Lung Adenocarcinoma and Squamous Cell Carcinoma Gene Expression Subtypes Demonstrate Significant Differences in Tumor Immune Landscape.

INTRODUCTION: Molecular subtyping of lung adenocarcinoma (AD) and lung squamous cell carcinoma (SCC) reveal biologically diverse tumors that vary in their genomic and clinical attributes. METHODS: Published immune cell signatures and several lung AD and SCC gene expression data sets, including The Cancer Genome Atlas, were used to examine immune response in relation to AD and SCC expression subtypes. Expression of immune cell populations and other immune related genes, including CD274 molecule gene (CD274) (programmed death ligand 1), was investigated in the tumor microenvironment relative to the expression subtypes of the AD (terminal respiratory unit, proximal proliferative, and proximal inflammatory) and SCC (primitive, classical, secretory, and basal) subtypes. RESULTS: Lung AD and SCC expression subtypes demonstrated significant differences in tumor immune landscape. The proximal proliferative subtype of AD demonstrated low immune cell expression among ADs whereas the secretory subtype showed elevated immune cell expression among SCCs. Tumor expression subtype was a better predictor of immune cell expression than CD274 (programmed death ligand 1) in SCC tumors but was a comparable predictor in AD tumors. Nonsilent mutation burden was not correlated with immune cell expression across subtypes; however, major histocompatibility complex class II gene expression was highly correlated with immune cell expression. Increased immune and major histocompatibility complex II gene expression was associated with improved survival in the terminal respiratory unit and proximal inflammatory subtypes of AD and in the primitive subtype of SCC. CONCLUSIONS: Molecular expression subtypes of lung AD and SCC demonstrate key and reproducible differences in immune host response. Evaluation of tumor expression subtypes as potential biomarkers for immunotherapy should be investigated.

Validation of the Lung Subtyping Panel in Multiple Fresh-Frozen and Formalin-Fixed, Paraffin-Embedded Lung Tumor Gene Expression Data Sets.

Context .- A histologic classification of lung cancer subtypes is essential in guiding therapeutic management. Objective .- To complement morphology-based classification of lung tumors, a previously developed lung subtyping panel (LSP) of 57 genes was tested using multiple public fresh-frozen gene-expression data sets and a prospectively collected set of formalin-fixed, paraffin-embedded lung tumor samples. Design .- The LSP gene-expression signature was evaluated in multiple lung cancer gene-expression data sets totaling 2177 patients collected from 4 platforms: Illumina RNAseq (San Diego, California), Agilent (Santa Clara, California) and Affymetrix (Santa Clara) microarrays, and quantitative reverse transcription-polymerase chain reaction. Gene centroids were calculated for each of 3 genomic-defined subtypes: adenocarcinoma, squamous cell carcinoma, and neuroendocrine, the latter of which encompassed both small cell carcinoma and carcinoid. Classification by LSP into 3 subtypes was evaluated in both fresh-frozen and formalin-fixed, paraffin-embedded tumor samples, and agreement with the original morphology-based diagnosis was determined. Results .- The LSP-based classifications demonstrated overall agreement with the original clinical diagnosis ranging from 78% (251 of 322) to 91% (492 of 538 and 869 of 951) in the fresh-frozen public data sets and 84% (65 of 77) in the formalin-fixed, paraffin-embedded data set. The LSP performance was independent of tissue-preservation method and gene-expression platform. Secondary, blinded pathology review of formalin-fixed, paraffin-embedded samples demonstrated concordance of 82% (63 of 77) with the original morphology diagnosis. Conclusions .- The LSP gene-expression signature is a reproducible and objective method for classifying lung tumors and demonstrates good concordance with morphology-based classification across multiple data sets. The LSP panel can supplement morphologic assessment of lung cancers, particularly when classification by standard methods is challenging.

Implementation of a pharmacogenomics service in a community pharmacy.

