Balasubramanian Narasimhan

Diagnosis of multiple cancer types by shrunken centroids of gene expression

We have devised an approach to cancer class prediction from gene expression profiling, based on an enhancement of the simple nearest prototype (centroid) classifier. We shrink the prototypes and hence obtain a classifier that is often more accurate than competing methods. Our method of “nearest shrunken centroids” identifies subsets of genes that best characterize each class. The technique is general and can be used in many other classification problems. To demonstrate its effectiveness, we show that the method was highly efficient in finding genes for classifying small round blue cell tumors and leukemias.

The Inference of Antigen Selection on Ig Genes

Analysis of somatic mutations in V regions of Ig genes is important for understanding various biological processes. It is customary to estimate Ag selection on Ig genes by assessment of replacement (R) as opposed to silent (S) mutations in the complementary-determining regions and S as opposed to R mutations in the framework regions. In the past such an evaluation was performed using a binomial distribution model equation, which is inappropriate for Ig genes in which mutations have four different distribution possibilities (R and S mutations in the complementary-determining region and/or framework regions of the gene). In the present work, we propose a multinomial distribution model for assessment of Ag selection. Side-by-side application of multinomial and binomial models on 86 previously established Ig sequences disclosed 8 discrepancies, leading to opposite statistical conclusions about Ag selection. We suggest the use of the multinomial model for all future analysis of Ag selection.

The relationship between human papillomavirus status and other molecular prognostic markers in head and neck squamous cell carcinomas.

PURPOSE To evaluate the relationship between human papillomavirus (HPV) status and known prognostic makers for head and neck cancers including tumor hypoxia, epidermal growth factor receptor (EGFR) expression and intratumoral T-cell levels and to determine the prognostic impact of these markers by HPV status. METHODS AND MATERIALS HPV status in 82 evaluable head and neck squamous cell carcinomas patients was determined by pyrosequencing and related to p16(INK4a) staining and treatment outcomes. It was correlated with tumor hypoxia (tumor pO(2) and carbonic anhydrase [CAIX] staining), EGFR status, and intratumoral lymphocyte expression (CD3 staining). RESULTS Forty-four percent of evaluable tumors had strong HPV signal by pyrosequencing. There was a significant relationship between strong HPV signal and p16(INK4a) staining as well as oropharynx location. The strong HPV signal group fared significantly better than others, both in time to progression (TTP, p = 0.008) and overall survival (OS, p = 0.004) for all patients and for the oropharyngeal subset. Positive p16(INK4a) staining was associated with better TTP (p = 0.014) and OS (p = 0.00002). There was no relationship between HPV status and tumor pO(2) or CAIX staining. However, HPV status correlated inversely with EGFR reactivity (p = 0.0006) and directly with CD3(+) T-lymphocyte level (p = 0.03). Whereas CAIX and EGFR overexpression were negative prognostic factors regardless of HPV status, CD3(+) T-cell levels was prognostic only in HPV(-) tumors. CONCLUSION HPV status was a prognostic factor for progression and survival. It correlated inversely with EGFR expression and directly with T-cell infiltration. The prognostic effect of CAIX and EGFR expression was not influenced by HPV status, whereas intratumoral T-cell levels was significant only for HPV(-) tumors.

β2-Microglobulin as a Biomarker in Peripheral Arterial Disease Proteomic Profiling and Clinical Studies

Background— Peripheral arterial disease (PAD) is common but commonly unrecognized. Improved recognition of PAD is needed. We used high-throughput proteomic profiling to find PAD-associated biomarkers. Methods and Results— Plasma was collected from PAD patients (ankle brachial index of <0.90; n=45) and subjects with risk factors but without PAD (n=43). Plasma was analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to quantify 1619 protein peaks. The peak intensity of a 12-kDa protein was higher in PAD patients. Western blot analyses and immunoaffinity studies confirmed that this protein was &bgr;2-microglobulin (B2M). In a validation study, B2M was measured by ELISA in plasma in age- and gender-matched PAD (n=20) and non-PAD (n=20) subjects. Finally, we studied a larger cohort of subjects (n=237) referred for coronary angiography but without known PAD. Plasma B2M levels were higher in PAD patients than in non-PAD patients with coronary artery disease. Plasma B2M correlated with ankle brachial index and functional capacity. Independent predictors of PAD were diabetes mellitus, age, and the combination of B2M and C-reactive protein level. Conclusions— In PAD patients, circulating B2M is elevated and correlates with the severity of disease independent of other risk factors. These findings might provide a needed biomarker for PAD and new insight into its pathophysiology. Further studies in other populations are needed to confirm the utility of measuring B2M in cardiovascular disease risk assessment.

