Cell-free DNA from nail clippings as source of normal control for genomic studies in hematologic malignancies.

Comprehensive genomic sequencing is becoming a critical component in the assessment of hematologic malignancies, with broad implications for patient management. In this context, unequivocally discriminating somatic from germline events is challenging but greatly facilitated by matched analysis of tumor:normal pairs. In contrast to solid tumors, conventional sources of normal control (peripheral blood, buccal swabs, saliva) could be highly involved by the neoplastic process, rendering them unsuitable. In this work we describe our real-world experience using cell free DNA (cfDNA) isolated from nail clippings as an alternate source of normal control, through the dedicated review of 2,610 tumor:nail pairs comprehensively sequenced by MSK-IMPACT-heme. Overall, we find nail cfDNA is a robust source of germline control for paired genomic studies. In a subset of patients, nail DNA may have tumor DNA contamination, reflecting unique attributes of the hematologic disease and transplant history. Contamination is generally low level, but significantly more common among patients with myeloid neoplasms (20.5%; 304/1482) compared to lymphoid diseases (5.4%; 61/1128) and particularly enriched in myeloproliferative neoplasms with marked myelofibrosis. When identified in patients with lymphoid and plasma-cell neoplasms, mutations commonly reflected a myeloid profile and correlated with a concurrent/evolving clonal myeloid neoplasm. For nails collected after allogeneic stem-cell transplantation, donor DNA was identified in 22% (11/50). In this cohort, an association with recent history of graft-vs-host disease was identified. These findings should be considered as a potential limitation for the use of nail as normal control but could also provide important diagnostic information regarding the disease process.

Validation of a High-Sensitivity Assay for Detection of Chimeric Antigen Receptor T-Cell Vectors Using Low-Partition Digital PCR Technology.

Although in vivo engraftment, expansion, and persistence of chimeric antigen receptor (CAR) T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital PCR assay for ultrasensitive detection of CAR constructs after treatment, circumventing known technical limitations of low-partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene, and Memorial Sloan Kettering CAR constructs, were employed to validate testing on the Bio-Rad digital PCR low-partitioning platform; results were compared with Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500 ng. Using dual-input reactions (20 and 500 ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1 × 10-5 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared with the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6 to 28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005% to 74% (vector versus reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (P < 0.005). Only three patients with undetectable constructs had disease progression at the time of sampling.

Clonal Characterization and Somatic Hypermutation Assessment by Next-Generation Sequencing in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: A Detailed Description of the Technical Performance, Clinical Utility, and Platform Comparison.

Somatic hypermutation status of the IGHV gene is essential for treating patients with chronic lymphocytic leukemia/small lymphocytic lymphoma. Unlike the conventional low-throughput method, assessment of somatic hypermutation by next-generation sequencing (NGS) has potential for uniformity and scalability. However, it lacks standardization or guidelines for routine clinical use. We critically assessed the performance of an amplicon-based NGS assay across 458 samples. Using a validation cohort (35 samples), the comparison of two platforms (Ion Torrent versus Illumina) and two primer sets [leader versus framework region 1 (FR1)] in their ability to identify clonotypic IGHV rearrangement(s) revealed 97% concordance. The mutation rates were identical by both platforms when using the same primer set (FR1), whereas a slight overestimation bias (+0.326%) was found when comparing FR1 with leader primers. However, for nearly all patients this did not affect the stratification into mutated or unmutated categories, suggesting that use of FR1 may provide comparable results if leader sequencing is not available and allowing for a simpler NGS laboratory workflow. In routine clinical practice (423 samples), the productive rearrangement was successfully detected by either primer set (leader, 97.7%; FR1, 94.7%), and a combination of both in problematic cases reduced the failure rate to 1.2%. Higher sensitivity of the NGS-based analysis also detected a higher frequency of double IGHV rearrangements (19.1%) compared with traditional approaches.

Clinical Utility and Performance of an Ultrarapid Multiplex RNA-Based Assay for Detection of ALK, ROS1, RET, and NTRK1/2/3 Rearrangements and MET Exon 14 Skipping Alterations.

