INTRODUCTION:The N-acetylation polymorphism has been the subject of comprehensive reviews describing the role of arylamine N-acetyltransferase 2 (NAT2) in the metabolism of numerous aromatic amine and hydrazine drugs.AREAS COVERED:We describe and review data that more clearly defines the effects of NAT2 haplotypes and genotypes on the expression of acetylator phenotype towards selected drugs within human hepatocytes in vitro, within human hepatocyte cultures in situ, and clinical measures such as bioavailability, plasma metabolic ratios of parent to N-acetyl metabolite, elimination rate constants and plasma half-life, and/or clearance determinations in human subjects. We review several drugs (isoniazid, hydralazine, sulfamethazine, amifampridine, procainamide, sulfasalazine, amonafide and metamizole) for which NAT2 phenotype-guided therapy may be important. The value of pharmacogenomics-guided isoniazid therapy for the prevention and treatment of tuberculosis is presented as a paradigm for NAT2 phenotype-dependent dosing strategies.EXPERT OPINION:Studies in human subjects and cryopreserved human hepatocytes show evidence for rapid, intermediate and slow acetylator phenotypes, with further data suggesting genetic heterogeneity within the slow acetylator phenotype. Incorporation of more robust NAT2 genotype/phenotypes relationships, including genetic heterogeneity within the slow acetylator phenotype, should lead to further advancements in both health outcomes and cost benefit for prevention and treatment of tuberculosis.

e12506 Background: Fluorescent in Situ Hybridization (FISH) is a method currently used for detection and assessment of HER2 gene amplification. Although clinical guidelines set forth by CAP/ASCO exist to ensure accuracy, limitations in HER2test results due to sample preparation, assay-conditions and tumor heterogeneity remain unresolved. We have successfully demonstrated analytical confidence in performing HER2 FISH on DEPArray™ sorted and recovered tumor cells. In this study, we aimed to evaluate inter-laboratory concordance of the DEPArray™ HER2-FISH assay. Methods: Three laboratories equipped with the DEPArray™ were designated as testing sites for this study. Positive control SKBr3 cells embedded in paraffin as well as 20 invasive breast carcinoma FFPE samples were blinded and evaluated by each of the three labs. Control and patient samples were processed through the DEPArray™ beginning with dissociation of the FFPE curls followed by single-cell image-based cell sorting to separate and recover pure distinct tumor cell populations prior to HER2 FISH analysis. Data was only obtained when ∼200 intact cytokeratin+/vimentin-/DAPI+ tumor cells from each sample were recovered and used for subsequent FISH using a standard dual-color HER2/CEP17 FISH procedure. Results: Overall, 80% concordance between DEPArray™-HER2 and conventional HER2 (6 HER2 negative and 10 HER2 positive) was observed between lab(s) and the conventional HER2 method. In each of 4 cases, a discordant HER2 result was reported by one of three sites. In three of these discordant cases, the DEPArray™ HER2 ratio was reported as amplified while the conventional result was negative. In the remaining discordant case, the converse was observed by one site; however, this case was initially evaluated 15 years ago. All three sites correctly scored the SKBr3 positive control cells. Conclusions: The results showed a high concordance rate of correct HER2 status classification. This data further supports the understanding that tissue heterogeneity can indeed give rise to discordant results that may consequently affect treatment options for patients. We demonstrate that sample preparation by DEPArray™ may aid in a more precise classification for tumor biomarker status.