Acute rejection and cardiac allograft vascular disease is reduced by suppression of subclinical cytomegalovirus infection

Background. Anticytomegalovirus (CMV) prophylaxis prevents the acute disease but its impact on subclinical infection and allograft outcome is unknown. We sought to determine whether CMV prophylaxis administered for three months after heart transplant would improve patient outcomes. Methods. This prospective cohort study of 66 heart transplant recipients compared aggressive CMV prophylaxis (n=21, CMV hyperimmune globulin [CMVIG] plus four weeks of intravenous ganciclovir followed by two months of valganciclovir); with standard prophylaxis (n=45, intravenous ganciclovir for four weeks). Prophylaxis was based on pretransplant donor (D) and recipient (R) CMV serology: R−/D+ received aggressive prophylaxis; R+ received standard prophylaxis. Outcome measures were: CMV infection assessed by DNA-polymerase chain reaction on peripheral blood polymorphonuclear leukocytes, acute rejection, and cardiac allograft vascular disease (CAV) assessed by intravascular ultrasound. All patients completed one year of follow-up. Results. CMV infection was subclinical in all but four patients (two in each group). Aggressively treated patients had a lower incidence of CMV infection (73±10% vs. 94±4%; P=0.038), and an independent reduced relative risk for acute rejection graded ≥3A (relative risk [95% CI]=0.55 [0.26–0.96]; P=0.03), as compared with the standard prophylaxis group. Aggressively prophylaxed patients also showed a slower progression of CAV, in terms of coronary artery lumen loss (lumen volume change=−21±13% vs. −10±14%; P=0.05); and vessel shrinkage (vessel volume change=−15±11% vs. −3±18%; P=0.03). Conclusions. Prolonged (val)ganciclovir plus CMVIG reduces viral levels, acute rejection, and allograft vascular disease, suggesting a role for chronic subclinical infection in the pathophysiology of the most common diseases affecting heart transplant recipients.

Characterizing the admixed African ancestry of African Americans

BackgroundAccurate, high-throughput genotyping allows the fine characterization of genetic ancestry. Here we applied recently developed statistical and computational techniques to the question of African ancestry in African Americans by using data on more than 450,000 single-nucleotide polymorphisms (SNPs) genotyped in 94 Africans of diverse geographic origins included in the HGDP, as well as 136 African Americans and 38 European Americans participating in the Atherosclerotic Disease Vascular Function and Genetic Epidemiology (ADVANCE) study. To focus on African ancestry, we reduced the data to include only those genotypes in each African American determined statistically to be African in origin.ResultsFrom cluster analysis, we found that all the African Americans are admixed in their African components of ancestry, with the majority contributions being from West and West-Central Africa, and only modest variation in these African-ancestry proportions among individuals. Furthermore, by principal components analysis, we found little evidence of genetic structure within the African component of ancestry in African Americans.ConclusionsThese results are consistent with historic mating patterns among African Americans that are largely uncorrelated to African ancestral origins, and they cast doubt on the general utility of mtDNA or Y-chromosome markers alone to delineate the full African ancestry of African Americans. Our results also indicate that the genetic architecture of African Americans is distinct from that of Africans, and that the greatest source of potential genetic stratification bias in case-control studies of African Americans derives from the proportion of European ancestry.

Minimal residual disease quantification using consensus primers and high-throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia

Quantification of minimal residual disease (MRD) following allogeneic hematopoietic cell transplantation (allo-HCT) predicts post-transplant relapse in patients with chronic lymphocytic leukemia (CLL). We utilized an MRD-quantification method that amplifies immunoglobulin heavy chain (IGH) loci using consensus V and J segment primers followed by high-throughput sequencing (HTS), enabling quantification with a detection limit of one CLL cell per million mononuclear cells. Using this IGH–HTS approach, we analyzed MRD patterns in over 400 samples from 40 CLL patients who underwent reduced-intensity allo-HCT. Nine patients relapsed within 12 months post-HCT. Of the 31 patients in remission at 12 months post-HCT, disease-free survival was 86% in patients with MRD <10−4 and 20% in those with MRD ⩾10−4 (relapse hazard ratio (HR) 9.0; 95% confidence interval (CI) 2.5–32; P<0.0001), with median follow-up of 36 months. Additionally, MRD predicted relapse at other time points, including 9, 18 and 24 months post-HCT. MRD doubling time <12 months with disease burden ⩾10−5 was associated with relapse within 12 months of MRD assessment in 50% of patients, and within 24 months in 90% of patients. This IGH–HTS method may facilitate routine MRD quantification in clinical trials.