Several kinase fusions are established targetable drivers in lung cancers. However, rapid and comprehensive detection remains challenging because of diverse partner genes and breakpoints. We assess the clinical utility and performance of a rapid microfluidic multiplex real-time PCR-based assay for simultaneous query of fusions involving ALK, ROS1, RET, and NTRK1/2/3, as well as MET exon 14 skipping, using a 3-hour automated process. Dual analytic strategies were utilized: fusion-specific amplification and 3' to 5' expression imbalance. One-hundred and forty-three independent, formalin-fixed, paraffin-embedded tumor samples (112 surgical specimens, 31 cytologic cell blocks) were analyzed: 133 with known kinase gene alterations and 10 negative samples based on clinically validated next-generation sequencing. Testing was successful in 142 (99%) cases. The assay demonstrated a sensitivity of 97% (28/29), 100% (31/31), 92% (22/24), 81% (22/27), and 100% (20/20) for ALK, RET, ROS1, and NTRK1/2/3 rearrangements and MET exon 14 skipping alterations, respectively, with 100% specificity for all. Concordant results were achieved in specimens aged up to 5 years, with >10% tumor, and inputs of at least 9 mm2 (surgical specimens) and 9000 cells (cytologic cell blocks). The assay enables rapid screening for clinically actionable kinase alterations with quicker turnaround and lower tissue requirements compared with immunohistochemistry and molecular methods, while also circumventing the infrastructure dependencies associated with next-generation sequencing and fluorescence in situ hybridization.

Rapid EGFR Mutation Detection Using the Idylla Platform: Single-Institution Experience of 1200 Cases Analyzed by an In-House Developed Pipeline and Comparison with Concurrent Next-Generation Sequencing Results.

Mutations in the epidermal growth factor receptor (EGFR) are the most common targetable alterations in lung adenocarcinoma. To facilitate rapid testing, the Idylla EGFR assay was incorporated as a screening method before next-generation sequencing (NGS). Validation and experience using an in-house developed analysis pipeline, enhanced with a manual review algorithm is described. Results are compared with corresponding NGS results. In all, 1249 samples were studied. Validation demonstrated 98.57% (69/70) concordance with the reference methods. The limit of detection varied from 2% to 5% variant allele frequency for total EGFR quantitation cycle between 20 and 23. Of the 1179 clinical cases, 23.41% were EGFR-positive by Idylla. Concurrent NGS was successfully performed on 94.9% (799/842) requests. Concordance of Idylla with NGS was 98.62% (788/799) and 98.50% (787/799) using our in-house and Idylla analysis pipelines, respectively. Discordances involved missed mutations by both assays associated with low tumor/low input. Incorporating a manual review algorithm to supplement automated calls improved accuracy from 98.62% to 99.37% and sensitivity from 94.68% to 97.58%. Overall reporting time, from receipt of material to official clinical report, ranged from 1 to 3 days. Therefore, Idylla EGFR testing enables rapid and sensitive screening without compromising subsequent comprehensive NGS, when required. Automated calling, enhanced with a manual review algorithm, reduces false-negative calls associated with low tumor/low input samples.

A Pan-Cancer Study of Somatic TERT Promoter Mutations and Amplification in 30,773 Tumors Profiled by Clinical Genomic Sequencing.

TERT gene promoter mutations are known in multiple cancer types. Other TERT alterations remain poorly characterized. Sequencing data from 30,773 tumors analyzed by a hybridization capture next-generation sequencing assay (Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets) were analyzed for the presence of TERT alterations. Promoter rearrangements (500 bases upstream of the transcriptional start site), hypermethylation (n = 57), and gene expression (n = 155) were evaluated for a subset of cases. Mutually exclusive and recurrent promoter mutations were identified at three hot spots upstream of the transcriptional start site in 11.3% of cases (-124: 74%; -146: 24%; and -138: <2%). Mutually exclusive amplification events were identified in another 2.3% of cases, whereas mutually exclusive rearrangements proximal to the TERT gene were seen in 24 cases. The highest incidence of TERT promoter mutations was seen in cutaneous melanoma (82%), whereas amplification events significantly outnumbered promoter mutations in well-differentiated/dedifferentiated liposarcoma (14.1% versus 2.4%) and adrenocortical carcinoma (13.6% versus 4.5%). Gene expression analysis suggests that the highest levels of gene expression are seen in cases with amplifications and rearrangements. Hypermethylation events upstream of the TERT coding sequence were not mutually exclusive with known pathogenic alterations. Studies aimed at defining the prevalence and prognostic impact of TERT alterations should incorporate other pathogenic TERT alterations as these may impact telomerase function.

