Impact of Using Median vs. Mean in Calculating ERBB2 FISH Results in Breast Cancer.

INTRODUCTION: Erb-b2 receptor tyrosine kinase 2 (ERBB2) testing is used to measure the status of ERBB2 gene expression and DNA amplification. Test results have been reported with a discrepancy as high as 20%. The purpose of this study was to improve ERBB2 fluorescence in situ hybridization (FISH) sensitivity by evaluating results generated by median as well as mean calculations. METHODS: We retrospectively identified breast cancer cases at our institution in which ERBB2 FISH testing was performed in-house from June 2018 to May 2020. FISH results were classified using the 2018 American Society of Clinical Oncology/College of American Pathologists guidelines: groups 1 and 5 are FISH positive and negative, respectively, and groups 2-4 are equivocal requiring additional work-up. FISH counting sheets were collected and regrouped by median ERBB2 copy number counts and median ERBB2/CEP17 ratio and compared with the mean ERBB2 and mean ERBB2/CEP17 ratio. Intra-tumor genetic heterogeneity and CEP17 copy number gain (CEP17 ≥3) were assessed to see if they affect the discrepancy between median and mean groups. RESULTS: Seventy-two breast cancer cases were collected and evaluated. Eleven cases (11 of 72 [15%]) had discrepant grouping by mean and median calculations. A significant number of discrepancies were found for CEP17 copy number gain (p = 0.027) but not for ERBB2 (p = 0.411), the ERBB2/CEP17 ratio (p = 0.445), FISH results (p = 0.194), or genetic heterogeneity (p = 0.465). Among the four cases regrouped to median group 1, 2 were from mean group 3 and underwent anti-ERBB2 targeted therapy and 2 were from mean groups 4 and 5 may have benefitted from targeted therapy with more than 30% amplified cells. The median may be better to reflect the monosomy subclone within tumor tissues for the case 387 moved from mean group 5 to median group 2. The 6 cases moved from mean group 5 to median group 4 with CEP17 copy number gain may have had a poor prognosis based on other study result. CONCLUSION: Including the median calculation may increase ERBB2 sensitivity and identification of CEP17 copy number gain. Further clinical studies are necessary to examine the outcome of including median in calculating ERBB2/CEP17 values.

Phenotypes Associated with 16p11.2 Copy Number Gains and Losses at a Single Institution.

Chromosome 16p11.2 is one of the susceptible sites for recurrent copy number variations (CNVs) due to flanking near-identical segmental duplications. Five segmental duplications, named breakpoints 1 to 5 (BP1-BP5), have been defined as recombination hotspots within 16p11.2. Common CNVs on 16p11.2 include a proximal ~593 kb between BP4 and BP5, and a distal ~220 kb between BP2 and BP3. We performed a search for patients carrying 16p11.2 CNVs, as detected using chromosome microarray (CMA), in the Molecular Diagnostic Laboratory at the University of Texas Medical Branch (UTMB), in Galveston. From March 2013 through April 2018, a total of 1200 CMA results were generated for germline testing, and 14 patients tested positive for 16p11.2 CNVs, of whom 7 had proximal deletion, 2 had distal deletion, 4 had proximal duplication, and 1 had distal duplication. Herein, we provide detailed phenotype data for these patients. Our study results show that developmental delay, abnormal body weight, behavioral problems, and hypotonia are common phenotypes associated with 16p11.2 CNVs.

Development and Application of Duplex Sequencing Strategy for Cell-Free DNA-Based Longitudinal Monitoring of Stage IV Colorectal Cancer.

Potential applications of cell-free DNA (cfDNA)-based molecular profiling have used in patients with diverse malignant tumors. However, capturing all cfDNA that originates from tumor cells and identifying true variants present in this minute fraction remain challenges to the widespread application of cfDNA-based liquid biopsies in the clinical setting. In this study, we evaluate a systematic approach and identify key components of wet bench and bioinformatics strategies to address these challenges. We found that concentration of enrichment oligonucleotides, elements of the library preparation, and the structure of adaptors are critical for achieving high enrichment of target regions, retaining variant allele frequencies accurately throughout all involved steps of library preparation, and obtaining high variant coverage. We developed a dual molecular barcode-integrated error elimination strategy to remove sequencing artifacts and a background error correction strategy to distinguish true variants from abundant false-positive variants. We further describe a clinical application of this cfDNA-based duplex sequencing approach that can be used to monitor disease progression in patients with stage IV colorectal cancer. The findings also suggest that cfDNA-based molecular testing observations are highly concordant with observations obtained by traditional imaging methods. Overall, the findings presented in this study have potential implications for early detection of cancer, identification of minimal residual disease, and evaluation of therapeutic responses in patients with cancer.