OBJECTIVE To determine the feasibility of implementing a pharmacogenomics service in a community pharmacy. SETTING A single community pharmacy that is part of a regional chain known for offering innovative pharmacy services. PRACTICE DESCRIPTION Community pharmacists at the project site routinely provide clinical pharmacy services, including medication therapy management, immunizations, point-of-care testing, blood pressure monitoring, and diabetes education. PRACTICE INNOVATION The implementation of a pharmacogenomic testing and interpretation service for the liver isoenzyme cytochrome P450 2C19. PARTICIPANTS 18 patients taking clopidogrel, a drug metabolized by CYP2C19. MAIN OUTCOME MEASURES Rate of patient participation, rate of prescriber acceptance of pharmacist recommendation, time to perform genetic testing service, and number of claims submitted to and paid by insurance. RESULTS Of 41 patients taking clopidogrel and meeting project criteria, 18 (43.9%) enrolled and completed testing and interpretation of pharmacogenomic results. The mean time pharmacists spent completing all stages of the project with each participant was 76.6 minutes. The mean time to complete participation in the project (time between person's first and second visit) was 30.1 days. Nine patients had wild-type alleles, and pharmacists recommended continuation of therapy as ordered. Genetic variants were found in the other nine patients, and all pharmacist recommendations for modifications in therapy were ultimately accepted by prescribers. Overall, 17 patients consented to filing of reimbursement claims with their insurers. Five were not able to be billed due to submission difficulties. Of the remaining 12, none was paid. CONCLUSION A pharmacogenomics service can be an extension of medication therapy management services in a community pharmacy. Prescribers are receptive to having community pharmacists conduct pharmacogenomics testing, but reimbursement is a challenge.

Cystic fibrosis carrier screening in a North American population.

PURPOSE: The aim of this study was to compare the mutation frequency distribution for a 32-mutation panel and a 69-mutation panel used for cystic fibrosis carrier screening. Further aims of the study were to examine the race-specific detection rates provided by both panels and to assess the performance of extended panels in large-scale, population-based cystic fibrosis carrier screening. Although genetic screening for the most common CFTR mutations allows detection of nearly 90% of cystic fibrosis carriers, the large number of other mutations, and their distribution within different ethnic groups, limits the utility of general population screening. METHODS: Patients referred for cystic fibrosis screening from January 2005 through December 2010 were tested using either a 32-mutation panel (n = 1,601,308 individuals) or a 69-mutation panel (n = 109,830). RESULTS: The carrier frequencies observed for the 69-mutation panel study population (1/36) and Caucasian (1/27) and African-American individuals (1/79) agree well with published cystic fibrosis carrier frequencies; however, a higher carrier frequency was observed for Hispanic-American individuals (1/48) using the 69-mutation panel as compared with the 32-mutation panel (1/69). The 69-mutation panel detected ~20% more mutations than the 32-mutation panel for both African-American and Hispanic-American individuals. CONCLUSION: Expanded panels using race-specific variants can improve cystic fibrosis carrier detection rates within specific populations. However, it is important that the pathogenicity and the relative frequency of these variants are confirmed.

Making pharmacogenetic testing a reality in a community pharmacy.

OBJECTIVE: To provide information for community pharmacies considering implementation of a pharmacogenetic testing service. SETTING: A single community pharmacy from a regional chain. PRACTICE DESCRIPTION: Community pharmacists at the study site routinely provide pharmacy services including medication therapy management, immunizations, point-of-care testing, blood pressure monitoring, and diabetes education. The pharmacy is a training site for post-graduate year 1 and 2 community-pharmacy residents and for introductory and advanced pharmacy practice experience students. PRACTICE INNOVATION: Implementation of a pharmacogenetics testing service in a community pharmacy. MAIN OUTCOME MEASURES: Feasibility of offering a pharmacogenetics testing service in a community pharmacy. RESULTS: Study investigators identified several internal and external barriers to the community pharmacy when initiating a pharmacogenetics service. This article shares experiences of the study team and solutions to the identified barriers. CONCLUSION: Community pharmacies interested in providing pharmacogenetic testing can overcome barriers by identifying practice partners and planning appropriately.

Exploratory planning and implementation of a pilot pharmacogenetic program in a community pharmacy.

AIM: To describe the exploratory planning and implementation of a pilot pharmacogenetic program in a community pharmacy. An institutional review board-approved protocol for a clopidogrel pharmacogenetic program in a community pharmacy was developed to address feasibility and evaluate the pilot program. STUDY CONCEPT: Subjects taking clopidogrel are asked to participate at the point of medication dispensing. A pharmacist schedules an appointment with subjects to discuss the study and collects a buccal swab sample for CYP2C19 testing. When the results are available, the pharmacist consults with the subject's prescriber regarding test result interpretation and associated recommendations, and schedules a second appointment with the participant to discuss results and review any physician-approved therapeutic changes. The intervention-associated consultation is then billed to the subject's insurance. RESULTS: Subject enrollment has begun. CONCLUSION: Community pharmacists may be valuable partners in pharmacogenetics.