Fluorescent in Situ Hybridization (FISH) guidelines defined by American Society of Clinical Oncology (ASCO) and the College of American Pathologists for determining HER2 status are set to improve accuracy and usefulness as a diagnostic marker in breast cancer. Despite these guidelines, many factors can influence HER2 testing results such as sample preparation, assay-conditions and interpretation of test results due to heterogeneous breast cancer samples. In this multi-site study, sample preparation was carried out using the DEPArray™ to recover pure tumor cell populations from formalin-fixed, paraffin-embedded (FFPE) breast tumor samples. We then compared HER2/CEP17 ratios obtained from the DEPArray™ processed samples from each laboratory to routine FISH on tissue sections. Methods: Eight breast FFPE tumor tissue biopsies were obtained from commercial tissue banks. From the paraffin tissue blocks, four consecutive tissue curls (each 50 microns thick) were prepared. One curl from each of the 8 patient samples was distributed to four different laboratories for analysis following DEPArray™ based sample preparation. After an initial disassociation of each curl into a single-cell suspension, intact cells were sorted and then recovered based on cytokeratin/ vimentin/DAPI staining using the DEPArray™. Cytokeratin+/Vimentin-/DAPI+ tumor (~250) and Cytokeratin-/Vimentin+/DAPI+ stromal (~250) recovered cells were then deposited onto glass slides prior to standard dual-color HER2/CEP17 FISH analysis for comparison to conventional HER2 FISH result. Results: Serially sectioned breast tumors from 8 negative/positive cases: 7 infiltrating ductal carcinoma (IDC) and 1 metastatic carcinoma were studied. All four sites demonstrated 100% concordance between FISH results compared to the conventional HER2 FISH result. Overall, u003e60% of DEPArray™ isolated cells were recovered from FFPE samples that ranged from 1- 15 years of age and reported to contain 60% to 80% tumor content. The use of pure sorted cells permitted the accurate determination of HER2 amplification status in only the tumor cells while the stromal cells consistently yielded a more normalized ratio of HER2 to centromere 17. Conclusion: The preliminary results of this multi-site study demonstrate that use of DEPArray™ for sorted pure populations is reproducible as well as reliable method for subsequent analysis of HER2 by FISH on FFPE derived tumor cells. Given that traditional FFPE-based HER2 FISH results may be influenced by the tissue sectioning procedure, tissue heterogeneity and/or the scattering of few HER2 amplified tumor cells among normal stromal cells. The DEPArray™ allows analysis of immunofluorescence images and DNA content to isolate and recover pure and intact cell populations. This isolation of pure cell populations prior to FISH analysis is attractive for achieving precise determination of HER2 status on equivocal cases. A more formal analytical validation of this approach through CLIA is currently underway. Citation Format: Gerber A, Konig L, Millner L, Strotoman L, Khurana A, Kasimir-Bauer S, Moore MW, Cotter PD, Bischoff F. Development of a novel HER2 testing strategy, using image-based cell-sorting to isolate pure cell populations from FFPE upstream of FISH [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-03-10.

Fluorescent in Situ Hybridization (FISH) is commonly used for assessment of chromosomal alterations. Guidelines for determining FISH-based classification of clinical biomarkers exist but are based on pre-analytical factors, including fixation/sectioning/thickness/age, that can greatly influence biomarker status determination. Here, we use single-cell image-based cell sorting by DEPArrayTM for the separation and recovery of pure distinct cell populations prior to FISH. Methods: A multi-center study to evaluate HER2-FISH based analysis on FFPE with and without DEPArrayTM pre-processing was conducted using breast tumors classified as infiltrating ductal carcinoma (n=12), metastatic (n=1) and ductal carcinoma (n=1). From each block, four 50-micron thick curls were sectioned. One curl from each sample was sent to each of four centers (3 US; 1 EU). Each site performed disassociation of curls to generate a single cell suspension. Cells were then stained and sorted using the DEPArrayTM platform for recovery of tumor (cytokeratin+/vimentin-/DAPI+) and stromal (cytokeratin-/vimentin+/DAPI+) cells. Dual-probe FISH for HER2 and centromere 17 was performed on the sorted cells and compared with conventional tissue section FISH. Results: Overall, ≥ 90% concordance between the sorted tumor cells and the conventional HER2 FISH result was observed. Among the 7 HER2+ cases, HER2 ratio scores for the sorted tumor cells ranged slightly higher, from 2.60 to 8.95, as compared to the conventional method (from 2.10 to 5.14). In all cases in which stromal cells were also recovered, an expected normal ratio was observed, thus verifying that the populations were efficiently separated. Discordance can be attributed to intra-tumoral heterogeneity and the fact that conventional FISH on FFPE requires only a 4-micron section for analysis. Conclusion: Today, a percentage of patients are likely misclassified for the biomarker of interest as result of pre-analytical factors. We demonstrate here the ability to overcome these pre-analytic factors and ultimately improve the accuracy in determining biomarker status using the DEPArrayTM Note: This abstract was not presented at the meeting. Citation Format: Amanda Gerber, Aditi Khurana, Lisa Koenig, Lindsay Strotoman, Lori Millner, Valeria Sero, Chiara Bolognesi, Sabine Kasimir-bauer, Gianni Medoro, Matthew Moore, Philip Cotter, Nicolo Manaresi, Farideh Bischoff. Image-based single cell-sorting to separate and recover distinct cell populations from complex heterogeneous mixed tissue: precise sample preparation upstream of FISH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2730. doi:10.1158/1538-7445.AM2017-2730