Prophylactic rituximab after allogeneic transplantation decreases B-cell alloimmunity with low chronic GVHD incidence

B cells are involved in the pathogenesis of chronic GVHD (cGVHD). We hypothesized that prophylactic anti-B-cell therapy delivered 2 months after transplantation would decrease allogeneic donor B-cell immunity and possibly the incidence of cGVHD. Therefore, in the present study, patients with high-risk chronic lymphocytic leukemia (n = 22) and mantle-cell lymphoma (n = 13) received a total lymphoid irradiation of 80 cGy for 10 days and antithymocyte globulin 1.5 mg/kg/d for 5 days. Rituximab (375 mg/m(2)) was infused weekly on days 56, 63, 70, and 77 after transplantation. The incidence of acute GVHD was 6%. The cumulative incidence of cGVHD was 20%. Nonrelapse mortality was 3%. Rituximab treatment after allogeneic transplantation significantly reduced B-cell allogeneic immunity, with complete prevention of alloreactive H-Y Ab development in male patients with female donors (P = .01). Overall survival and freedom from progression at 4 years for chronic lymphocytic leukemia patients were 73% and 47%, respectively; for mantle-cell lymphoma patients, they were 69% and 53%, respectively.

Mouse Strain–Specific Differences in Vascular Wall Gene Expression and Their Relationship to Vascular Disease

Objective— Different strains of inbred mice exhibit different susceptibility to the development of atherosclerosis. The C3H/HeJ and C57Bl/6 mice have been used in several studies aimed at understanding the genetic basis of atherosclerosis. Under controlled environmental conditions, variations in susceptibility to atherosclerosis reflect differences in genetic makeup, and these differences must be reflected in gene expression patterns that are temporally related to the development of disease. In this study, we sought to identify the genetic pathways that are differentially activated in the aortas of these mice. Methods and Results— We performed genome-wide transcriptional profiling of aortas from C3H/HeJ and C57Bl/6 mice. Differences in gene expression were identified at baseline as well as during normal aging and longitudinal exposure to high-fat diet. The significance of these genes to the development of atherosclerosis was evaluated by observing their temporal pattern of expression in the well-studied apolipoprotein E model of atherosclerosis. Conclusion— Gene expression differences between the 2 strains suggest that aortas of C57Bl/6 mice have a higher genetic propensity to develop inflammation in response to appropriate atherogenic stimuli. This study expands the repertoire of factors in known disease-related signaling pathways and identifies novel candidate genes for future study.

Effects of Exercise Training in Patients With Abdominal Aortic Aneurysm: Preliminary Results From a Randomized Trial

OBJECTIVE: No effective medical therapy exists for early abdominal aortic aneurysm (AAA) disease. Lower extremity exercise improves aortic hemodynamics and reduces inflammation, but the safety and efficacy of exercise training in AAA disease is unknown. As an interim analysis of our prospective, randomized, longitudinal trial of exercise for AAA suppression, we investigated whether subjects with early disease could safely achieve target metabolic and hemodynamic goals. METHODS: One hundred eight participants were randomized to exercise training (EX) or usual care (UC). EX subjects participated in a combination of in-house and home exercise training, with efforts directed toward moderate daily exercise participation. Comparisons were made between EX and UC subjects who completed 1 year of follow-up (n = 26 and 31, respectively, mean age 72 ± 8 years). EX and UC groups were compared for safety, cardiopulmonary exercise test responses, weekly energy expenditure, and biometric indices. RESULTS: No paradoxical increase in AAA growth rate or adverse clinical events occurred as a consequence of exercise training. EX participants expended an average of 2269 ± 1207 kcal/wk and increased exercise capacity (42% increase in treadmill time, 24% increase in estimated metabolic equivalents, P = .01 and .08 between groups, respectively). EX participants demonstrated a significant reduction in C-reactive protein and tended to reduce waist circumference and waist-to-hip ratio (P = .06 and .07, respectively). CONCLUSIONS: Preliminary analyses suggest that exercise training is well tolerated and sustainable in small AAA subjects over 1 year. Despite age and comorbidities, exercising AAA subjects achieve meaningful exercise targets and significantly modify activity-dependent variables.