS-TRAM) and deep inferior epigastric perforator (DIEP) flaps involve transferring skin and subcutaneous tissue from the lower abdominal area and have many features that make them well suited for breast reconstruction. The robust blood supply of the free flap reduces the risk of fat necrosis and also enables aggressive shaping of the flap for breast reconstruction to optimize the aesthetic outcome. In addition, the free MS-TRAM flap and DIEP flap require minimal donor-site sacrifice in most cases. With proper patient selection and safe surgical technique, the free MS-TRAM flap and DIEP flap can transfer the lower abdominal skin and subcutaneous tissue to provide an aesthetically pleasing breast reconstruction with minimal donor-site morbidity.
Free tissue flaps; Mammaplasty; Mastectomy
To identify the genetic factors that influence overall survival in never smokers who have non-small cell lung cancer (NSCLC), we performed a consistency meta-analysis study utilizing genome-wide association approaches for overall survival in 327 never smoker NSCLC patients from the MD Anderson Cancer Center and 293 cases from the Mayo Clinic. We then performed a two-pronged validation of the top 25 variants that included additional validation in 1,256 NSCLC patients from Taiwan and assessment of expression quantitative trait loci (eQTL) and differential expression of genes surrounding the top loci in 70 tumors and matched normal tissues. A total of 94 loci were significant for overall survival in both MD Anderson and Mayo studies in the consistency meta-analysis phase, with the top 25 variants reaching a p-value of 10−6. Two variants of these 25 were also significant in the Taiwanese population: rs6901416 (HR:1.44, 95%CI:1.01-2.06) and rs10766739 (HR:1.23, 95%CI:1.00-1.51). These loci resulted in a reduction in median survival time of at least 8 and 5 months in three populations, respectively. An additional six variants (rs4237904, rs7976914, rs4970833, rs954785, rs485411, and rs10906104) were validated through eQTL analysis that identified significant correlations with expression levels of six genes (LEMD3, TMBIM, ATXN7L2, SHE, ITIH2, and NUDT5, respectively) in normal lung tissue. These genes were also significantly differentially expressed between the tumor and normal lung. These findings identify several novel, candidate prognostic markers for NSCLC in never smokers, with eQTL analysis suggesting a potential biological mechanism for a subset of these observed associations.
Tobacco-induced lung cancer is characterized by a deregulated inflammatory microenvironment. Variants in multiple genes in inflammation pathways may contribute to risk of lung cancer.
We therefore conducted a three-stage comprehensive pathway analysis (discovery, replication and meta-analysis) of inflammation gene variants in ever smoking lung cancer cases and controls. A discovery set (1096 cases; 727 controls) and an independent and non-overlapping internal replication set (1154 cases; 1137 controls) were derived from an ongoing case-control study. For discovery, we used an iSelect BeadChip to interrogate a comprehensive panel of 11737 inflammation pathway SNPs and selected nominally significant (p<0.05) SNPs for internal replication.
There were 6 SNPs that achieved statistical significance (p<0.05) in the internal replication dataset with concordant risk estimates for former smokers and 5 concordant and replicated SNPs in current smokers. Replicated hits were further tested in a subsequent meta-analysis using external data derived from two published GWAS and a case-control study. Two of these variants (a BCL2L14 SNP in former smokers and a SNP in IL2RB in current smokers) were further validated. In risk score analyses, there was a 26% increase in risk with each additional adverse allele when we combined the genotyped SNP and the most significant imputed SNP in IL2RB in current smokers and a 36% similar increase in risk for former smokers associated with genotyped and imputed BCL2L14 SNPs.
Before they can be applied for risk prediction efforts, these SNPs should be subject to further external replication and more extensive fine mapping studies.