There is a profound need in oncology to detect cancer earlier, guide individualized therapies, and better monitor progress during treatment. Currently, some of this information can be achieved through solid tissue biopsy and imaging. However, these techniques are limited because of the invasiveness of the procedure and the size of the tumor. A liquid biopsy can overcome these barriers as its non-invasive nature allows samples to be collected over time. Liquid biopsies may also allow earlier detection than traditional imaging. Liquid biopsies include the analysis of circulating tumor cells (CTCs), cell-free nucleic acid (cfNA), or extracellular vesicles obtained from a variety of biofluids, such as peripheral blood. In this review, we discuss different liquid biopsy types and how they fit into the current regulatory landscape.

Precision medicine in oncology focuses on identifying which therapies are most effective for each patient based on genetic characterization of the cancer. Traditional chemotherapy is cytotoxic and destroys all cells that are rapidly dividing. The foundation of precision medicine is targeted therapies and selecting patients who will benefit most from these therapies. One of the newest aspects of precision medicine is liquid biopsy. A liquid biopsy includes analysis of circulating tumor cells, cell-free nucleic acid, or exosomes obtained from a peripheral blood draw. These can be studied individually or in combination and collected serially, providing real-time information as a patient's cancer changes.

The ability to isolate, phenotypically characterize, and profile the gene signature of single circulating tumor cells (CTCs) will provide deeper insight into cancer metastasis and will lead to improved diagnosis and treatment of cancer patients. Using current methods based on positive selection of epithelial markers, up to 40% of patients with highly aggressive metastatic breast cancer have no CTCs detected. This may be due to CTCs undergoing the process of epithelial to mesenchymal transition (EMT). This process causes epithelial characteristics to be down regulated as more mesenchymal features develop allowing the cells to have higher motility and increased ability to evade immune detection. Methods to enable capture and characterization of heterogeneous CTCs including those with non-epithelial phenotypes are needed. Additionally, having the ability to isolate pure, single CTCs allows for individual cell analysis providing information that is masked by bulk analysis. Here we provide details on an integrated method for isolating and characterizing heterogeneous single breast cancer cells based on a multiantigen, negative depletion strategy followed by sorting with dielectrophoresis (DEPArray). This negative depletion method removes unwanted leukocytes and allows heterogeneous, epithelial and non-epithelial cells to be retained. We were able to visually verify the isolation of single tumor cells with 100% purity. Following sorting, next generation sequencing and real-time PCR of single cells is described. This method allows single, heterogeneous CTCs to be analyzed which will have implications for prognosis and treatment options.