Ultrarapid EGFR Mutation Screening Followed by Comprehensive Next-Generation Sequencing: A Feasible, Informative Approach for Lung Carcinoma Cytology Specimens With a High Success Rate.

INTRODUCTION: For patients with advanced NSCLC, cytologic samples may be the only diagnostic specimen available for molecular profiling. Although both rapid and comprehensive assessment are essential in this setting, an integrated multitest approach remains an important strategy in many laboratories, despite the risks and challenges when working with scant samples. In this study, we describe our experience and high success rate in using a multitest approach, focusing on the clinical validation and incorporation of ultrarapid EGFR testing using the Idylla system followed by comprehensive next-generation sequencing (NGS). METHODS: Cytology samples received for routine molecular testing were included in this study. The performance characteristics of the EGFR Idylla assay were assessed; tissue suitability parameters and interpretation criteria to supplement automated mutation calling were established. The assay performance was monitored for 1 year, comparing the results with those of concurrent NGS testing by MSK-IMPACT (primarily) or MSK-AmpliSeq and MSK-Fusion solid panel in a subset of cases. RESULTS: Overall, 301 samples were studied; 83 samples were included in validation (60.2% [50 of 83] were positive for EGFR mutations). Concordance with the reference method was 96.4% (80 of 83) of the samples with excellent reproducibility. The limit of detection was variable depending on the total tissue input and the specific mutation tested. Unextracted tissue inputs that maintained total EGFR cycle of quantification at less than 23 allowed all mutations to be detected if present at greater than 5% variant allele frequency. Mutations could be detected at 1% variant allele frequency with total EGFR cycle of quantification of 18. During the clinical implementation phase, 218 NSCLC samples were tested by Idylla (24.3% [53 of 218] were EGFR mutation positive). Concurrent NGS testing was requested on 165 samples and successfully performed on 96.4% (159 of 165) of the samples. The Idylla automated results were concordant with those obtained by NGS in 96.2% (153 of 159) of cases and improved to 98.7% (157 of 159) after incorporation of manual review criteria to supplement automated calling, resulting in a diagnostic sensitivity of 95.6% (95% confidence interval: 84.9%-99.5%). In general, 9% (14 of 159) of the cases tested by NGS had EGFR mutations not covered by the Idylla assay, primarily insertions in exon 19 and 20 and minor mutations cooccurring with canonical sensitizing mutations. CONCLUSIONS: Comprehensive molecular testing is feasible and has a high success rate in NSCLC cytology samples when using a multitest approach. Testing with the Idylla system enables rapid and accurate determination of the EGFR status without compromising subsequent NGS testing.

TFEB Expression Profiling in Renal Cell Carcinomas: Clinicopathologic Correlations.

TFEB is overexpressed in TFEB-rearranged renal cell carcinomas as well as in renal tumors with amplifications of TFEB at 6p21.1. As recent literature suggests that renal tumors with 6p21.1 amplification behave more aggressively than those with rearrangements of TFEB, we compared relative TFEB gene expression in these tumors. This study included 37 TFEB-altered tumors: 15 6p21.1-amplified and 22 TFEB-rearranged (including 5 cases from The Cancer Genome Atlas data set). TFEB status was verified using a combination of fluorescent in situ hybridization (n=27) or comprehensive molecular profiling (n=13) and digital droplet polymerase chain reaction was used to quantify TFEB mRNA expression in 6p21.1-amplified (n=9) and TFEB-rearranged renal tumors (n=19). These results were correlated with TFEB immunohistochemistry. TFEB-altered tumors had higher TFEB expression when normalized to B2M (mean: 168.9%, n=28), compared with non-TFEB-altered controls (mean: 7%, n=18, P=0.005). Interestingly, TFEB expression in tumors with rearrangements (mean: 224.7%, n=19) was higher compared with 6p21.1-amplified tumors (mean: 51.2%, n=9; P=0.06). Of note, classic biphasic morphology was only seen in TFEB-rearranged tumors and when present correlated with 6.8-fold higher TFEB expression (P=0.00004). Our results suggest that 6p21.1 amplified renal tumors show increased TFEB gene expression but not as much as t(6;11) renal tumors. These findings correlate with the less consistent/diffuse expression of downstream markers of TFEB activation (cathepsin K, melan A, HMB45) seen in the amplified neoplasms. This suggests that the aggressive biological behavior of 6p21.1 amplified renal tumors might be secondary to other genes at the 6p21.1 locus that are co-amplified, such as VEGFA and CCND3, or other genetic alterations.