Inflammation SNPS; lung cancer; smokers
Curatively treated patients with early-stage head and neck squamous cell carcinoma (HNSCC) are at high risks for second primary tumor (SPT) and recurrence. The regulator of G-protein signaling (RGS) is important in essential signaling transduction and cellular activities. We hypothesize that genetic variations of RGS may modulate the risk of SPT/recurrence in patients with early-stage HNSCC. In a nested case–control study, we evaluated 98 single-nucleotide polymorphisms (SNPs) in 17 RGS genes for the risk of SPT/recurrence among 450 HNSCC patients. Eight SNPs showed significant associations with the risk of SPT/recurrence, with the most significant one of rs2179653, which is located in the 5′-flanking region of RGS2 gene. Under a recessive genetic model, the homozygous variant genotype of this SNP was associated with 2.95-fold [95% confidence interval (CI): 1.52–5.74] increased risk of SPT/recurrence. This association remained significant after the adjustment for multiple comparisons. Cumulative effects analysis revealed that the risk increased significantly with the increasing numbers of unfavorable genotypes. Compared with subjects carrying 0–2 unfavorable genotypes, the hazard ratios (95% CIs) for those carrying 3 or 4+ were 1.73 (1.10–2.70) and 3.05 (1.92–4.83), respectively. Furthermore, survival tree analysis revealed potential higher order gene–gene interactions and indicated different outcomes based on distinct genotype profiles. Genetic variations of RGS genes may modulate the susceptibility to SPT/recurrence in early-stage HNSCC patients individually and cumulatively. Our results stressed the importance of taking a polygenic approach to evaluate the cumulative and interaction effects of genetic variations in the prediction of cancer risk and prognosis.
The transforming growth factor-β (TGF-β) signaling pathway is involved in a diverse array of cellular processes responsible for tumorigenesis. In this case-control study, we applied a pathway-based approach to evaluate single-nucleotide polymorphisms (SNPs) in the TGF-β signaling pathway as predictors of ovarian cancer risk. We systematically genotyped 218 SNPs from 21 genes in the TGF-β signaling pathway in 417 ovarian cancer cases and 417 matched control subjects. We analyzed the associations of these SNPs with ovarian cancer risk, performed haplotype analysis and identified potential cumulative effects of genetic variants. We also performed analysis to identify higher-order gene-gene interactions influencing ovarian cancer risk. Individual SNP analysis showed that the most significant SNP was SMAD6: rs4147407, with an adjusted odds ratio (OR) of 1.60 (95% confidence interval [CI], 1.14–2.24, P = 0.0066). Cumulative genotype analysis of 13 SNPs with significant main effects exhibited a clear dose-response trend of escalating risk with increasing number of unfavorable genotypes. In gene-based analysis, SMAD6 was identified as the most significant gene associated with ovarian cancer risk. Haplotype analysis further revealed that two haplotype blocks within SMAD6 were significantly associated with decreased ovarian cancer risk, as compared to the most common haplotype. Gene-gene interaction analysis further categorized the study population into subgroups with different ovarian cancer risk. Our findings suggest that genetic variants in the TGF-β signaling pathway are associated with ovarian cancer risk and may facilitate the identification of high-risk subgroups in the general population.
Tumors of the skull base are rare in children and adolescents and present a complicated management problem for oncologists and surgeons alike. Surgical resection is an integral component of the management of many pediatric neoplasms, especially those that are benign or, though not frankly malignant, are locally invasive. The general principles of skull base reconstruction following tumor ablation are applicable to nearly all patients; the reconstructive algorithm, however, is particularly complex in the pediatric population and the potential benefits of therapy must be balanced against the cumulative impact on craniofacial growth and maturity and the donor site morbidity. A retrospective analysis of all patients less than 19 years of age who underwent resection of a skull base tumor was performed. Particular emphasis was placed on the 12 patients who required complex reconstruction by the plastic surgical service. This represents approximately a third of the operated patients. Data were recorded on patient age, tumor pathology and location, prior therapies, surgical approach, extent of resection, margin status, defect components, details of reconstructive methods employed, complications, additional procedures or interventions, and the use and timing of adjuvant therapies. Patient outcome at most recent follow-up was recorded. All patients were followed clinically and by MRI and/or CT scan of the skull base. The reconstructive details recorded included flap choice, recipient vessels, and any concomitant procedures performed. The indications for and details of any staged surgical revisions or prosthetics were also noted. Complications recorded included partial or total flap loss, cerebrospinal fluid leakage, meningitis, infection, abscess, hematoma or seroma formation, delayed healing, and donor site dysfunction. The vertical rectus abdominis myocutaneous free flap was the most common means of reconstruction utilized in this series. Three of 12 patients had reconstruction related complications. Delayed reconstructive procedures or prosthetic interventions have been performed in 6 of the 12 patients who underwent complex reconstructions. On the basis of our experience and previous reports in the literature, we offer the following guidelines for the successful multidisciplinary care of children and adolescents undergoing skull base reconstruction after tumor resection: (1) skull base reconstruction may be safely performed in children and adolescents using free tissue transfer or local flaps; (2) larger defects and those involving more than one anatomic region of the skull base should be repaired with soft-tissue free flaps; and (3) because of the versatility and reliability of free flaps, pedicled flaps should be reserved for limited defects. Because of the potentially synergistic effects of multimodality treatment for skull base malignancies on craniofacial growth and development, we advocate soft-tissue reconstruction as the primary technique, reserving bony flaps for definitive procedures in survivors who have reached skeletal maturity.