The measurement and characterization of circulating tumor cells (CTCs) hold promise for advancing personalized therapeutics. CTCs are the precursor to metastatic cancer and thus have the potential to radically alter patient treatment and outcome. Currently, clinical information provided by the enumeration of CTCs is limited to predicting clinical outcome. Other areas of interest in advancing the practice of pathology include: using CTCs for early detection of potential metastasis, determining and monitoring the efficacy of individualized treatment regimens, and predicting site-specific metastasis. Important hurdles to overcome in obtaining this type of clinical information involve present limitations in defining, detecting, and isolating CTCs. Currently, CTCs are detected using epithelial markers. The definition of what distinguishes a CTC should be expanded to include CTCs with heterogeneous phenotypes, and markers should be identified to enable a more comprehensive capture. Additionally, most methods available for detecting CTCs do not capture functionally viable CTCs. Retaining functional viability would provide a significant advantage in characterizing CTC-subtypes that may predict the site of metastatic invasion and thus assist in selecting effective treatment regimens. In this review we describe areas of clinical interest followed by a summary of current circulating cell-separation technologies and present limitations. Lastly, we provide insight into what is required to overcome these limitations as they relate to applications in advancing the practice of pathology and laboratory medicine.

N-acetyltransferase 1 (NAT1) is a phase II metabolic enzyme responsible for the biotransformation of aromatic and heterocyclic amine carcinogens such as 4-aminobiphenyl (ABP). NAT1 catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5'-untranslated regions (UTR). NATa mRNA is expressed primarily in the kidney, liver, trachea, and lung while NATb mRNA has been detected in all tissues studied. To determine if differences in 5'-UTR have functional effect upon NAT1 activity and DNA adducts or mutations following exposure to ABP, pcDNA5/FRT plasmid constructs were prepared for transfection of full-length human mRNAs including the 5'-UTR derived from NATa or NATb, the open reading frame, and 888 nucleotides of the 3'-UTR. Following stable transfection of NATb/NAT1*4 or NATa/NAT1*4 into nucleotide excision repair (NER) deficient Chinese hamster ovary cells, N-acetyltransferase activity (in vitro and in situ), mRNA, and protein expression were higher in NATb/NAT1*4 than NATa/NAT1*4 transfected cells (P?<?0.05). Consistent with NAT1 expression and activity, ABP-induced DNA adducts and hypoxanthine phosphoribosyl transferase mutants were significantly higher (P?<?0.05) in NATb/NAT1*4 than in NATa/NAT1*4 transfected cells following exposure to ABP. These differences observed between NATa and NATb suggest that the 5'-UTRs are differentially regulated. (c) 2011 Wiley Periodicals, Inc.

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II cytosolic enzyme responsible for the activation or deactivation of many arylamine compounds including pharmaceuticals and environmental carcinogens. NAT1 is highly polymorphic and has been associated with altered risk toward many cancers. NAT1*14B is characterized by a single nucleotide polymorphism in the coding region (rs4986782; 560G>A; R187Q). NAT1*14B is associated with higher frequency of smoking-induced lung cancer and is the most common "slow acetylator" arylamine NAT1 genetic variant. Previous studies have reported decreased N- and O-acetylation capacity and increased proteasomal degradation of NAT1 14B compared with the referent, NAT1 4. The current study is the first to investigate NAT1*14B expression using constructs that completely mimic NAT1 mRNA by including the 5'- and 3'-untranslated regions, together with the open reading frame of the referent, NAT1*4, or variant, NAT1*14B. Our results show that NAT1 14B is not simply associated with "slow acetylation." NAT1 14B-catalyzed acetylation phenotype is substrate-dependent, and NAT1 14B exhibits higher N- and O-acetylation catalytic efficiency as well as DNA adducts after exposure to the human carcinogen 4-aminobiphenyl.