Application of a pharmacogenetic test adoption model to six oncology biomarkers.

The ability of genomics to match precise information about the molecular biology of a cancer with the available present and future therapeutics offers tremendous promise for cancer patients. Unfortunately, few genomic-based tests or treatments are available today to benefit these patients. Using a pharmacogenetic test adoption model, previously introduced to model the adoption of HLA-B*5701 testing for abacavir hypersensitivity, six oncology biomarkers, HER2, BCR-ABL quantitation, KRAS mutation, UGT1A1, CYP2D6 for tamoxifen and EGFR expression, test adoption patterns are explored. Developmental milestones and emerging scientific knowledge relating to each of the biomarkers are discussed in the context of their impact on test ordering patterns. Through analysis of the adoption patterns of multiple cancer biomarkers, a pharmacogenetic model emerges which appears to be applicable in five of the six biomarkers. This model may be useful in predicting adoption patterns of new markers and in providing guidance to drug and test developers introducing personalized medicine applications.

Abacavir hypersensitivity: a model system for pharmacogenetic test adoption.

A pharmacogenetic marker for abacavir hypersensitivity is rapidly being incorporated into routine medical practice following demonstration of strong clinical utility in pivotal clinical studies. As one of the few pharmacogenetic markers that have crossed from research tools to clinical adoption and utilization, the abacavir hypersensitivity pharmacogenetic marker provides a great model for demonstration of factors that are critical to successful pharmacogenetic test adoption. Several examples of novel diagnostic test implementation are reviewed with focus on factors that are critical to translation into clinical practice. Other pharmacogenetic markers that have not yet been integrated into routine clinical care are discussed and reasons for their lack of acceptance are suggested.

HLA-B*5701 screening for hypersensitivity to abacavir.

Evaluation of: Mallal S, Phillips E, Carosi G et al.: HLA-B*5701 screening for hypersensitivity to abacavir. N. Engl. J. Med. 358, 568-579 (2008). The field of pharmacogenetics and personalized medicine took a leap forward with publication of a recent multicenter global study, in which prospective trial results clearly validated the use of pharmacogenetic testing in avoiding a drug toxicity reaction. In a study of over 1900 HIV-1-infected patients, HLA-B*5701 screening prior to administering an abacavir-containing regimen, with avoidance of the antiretroviral abacavir in HLA-B*5701-positive patients, was shown to be a highly effective pharmacogenetic test. The screening test provided a clear actionable result with a perfect negative predictive value - 100% of hypersensitivity reactions were prevented by the application of the screening test. This article summarizes the study, compares the findings to previous publications on this topic and discusses factors that impact translating research developments in pharmacogenetics into widespread community practice.

HLA-B*5701 clinical testing: early experience in the United States.

HLA-B*5701 testing to provide risk stratification for abacavir hypersensitivity has the potential to reduce incidence of hypersensitivity reactions in susceptible individuals. Early experience with clinical HLA-B*5701 testing of the first 100 specimens, from a large clinical reference laboratory in the United States, is presented. Patient samples were tested using a two-step approach. The first step allowed rapid identification of most HLA-B*5701-negative samples in a high throughput mode. The second step involved resolution of putative positives by DNA sequencing to identify B*5701 specifically as well as other B57 subtypes. Test reporting included a phone call from a genetic counselor to obtain the ethnic background and indication for testing and to provide a patient-specific interpretation. The patients population was comprised of Caucasians, 84%; Hispanics, 13%; and African Americans, 3%. Among the 100 samples tested, 92% were HLA-B*5701-negative and 8% were positive for the HLA-B*5701 allele. All HLA-B*5701 allele positives were identified in Caucasian patients. Where the indication for testing was obtainable (57 patients), pre-abacavir therapy screening was the indication 67% of the time. Clarification of previous suspected history of hypersensitivity was the indication 33% of the time. Among samples tested to help clarify a previous history of hypersensitivity, 16/19 or 84% did not carry the HLA-B*5701 allele whereas 3/19 (16%) were carriers of the HLA-B*5701 allele. Early utilization of HLA-B*5701 testing in community practice was not always consistent with the clinical indications for testing. Post-test communication assisted in providing physician education and interpretation of patient-specific results.