Skull base; pediatric; microvascular; reconstruction
Clinical experience supports a role for palliative procedures in patients with locally advanced or recurrent breast cancer, yet numerous challenges are entailed in both the extirpation and reconstruction of the chest wall in these cases. The defects may be profound and complicated by prior surgery, radiation therapy, or patient-related variables. The reconstructive techniques employed must neither encumber nor delay any necessary postoperative therapy and must not result in unacceptable morbidity or compromise quality of life. Our surgical approach to these cases incorporates a team of specialists from a broad spectrum of medical and surgical disciplines. Each operative plan is tailored to the specific needs and requirements of the individual patient.
Chest wall reconstruction; advanced breast cancer
We have assayed the oncogenic, proliferative, and apoptotic activities of the frequent mutations that occur in the c-myc gene in Burkitt's lymphomas. Some alleles have a modest (50 to 60%) increase in transforming activity; however, the most frequent Burkitt's lymphoma allele (T58I) had an unexpected substantial decrease in transforming activity (85%). All alleles restored the proliferation function of c-Myc in cells that grow slowly due to a c-myc knockout. There was discordance for some alleles between apoptotic and oncogenic activities, but only the T58A allele had elevated transforming activity with a concomitant reduced apoptotic potential. We discovered a novel missense mutation, MycS71F, that had a very low apoptotic activity compared to wild-type Myc, yet this mutation has never been found in lymphomas, suggesting that there is no strong selection for antiapoptotic c-Myc alleles. MycS71F also induced very low levels of cytochrome c release from mitochondria, suggesting a mechanism of action for this mutation. Phosphopeptide mapping provided a biochemical basis for the dramatically different biological activities of the transformation-defective T58I and transformation-enhanced T58A c-Myc alleles. Furthermore, the antiapoptotic survival factor insulin-like growth factor 1 was found to suppress phosphorylation of T58, suggesting that the c-Myc transactivation domain is a direct target of survival signals.
Genetic polymorphisms in oxidative stress pathway genes may contribute to carcinogenesis, disease recurrence, treatment response, and clinical outcomes. We applied a pathway-based approach to determine the effects of multiple single nucleotide polymorphisms (SNPs) within this pathway on clinical outcomes in non-muscle-invasive bladder cancer (NMIBC) patients treated with Bacillus Calmette-Guérin (BCG). We genotyped 276 SNPs in 38 genes and evaluated their associations with clinical outcomes in 421 NMIBC patients. Twenty-eight SNPs were associated with recurrence in the BCG-treated group (P<0.05). Six SNPs, including five in NEIL2 gene from the overall and BCG group remained significantly associated with recurrence after multiple comparison adjustments (q<0.1). Cumulative unfavorable genotype analysis showed that the risk of recurrence increased with increasing number of unfavorable genotypes. In the analysis of risk factors associated with progression to disease, rs3890995 in UNG, remained significant after adjustment for multiple comparison (q<0.1). These results support the hypothesis that genetic variations in host oxidative stress genes in NMIBC patients may affect response to therapy with BCG.
We conducted a genome-wide association study on 969 bladder cancer cases and 957 controls from Texas. For fast-track validation, we evaluated 60 SNPs in three additional US populations and validated the top SNP in nine European populations. A missense variant (rs2294008) in the PSCA gene showed consistent association with bladder cancer in US and European populations. Combining all subjects (6,667 cases, 39,590 controls), the overall P-value was 2.14 × 10−10 and the allelic odds ratio was 1.15 (95% confidence interval 1.10–1.20). rs2294008 alters the start codon and is predicted to cause truncation of nine amino acids from the N-terminal signal sequence of the primary PSCA translation product. In vitro reporter gene assay showed that the variant allele significantly reduced promoter activity. Resequencing of the PSCA genomic region showed that rs2294008 is the only common missense SNP in PSCA. Our data identify rs2294008 as a new bladder cancer susceptibility locus.