N-acetyltransferase 1 (NAT1) catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1*10 is the most common variant haplotype and is associated with increased risk for numerous cancers. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in messenger RNAs (mRNAs) with distinct 5'-untranslated regions (UTRs). To best mimic in vivo metabolism and the effect of NAT1*10 polymorphisms on polyadenylation usage, pcDNA5/Flp recombination target plasmid constructs were prepared for transfection of full-length human mRNAs including the 5'-UTR derived from NATb, the open reading frame and 888 nucleotides of the 3'-UTR. Following stable transfection of NAT1*4, NAT1*10 and an additional NAT1*10 variant (termed NAT1*10B) into nucleotide excision repair-deficient Chinese hamster ovary cells, N- and O-acetyltransferase activity (in vitro and in situ), mRNA and protein expression were higher in cells transfected with NAT1*10 and NAT1*10B than in cells transfected with NAT1*4 (P < 0.05). Consistent with NAT1 expression and activity, cytotoxicity and hypoxanthine phosphoribosyl transferase mutants following 4-aminobiphenyl exposures were higher in NAT1*10 than in NAT1*4 transfected cells. Ribonuclease protection assays showed no difference between NAT1*4 and NAT1*10. However, protection of one probe by NAT1*10B was not observed with NAT1*4 or NAT1*10, suggesting additional mechanisms that regulate NAT1*10B. The higher mutants in cells transfected with NAT1*10 and NAT1*10B are consistent with an increased cancer risk for individuals possessing NAT1*10 haplotypes.

: activation or inactivation. NAT1*10 and NAT1*14, common variant alleles have been associated with increased risk for numerous cancers including breast. NAT1 is also upregulated in breast cancer. We employed a novel approach to study functional differences caused by NAT1*10 and NAT1*14 polymorphisms by using constructs that mimic complete human mRNAs by including the 5 -UTR, coding region and 3 -UTR. Significantly more enzymatic activity, protein expression, mRNA levels and 4-aminobiphenyl-induced DNA adducts and mutants were observed in all constructs containing the NATb 5 -UTR compared to those containing the NATa 5 -UTR. After treatment with 4-aminobiphenyl(ABP), more DNA adducts and mutagenesis was observed in cells transfected with NATb constructs than cells transfected with NATa constructs. Kinetic parameters for NAT1*14B compared to NAT1*4 were determined. The NAT1*14B v(max) for PABA, ABP, and N-OH-ABP was less than NAT1*4 vmax. The NAT1*14B v(max)/km, or instrinsic clearance, was lower for PABA when compared to NAT1*4 v(max)/km. The NAT1*14B v(max)/km was not different compared to NAT1*4 v(max)/km for ABP, but the NAT1*14B v(max)/km was higher for N -OH-ABP compared to vmax/km NAT1*4 . This indicates that clearance for the NAT1 variant, NAT1*14B, is substrate dependent. Consequently, cancer risk related to NAT1*14B is likely also substrate dependent. NATb/NAT1*10 and NATb/NAT1*10B transiently and stably transfected cells resulted in higher mRNA levels, protein expression, ABP induced cytotoxicity and hprt -mutants. This indicates that individuals possessing a NAT1*10 or NAT1*10B genotype are associated with an increased cancer risk than individuals who possess a NAT1*4 genotype.

Aromatic amines such as 4-aminobiphenyl (ABP) require biotransformation to exert their carcinogenic effects. Genetic polymorphisms in biotransformation enzymes such as N-acetyltransferase 2 (NAT2) may modify cancer risk following exposure. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator), NAT2*5B (common Caucasian slow acetylator), or NAT2*7B (common Asian slow acetylator) alleles (haplotypes) were treated with ABP to test the effect of NAT2 polymorphisms on DNA adduct formation and mutagenesis. ABP N-acetyltransferase catalytic activities were detectable only in cell lines transfected with NAT2 and were highest in cells transfected with NAT2*4, lower in cells transfected with NAT2*7B, and lowest in cells transfected with NAT2*5B. Following ABP treatment, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) was the primary adduct formed. Cells transfected with both CYP1A1 and NAT2*4 showed the highest concentration-dependent cytotoxicity, hypoxanthine phosphoribosyl transferase (hprt) mutants, and dG-C8-ABP adducts. Cells transfected with CYP1A1 and NAT2*7B showed lower levels of cytotoxicity, hprt mutagenesis, and dG-C8-ABP adducts. Cells transfected with CYP1A1 only or cells transfected with both CYP1A1 and NAT2*5B did not induce cytotoxicity, hprt mutagenesis or dG-C8-ABP adducts. ABP-DNA adduct levels correlated very highly (r>0.96) with ABP-induced hprt mutant levels following each treatment. The results of the present study suggest that investigations of NAT2 genotype or phenotype associations with disease or toxicity could be more precise and reproducible if heterogeneity within the "slow" NAT2 acetylator phenotype is considered and incorporated into the study design.