Establishment of Immunoglobulin Heavy (IGH) Chain Clonality Testing by Next-Generation Sequencing for Routine Characterization of B-Cell and Plasma Cell Neoplasms.

Immunoglobulin heavy chain (IGH) clonality testing by next-generation sequencing (NGS) offers unique advantages over current low-throughput methods in the assessment of B-cell lineage neoplasms. Clinical use remains limited because assays are not standardized and validation/implementation guidelines are not yet developed. Herein, we describe our clinical validation and implementation of NGS IGH clonality testing and summarize our experience based on extensive routine use. NGS-based clonality testing targeting IGH FR1, FR2, FR3, and the conserved leader sequence upstream of FR1 was validated using commercially available kits. Data were analyzed by commercial and in-house-developed bioinformatics pipelines. Performance characteristics were evaluated directly comparing with capillary electrophoresis (CE) assays (BIOMED-2 primers). Assays were monitored after implementation (>1.5 years), concurrently testing by CE methods. A total of 1189 clinical samples were studied (94 validation, 1095 postimplementation). NGS showed superior performance compared with CE assays. For initial assessment, clonality detection rate was >97% for all malignancy types. Concordance with CE was 96%; discordances were related to higher sensitivity/resolution of NGS and improved detection in cases with high somatic hypermutation. Routine NGS clonality assessment is feasible and superior to existing assays, enabling accurate and specific index clone assessment and future tracking of all rearrangements in a patient sample. Successful implementation requires new standardization, validation, and implementation processes, which should be performed as a multicenter and multidisciplinary collaboration.

Clinical relevance of 1p and 19q deletion for patients with WHO grade 2 and 3 gliomas.

PURPOSE: To assess the frequency of chromosomes 1p and 19q deletions in gliomas and to correlate 1p deletion with prognosis in patients with grade 2 and grade 3 gliomas independently of histologic subtype. METHODS: We retrospectively evaluated 208 patients with WHO grade 2 and 3 gliomas who had 1p/19q molecular studies performed between 2000 and 2004. DNA was extracted from tumor tissue and germline material and evaluated by PCR using microsatellite markers for each chromosome. RESULTS: There were 113 men and 95 women with a median age at diagnosis of 40. Thirty-eight patients had a low-grade astrocytoma (A2), 58 low-grade oligodendroglioma (O2), 31 low-grade oligoastrocytoma (OA2), 21 anaplastic astrocytoma (A3), 37 anaplastic oligodendroglioma (O3), and 23 had an anaplastic oligoastrocytoma (OA3). Chromosome 1p analysis was performed in all patients and showed deletions in 105 patients (76% of O2, 42% of OA2, 21% of A2, 89% of O3, 17% of AO3, and 14% of A3). Chromosome 19q studies were performed in 118 patients and showed deletions in 46 (56% of O2, 45% of OA2, 27% of A2, 76% of O3, 11% of OA3 and 0% of A3). On multivariate analyses, chromosome 1p was a prognostic factor for prolonged PFS (HR = 1.75, P = 0.03) and OS (HR = 3.59, P = 0.02) in grade 2 gliomas but not for grade 3 (HR = 0.81, P = 0.7 for PFS; HR = 1.31, P = 0.7 for OS). CONCLUSION: Chromosome 1p deletion is a significant positive prognostic marker in diffuse, grade 2 gliomas regardless of histologic subtype.

Efeito dos ácidos lático e butírico, isolados e associados, sobre o desempenho e morfometria intestinal em frangos de corte / Effect of lactic and butiric acids , isolated and associated, on performance and intestinal morfometric in broilers