Personalized medicine in non-small cell lung cancer: a review from a pharmacogenomics perspective.

Non-small cell lung cancer is a prevalent and rapidly-expanding challenge to modern medicine. While generalized medicine with traditional chemotherapy yielded comparatively poor response rates and treatment results, the cornerstone of personalized medicine using genetic profiling to direct treatment has exalted the successes seen in the field and raised the standard for patient treatment in lung and other cancers. Here, we discuss the current state and advances in the field of personalized medicine for lung cancer, reviewing several of the mutation-targeting strategies that are approved for clinical use and how they are guided by patient genetic information. These classes include inhibitors of tyrosine kinase (TKI), anaplastic lymphoma kinase (ALK), and monoclonal antibodies. Selecting from these treatment plans and determining the optimal dosage requires in-depth genetic guidance with consideration towards not only the underlying target genes but also other factors such as individual metabolic capability and presence of resistance-conferring mutations both directly on the target gene and along its cascade(s). Finally, we provide our viewpoints on the future of personalized medicine in lung cancer, including target-based drug combination, mutation-guided drug design and the necessity for data of population genetics, to provide rough guidance on treating patients who are unable to get genetic testing.

Draft Genome Sequences of Four Genetically Distinct Human Isolates of Streptococcus dysgalactiae subsp. equisimilis.

ß-Hemolytic group C and group G streptococci (GCS-GGS; Streptococcus dysgalactiae subsp. equisimilis) emerged as human pathogens in the late 1970s. We report here the draft genome sequences of four genetically distinct human strains of GCS-GGS isolated between the 1960s and 1980s. Comparative analysis of these genomes may provide a deeper understanding of GCS-GGS genome and virulence evolution.

Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma.

BACKGROUND: Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%-80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy. METHODS: We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. RESULTS: Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. CONCLUSIONS: Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.

Lack of correlation between HERV-K expression and HIV-1 viral load in plasma specimens.

HERV-K viral RNA has been reported in plasma specimens of HIV-1 infected individuals. Emerging data support the regulation and functional interaction between HERV-K and HIV-1, which warrant development of accurate HERV-K assays to evaluate HERV-K activation. In this study, we examined HERV-K RNA expression after careful removal of "contaminating" cellular DNA using DNase I. We found that DNase I digestion effectively reduced HERV-K RT-PCR positive signal. We also found that levels of HERV-K expression did not correlate with HIV-1 viral load. Our study is in agreement with the published studies on HERV-K activation in HIV-1 positive plasma specimens, and in addition, calls for careful removal of cellular DNA to accurately evaluate HERV-K RNA expression.

Defining causative factors contributing in the activation of hedgehog signaling in diffuse large B-cell lymphoma.

Hedgehog (Hh) signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL). Genetic abnormalities that explain activation of Hh signaling in DLBCL are unknown. We investigate the presence of amplifications of Hh genes that might result in activation of this pathway in DLBCL. Our data showed few extra copies of GLI1 and SMO due to chromosomal aneuploidies in a subset of DLBCL cell lines. We also showed that pharmacologic inhibition of PI3K/AKT and NF-κB pathways resulted in decreased expression of GLI1 and Hh ligands. In conclusion, our data support the hypothesis that aberrant activation of Hh signaling in DLBCL mainly results from integration of deregulated oncogenic signaling inputs converging into Hh signaling.

A professional development model for medical laboratory scientists working in the Core Laboratory.

The Division of Pathology and Laboratory Medicine at The University of Texas MD Anderson Cancer Center has implemented a professional development model designed to further the education, expertise, and experiences of medical laboratory scientists in the core laboratory. The professional development model (PDM) has four competency levels: Discovery, Application, Maturation and Expert. All levels require the medical laboratory scientist to learn new skill sets, complete task and projects, and meet continuing education and certification requirements. Each level encourages personal development, recognizes increased competencies, and sets high standards for all services provided. Upon completion of a level within a given timeframe, the medical laboratory scientist receives a salary adjustment based on the competency level completed.

A professional development model for medical laboratory scientists working in the microbiology laboratory.