N‐acetyltransferase 1 (NAT1) catalyzes N‐acetylation of aromatic and heterocyclic amine carcinogens resulting in their activation or inactivation. NAT1*10, a common NAT1 variant in many ethnic groups, is associated with increased risk for numerous cancers and congenital defects. NAT1*10 has putatively been described as a rapid acetylator allele (haplotype) and is characterized by two polymorphisms located in the 3' UTR, one of which disrupts a polyadenylation (polyA) signal. We determined polyA patterns of NAT1*10 compared to referent NAT1*4 using RNase protection assays (RPAs). We employed a novel approach to study functional differences caused by NAT1*10 polymorphisms by using constructs that mimic complete human mRNAs. Plasmid constructs of NAT1*10 and NAT1*4 contained full length human mRNAs including either the NATa (alternative promoter) or NATb (major promoter) 5′‐UTR, the ORF, and 885 base pairs of the 3'UTR region. Following transient transfection into Chinese hamster ovary cells, NAT1‐catalyzed N‐acetylation of p‐aminobenzoic acid was measured by HPLC and NAT1 protein expression was measured by Western blot. mRNA levels were determined by RT‐PCR and polyadenylation patterns by RPA. No differences were observed between NAT1*10 and NAT1*4 in levels of N‐acetyltransferase activity, NAT1 protein and mRNA, or polyA pattern. Supported by USPHS grants CA034627, ES011564, and ES014443 and DOD BC083107.

Abstract B77 N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of many aromatic amine carcinogens such as 2-aminofluorene and 4-aminobiphenyl. Following N-oxidation by cytochrome P450’s, NAT1 catalyzes the O-acetylation of the N-hydroxy metabolites of many aromatic and heterocyclic amine carcinogens. NAT1 genetic polymorphisms have been associated with differential risks to various cancers related to aromatic and heterocyclic amine carcinogens, but the findings have been inconsistent. Individual susceptibility to these carcinogenic compounds could be modified by genetic polymorphism or by other factors causing differences in NAT1 expression. Our laboratory and others have reported that NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5’-UTR regions. The 117 nucleotide NATb 5’-UTR contains two noncoding exons, while the 371 nucleotide NATa 5’UTR contains four noncoding exons. The two 5’UTRs share a 79 nucleotide exon immediately upstream of the open reading frame (ORF). We hypothesized that mRNAs including either the NATa or NATb 5’-UTR may be associated with differences in protein expression and that SNPs included in variant NAT1 alleles may modify this effect. We therefore investigated effects on protein expression due to interaction of the different 5’-UTRs with the ORF and region 3’ to the ORF of the reference NAT1*4 allele, and two variant alleles: NAT1*10 and NAT1*11 that are associated with modified risk to arylamine-induced cancers. pcDNA5/FRT plasmid constructs were prepared for transient transfection of full length human mRNAs including either the NATa or NATb 5’-UTR, the ORF, and 885 nucleotides of the region 3’ to the ORF. For each allele of interest, two constructs were made, one including the NATa 5’-UTR and one including the NATb 5’-UTR. Following transient transfection into Chinese hamster ovary cells, NAT1-catalyzed N-acetylation of p-aminobenzoic was measured by HPLC and NAT1 protein expression was measured by Western blot. A difference of approximately 10 fold (p<0.05) in both NAT1 catalytic activity and protein was observed between the NATa and NATb mRNA forms but there was no evidence for allele specific 5’-UTR interactions. These results suggest that differences in NAT1 expression are modified by factors other than polymorphisms in the ORF. Further experiments to examine the possible roles of mRNA production, mRNA stability or differences in mRNA translation efficiency on NAT1 expression are needed. Partially supported by USPHS grants CA034627, ES014559, ES011564, and ES014443. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B77.