A avicultura nacional tem sofrido constantes desafios, tendo como objetivo ampliar o mercado consumidor do frango de corte ao redor do mundo. Cada mercado apresenta exigências diferentes, como no caso do mercado europeu que estabelece a exclusão dos antibióticos como promotores de crescimento na alimentação dos frangos. Em função deste fato, realizou-se este estudo, com o objetivo de avaliar o uso dos ácidos lático e butírico, isolados e associados, como aditivos em dietas de frangos de corte, em relação ao promotor de crescimento usualmente utilizado nas dietas dos mesmos. Foram avaliados o desempenho e a morfometria intestinal das aves. Utilizaram-se 1400 pintinhos machos da linhagem comercial Ross, divididos em cinco tratamentos (controle - sem aditivo; com adição de ácido butírico; com adição de ácido lático; com adição de ácido butírico+ ácido láctico; com adição do antibiótico avilamicina, promotor de crescimento tradicionalmente utilizado na criação de frangos de corte. Os resultados de desempenho indicaram que a interação dos ácidos foi significativa na fase inicial, entretanto não ocorreu um efeito aditivo dos ácidos, sendo o uso do ácido butírico mais recomendável durante essa fase. Já na fase de crescimento, a interação foi significativa com um efeito aditivo, recomendando seu uso nas rações de crescimento. Os resultados obtidos nas analises de morfometria intestinal não foram conclusivos. Em termos gerais, requerem-se mais estudos quanto ao uso dos ácidos orgânicos como promotores de crescimento.

During the last years, the national poultry keeping has suffered constant challenges, having as objective to diversify the consuming market of the broiler to around of the world. However, each market presents different requirements, as in the case of the European market that establishes the exclusion of antibiotics as promotional of growth in the feeding of the broilers. Aiming at this problematic one, this study was become fullfilled, with the objective to analyze the results of the association of the butyric and lactic acids as additives in the ration of broilers, in comparison to the results gotten for the usually used promoters of growth in the diets of broilers. They had been evaluated: the animal performance, humoral immunology and intestinal morfometric of the birds. 1400 male chickens of the commercial ancestry Ross had been used, dividing them in five groups with different treatments to that if it relates to the additive use. Being a group it has controlled absent of additive, a group with butyric acid, a group with lactic acid, the fourth group with the association of butyric and lactic acids, and the fifth group with avilamicina (antibiotic) as promotional of traditionally used growth in the production of broilers. The performance results had indicated that the interaction of acid ones was significant in the initial phase, mean while did not occur an additive effect of acid ones, being the use of the butyric acid separately more recommendable during this phase. Already in the growth phase, the interaction was significant with an additive effect, recommending its use in the rations of growth. In accordance with the average headings of antibodies gotten in the study, the interaction was significant in the third sampling and showed a synergic effect of acid ones, being the combination of acids in question a powerful modulator of the humoral immunity. Meanwhile, the results gotten in the analyses of intestinal morfometric had not been conclusive. In general terms, one requires more organic acid studies that confirm the use of as the promotional ones of growth.

Glioneuronal tumor with neuropil-like islands (GTNI): a report of 8 cases with chromosome 1p/19q deletion analysis.

Glioneuronal tumor with neuropil-like islands (GTNI) is a rare neoplasm harboring circumscribed loci of neuronal differentiation and diffusely infiltrating astroglial and oligodendrocytelike components. We report 8 previously unpublished examples of GTNI, specifically studied for chromosome 1p and 19q allelic losses. All tumors showed characteristic histologic features and immunoprofile. One primary tumor displayed frankly malignant histology with frequent mitoses, microvascular proliferation, and necrosis. This tumor progressed within months of the initial resection. Three other tumors (2 low-grade and 1 showing only focal microvascular proliferation) recurred at 2 years, 3 years, and 1 year, respectively. All cases were evaluated for 1p/19q allelic losses by standard polymerase chain reaction-based loss of heterozygosity assays. No evidence of 1p/19q losses was found in 7 of 8 tumors. One tumor demonstrated small interstitial deletions at 1p36 (at D1S1612 and D1S513, but not at D1S548 or D1S1592) and a small interstitial deletion at 19q13 (at D19S219 and D19S412, but not at PLA2G4C). The lack of large, whole-arm 1p/19q losses (such as those found in oligodendroglial tumors), aberrant p53 expression, and the predominance of astroglial components may indicate a biologic relationship of the GTNI to diffuse astrocytoma. Although GTNI shares some morphologic features with recently reported cases of oligodendroglioma with neurocytic differentiation, the 2 tumors appear different at the molecular genetic level.

Allelic losses at 1p36 and 19q13 in gliomas: correlation with histologic classification, definition of a 150-kb minimal deleted region on 1p36, and evaluation of CAMTA1 as a candidate tumor suppressor gene.