Antagonism between the HIV-1 reverse-transcriptase mutation K65R and thymidine-analogue mutations at the genomic level.

Prior virologic and biochemical studies have shown phenotypic antagonism between K65R and multiple thymidine-analogue mutations (TAMs) in site-directed mutants tested in vitro. We hypothesized, on the basis of this observed antagonism, that K65R and T215Y/F with multiple TAMs would not be selected on the same human immunodeficiency virus type 1 genome in vivo. We searched a large database of patient genotypes (n=59,262) for the frequency of K65R in combination with >or=3 TAMs as determined by standard population sequencing. K65R and multiple TAMs were rarely detected (<0.1%) in the same plasma sample. Samples with both K65R and >or=3 TAMs (n=21) were further analyzed by use of single-genome sequencing. K65R was never found on the same genome with T215F/Y and >or=2 other TAMs, except in the presence of the Q151M multiple nucleoside reverse-transcriptase inhibitor (NRTI)--resistance complex. These results indicate that antagonism between the K65R and T215Y/F pathways of NRTI resistance occurs at the genomic level. Therapy with NRTI combinations that select both pathways simultaneously may delay the emergence of NRTI resistance and prolong treatment response.

Progression of liver fibrosis among injection drug users with chronic hepatitis C.

Although most hepatitis C virus (HCV) infections are acquired by injection drug use, prospective data on the progression of liver fibrosis are sparse. Baseline liver biopsies were obtained (1996-1998) on a random sample of 210 out of 1667 HCV-positive injection drug users (IDUs). Subjects were followed biannually, with a second biopsy offered to those eligible. Paired biopsies were scored 0 to 6 (modified Ishak score), significant fibrosis was defined as score 3 or greater, and progression of fibrosis was defined as an increase 2 or more units or clinical evidence of end-stage liver disease. Predictive values of blood markers [FibroSURE, aspartate aminotransferase-to-platelet-ratio index (APRI) and alanine aminotransferase (ALT)] were assessed for detection of contemporaneous and future liver fibrosis. Among 119 prospectively followed IDUs, 96% were African American; 97% HCV genotype 1a/b; 27% HIV-infected, and median age was 42 years. Most (90.7%) did not have significant liver fibrosis at first biopsy. Although predictive value for detecting insignificant fibrosis at first biopsy was greater than 95% for FibroSURE, APRI, and ALT, specificities were 88.9%, 72.7%, and 72.7%, respectively. After 4.2 years median follow-up, 21% had progression of fibrosis, which was significantly associated with serum level of HCV RNA and ALT. No serological test had predictive value greater than 40% for contemporaneous or future significant fibrosis. Even initial biopsy result had only a 30.4% value for predicting future significant fibrosis. In conclusion, significant liver fibrosis and progression were detected in some, but not most, IDUs in this cohort. In this setting with low fibrosis prevalence, FibroSURE, ALT, and APRI tests predict insignificant fibrosis; however, further work is needed to find noninvasive markers of significant liver fibrosis.

Update on HIV resistance and resistance testing.

The introduction of highly active antiretroviral therapy, including a combination of antivirals directed at various steps in the viral life cycle, has led to significant decreases in morbidity and mortality associated with human immunodeficiency virus (HIV-1) infections. Despite the availability of numerous antivirals, many extensively treated patients gradually loose the ability to control viral replication because of development of antiviral resistance. Laboratory tests have been developed and validated to assist in recognizing such resistance and to help predict which antivirals may be more likely to control viral replication in a given patient. Both genotypic and phenotypic assays have been developed to assess HIV-1 antiviral resistance. The assay methodologies, including the advantages and disadvantages of each method, as well as the limitations of each method are reviewed. The ability to predict likely drug response from a genotype or a phenotype is continually evolving, and the more recently discovered mutation/drug resistance associations are discussed in terms of their implications for HIV resistance assays. To provide additional options for those who have developed resistance to all currently available drugs, new antivirals, such as the fusion inhibitors, are being developed. These new classes of antivirals block the HIV viral life cycle at sites other than reverse transcriptase and protease. Unique and novel resistance assays are being developed to measure HIV resistance to these new drugs.