The University of Texas M.D. Anderson Cancer Center, Division of Pathology and Laboratory Medicine is committed to providing the best pathology and medicine through: state-of-the art techniques, progressive ground-breaking research, education and training for the clinical diagnosis and research of cancer and related diseases. After surveying the laboratory staff and other hospital professionals, the Department administrators and Human Resource generalists developed a professional development model for Microbiology to support laboratory skills, behavior, certification, and continual education within its staff. This model sets high standards for the laboratory professionals to allow the labs to work at their fullest potential; it provides organization to training technologists based on complete laboratory needs instead of training technologists in individual areas in which more training is required if the laboratory needs them to work in other areas. This model is a working example for all microbiology based laboratories who want to set high standards and want their staff to be acknowledged for demonstrated excellence and professional development in the laboratory. The PDM model is designed to focus on the needs of the laboratory as well as the laboratory professionals.

BK virus as a potential co-factor for HPV in the development of cervical neoplasia.

Cervical cancer is the third most common type of cancer in women worldwide. A persistent infection with high risk (HR) human papillomavirus (HPV) is necessary for cervical cancer to occur. However, the great majority of women that are infected with HR-HPV will not develop cervical cancer, indicating that HR-HPV alone is not adequate to drive the development of cervical cancer, suggesting the involvement of cofactors. The BK polyomavirus (BKV) establishes latency near cervical tissue in the urogenital tract and is frequently detected in the urine, especially in immunosuppressed patients, and hence may coexist with HR-HPV. Current experimental evidence indicates that both HR-HPV and BKV are capable of altering cell-cycle control and inhibit apoptosis. Therefore, they may act additively or synergistically to promote malignant transformation. We hypothesize that BKV is a co-factor for HR-HPV in cervical cancer. In this study, we examined 249 cervical swabs that were submitted for routine HR-HPV screening test in the Molecular Diagnostics Laboratory at the University of Texas Medical Branch (UTMB). Our results showed that 107 samples contained HR-HPV at an overall rate of 43% (107/249); BKV was present in 4 (3.7%) of the 107 HR-HPV positive specimens and in 12 (8.5%) of the 142 HR-HPV negative samples with an overall positive rate of 6.4% (16/249). Although there was no statistical significance between HR-HPV and BKV co-infection (P=0.19, Fisher's exact test), our results support the hypothesis that BKV can co-exist with HR-HPV in cervical specimens.

A professional development model for medical laboratory scientists working in the immunohematology laboratory.

Transfusion medicine, a section of the Department of Laboratory Medicine at The University of Texas MD Anderson Cancer Center is committed to the education and advancement of its health care professionals. It is our belief that giving medical laboratory professionals a path for advancement leads to excellence and increases overall professionalism in the Immunohematology Laboratory. As a result of this strong commitment to excellence and professionalism, the Immunohematology laboratory has instituted a Professional Development Model (PDM) that aims to create Medical Laboratory Scientists (MLS) that are not only more knowledgeable, but are continually striving for excellence. In addition, these MLS are poised for advancement in their careers. The professional development model consists of four levels: Discovery, Application, Maturation, and Expert. The model was formulated to serve as a detailed path to the mastery of all process and methods in the Immunohematology Laboratory. Each level in the professional development model consists of tasks that optimize the laboratory workflow and allow for concurrent training. Completion of a level in the PDM is rewarded with financial incentive and further advancement in the field. The PDM for Medical Laboratory Scientists in the Immunohematology Laboratory fosters personal development, rewards growth and competency, and sets high standards for all services and skills provided. This model is a vital component of the Immunohematology Laboratory and aims to ensure the highest quality of care and standards in their testing. It is because of the success of this model and the robustness of its content that we hope other medical laboratories aim to reach the same level of excellence and professionalism, and adapt this model into their own environment.

Circulating endothelial cells and endothelial progenitors as surrogate biomarkers in vascular dysfunction.

An increase in the number of circulating endothelial cells (CECs) and of bone marrow derived endothelial progenitors (CEPs) in the peripheral blood (PB) is normally associated with vascular injury, repair, and neovascularization. These cells rarely exist in the PB of healthy individuals. Therefore, when they are present in the PB of individuals, their phenotypes and quantity in the PB may serve as surrogate diagnostic or prognostic parameters of vascular injury and/or as an indication of tumor growth. An elevated level of CEPs may suggest an ongoing repair of ischemic vascular injuries and/or angiogenesis. Recently, more advanced techniques for CEC isolation and CEP enumeration have become available. In particular, immunobeads isolation and fluorescence-activated cell sorting (FACS) techniques have been employed with success in evaluation of vascular dysfunctions. Therefore, CECs and CEPs may serve as potential surrogate markers for monitoring various vascular diseases, which could help to determine pathological process and clinical treatment. In this article, we will present an overview of CECs and CEPs by discussing their origins, reviewing methodologies adapted to the measurement of rare events, describing pathological situations associated with CECs/CEPs, and correlating them with a broad spectrum of disease processes.