PURPOSE: Allelic loss at 1p is seen in 70% to 85% of oligodendrogliomas (typically in association with 19q allelic loss) and 20-30% of astrocytomas. Because most 1p deletions in gliomas involve almost the entire chromosome arm, narrowing the region of the putative tumor suppressor gene has been difficult. To better define the histologic correlates of different patterns of 1p and 19q loss, we evaluated 1p/19q status in a large group of gliomas. This also allowed us to define a very small minimal deleted region (MDR) on 1p36. EXPERIMENTAL DESIGN: Among 205 consecutive cases of glioma studied for 1p loss of heterozygosity (LOH), 112 tumors were evaluated for both 1p and 19q LOH using at least three polymorphic markers on 1p and 19q each. The latter group included both low-grade tumors (oligodendroglioma, diffuse astrocytoma, and "oligoastrocytoma") and high-grade tumors (anaplastic oligodendrogliomas, anaplastic astrocytomas, anaplastic oligoastrocytomas). Tumors with small segmental 1p losses (defined as LOH at some loci with retention of heterozygosity at other loci) were studied using a more extensive panel of markers to define the 1p MDR. The candidate gene was screened for mutations and its expression was studied by qualitative and quantitative reverse transcriptase-PCR and Northern blotting. RESULTS: Allelic losses on 1p and 19q, either separately or combined, were more common in classic oligodendrogliomas than in either astrocytomas or oligoastrocytomas (P < 0.0001). Classic oligodendrogliomas showed 1p loss in 35 of 42 (83%) cases, 19q loss in 28 of 39 (72%), and these were combined in 27 of 39 (69%) cases. There was no significant difference in 1p/19q LOH status between low-grade and anaplastic oligodendrogliomas. In contrast, no astrocytomas and only 6 of 30 (20%) oligoastrocytic tumors had combined 1p/19q loss. Although rare, 1p deletions were more often segmental in astrocytomas (5 of 6, 83%) than in oligodendrogliomas (3 of 35, 9%; P = 0.006). Eleven tumors (6 oligodendrogliomas or having oligodendroglial components, 5 purely astrocytic) with small segmental 1p losses underwent further detailed LOH mapping. All informative tumors in the oligodendroglial group and 2 of 3 informative astrocytomas showed LOH at 1p36.23, with a 150-kb MDR located between D1S2694 and D1S2666, entirely within the CAMTA1 transcription factor gene. Mutation analysis of the exons encoding conserved regions of CAMTA1 showed no somatic mutations in 10 gliomas, including 6 cases with and 4 cases without 1p LOH. CAMTA1 is normally expressed predominantly in non-neoplastic adult brain tissue. Relative to the latter, the expression level of CAMTA1 was low in oligodendroglial tumors and was further halved in cases with 1p deletion compared with those without 1p deletion (Mann-Whitney, P = 0.03). CONCLUSIONS: Our data confirm the strong association of combined 1p/19q loss with classic oligodendroglioma histology and identify a very small segment of 1p36 located within CAMTA1 that was deleted in all oligodendroglial tumors with 1p LOH. This MDR also overlaps the neuroblastoma 1p36 MDR. CAMTA1 shows no evidence of inactivation by somatic mutations but its expression is reduced by half in cases with 1p LOH, suggesting that the functional effects of CAMTA1 haploinsufficiency warrant further investigation.

Molecular analysis of gastric washings in the diagnosis and monitoring of gastric lymphomas.

The monitoring of gastric lymphomas is often hampered by the inherently limited sampling provided by small endoscopic biopsy specimens. To investigate the feasibility of using gastric washing fluid for monitoring patients with known gastric lymphoma and for diagnosing gastric involvement in patients with extranodal nongastric lymphoma, we collected 49 gastric washings from 39 patients (29 patients with gastric lymphoma and 10 patients with nongastric extranodal lymphoma). Collection was done at the time of follow-up biopsy and when no endoscopic abnormalities were found. DNA was extracted from the washing fluid and analyzed for clonal IgH gene rearrangement by Southern blotting (J6 probe) and/or polymerase chain reaction (PCR) (using VH-FR3 and JH primers). Forty-one of 49 samples (84%) yielded sufficient DNA for molecular analysis. Sixteen of 41 analyzable gastric washing samples (39%) failed Southern blot analysis due to degraded or insufficient DNA. Concordance between the results of Southern blot analysis of the washing and histology of the simultaneous biopsy specimen was found in 20 (80%) of the remaining 25 samples. The IgH PCR result was concordant with biopsy histology in 33 out of 41 washing samples (80%). The overall concordance between molecular clonality studies of washings (Southern blotting and/or PCR) and biopsy histology was 83% (34 of 41). Of the 7 (18%) discrepant specimens, 2 were diagnosed histologically as lymphoma, but the simultaneous washings were negative by molecular studies. Five biopsy specimens were histologically benign, but the corresponding washings demonstrated clonal IgH gene rearrangement (3 cases by PCR and 2 cases by Southern blotting). This study demonstrates the diagnostic utility of molecular clonality analysis of gastric washings.

HER-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization: a single-institution experience of 2,279 cases and comparison of dual-color and single-color scoring.

We analyzed concordance between immunohistochemical analysis and fluorescence in situ hybridization (FISH) in HER-2 status and studied the effect of dual-color (D-FISH) vs single-color FISH (S-FISH) scoring on the assignment of tumors to amplified or nonamplified categories. The assays were performed on formalin-fixed, paraffin-embedded sections of 2,279 invasive breast carcinomas. Immunohistochemical results were interpreted as negative (0, 1+) or positive (2+, 3+). For FISH analyses, a ratio for HER-2/chromosome 17 of 2.0 or more (D-FISH) or an absolute HER-2 copy number per nucleus of more than 4.0 (S-FISH) were interpreted as positive gene amplification. We found 547 (24.0%) cases positive immunohistochemically, 326 (14.3%) by D-FISH, and 351 (15.4%) by S-FISH. Overall concordance in HER-2 status with immunohistochemical analysis was 87% for D-FISH and 86% for S-FISH. Excellent concordance was found among groups scored immunohistochemically as 0, 1+, and 3+ (with D-FISH, 97%; with S-FISH, 96%). The most discordant category was the group scored 2+ immunohistochemically, in which only a quarter of the 2+ tumors were FISH(+). D-FISH and S-FISH scoring results were discordant in 89 tumors (4%), of which 8 (9%) had 3+ immunohistochemical staining and none showed high-level HER-2 amplification. Among all FISH(+) tumors, 10% were negative by immunohistochemical analysis, and notably almost half (47%) showed borderline to low HER-2 amplification (D-FISH score, 2.0-3.9); the clinical significance of these findings warrants further investigation.

Impact of polysomy 17 on HER-2/neu immunohistochemistry in breast carcinomas without HER-2/neu gene amplification.

Her-2/neu, a proto-oncogene located on chromosome 17, is an important biomarker in breast carcinoma. Immunohistochemistry (IHC) is currently the most widely used method for assessing Her-2/neu status. Some IHC-positive cases do not show Her-2/neu gene amplification by fluorescence in situ hybridization (FISH). It has been suggested that some of these IHC "false positive" results may in part be due to increased copy number of chromosome 17 resulting in increased Her-2/neu protein expression. We analyzed IHC and FISH data from 561 cases of invasive breast carcinoma to test this hypothesis. IHC and FISH for Her-2/neu were performed on formalin-fixed, paraffin-embedded sections of 561 invasive breast carcinomas. The IHC results were interpreted as 0, 1+, 2+, or 3+ according to the manufacturer's recommended criteria. The FISH results were expressed as a ratio of Her-2/neu/chromosome 17 and were interpreted as positive (> = 2.0) or negative (<2.0) for gene amplification according to the manufacturer's recommended scoring system. We found that in IHC 3+/FISH-negative cases (n = 15) both the average chromosome 17 copy number and the average Her-2/neu copy number were significantly higher than that in IHC (0 to 2+)/FISH-negative cases (n = 411) (2.45 vs. 1.68; P < 0.0001, and 3.19 vs. 1.95; P < 0.0001, respectively). In contrast, the IHC 2+/FISH-negative cases did not exhibit a significantly increased number of chromosome 17 compared to IHC 0 to 1+ cases. In addition, the average copy number of chromosome 17 in FISH-positive cases (n = 135) was significantly higher than that in FISH-negative cases (n = 426) (2.27 vs. 1.70; P < 0.0001), indicating a general association of increased chromosome 17 copy number with Her-2/neu gene amplification. Thus, our data suggest that IHC 3+ immunostaining without scorable gene amplification may indeed be, at least in some cases, the result of increased Her-2/neu protein expression secondary to an increased copy number of chromosome 17, associated with an increased total number of Her-2/neu gene copies per tumor cell.