Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the ... more Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity, and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments, and proficiency testing on standardized cell line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas below this limit detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false-negatives) were more common than erroneous candidates (false-positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best-practice guidelines and provides a resource for precision oncology. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
1773 Risk stratification in chronic lymphocytic leukemia (CLL) is highly desirable and should com... more 1773 Risk stratification in chronic lymphocytic leukemia (CLL) is highly desirable and should comprise not only evaluation of clinical features but also molecular prognostic markers. Currently such molecular markers include loss of 17p13, 11q22, 13q14, 6q22, and gain of chromosome 12 as assessed by fluorescence in situ hybridization (FISH) and mutation status of the variable region of the IGH gene (IGHV) by sequencing. In recent years, genome-wide scanning technologies such as array-comparative genomic hybridization (array-CGH) have revealed novel and refined known copy number alterations (CNAs) in the CLL genome. In order to evaluate the potential of array-CGH in prognostication in mature B-cell neoplasms, including CLL, and implement array-CGH in a clinical diagnostic laboratory, a targeted oligonucleotide-based microarray was custom designed to represent genomic regions exhibiting gain/loss in these lymphoid neoplasms. The 4 × 44K formatted array included 2 × 17,348 probes for th...
Background: Recent advancements in comprehensive sequencing technologies has enabled researchers ... more Background: Recent advancements in comprehensive sequencing technologies has enabled researchers to uncover numerous somatic variants within many disease types, but their respective contributions to disease pathogenesis and potential roles as outcome biomarkers are less well-defined. Even fewer studies have integrated somatic mutation events with genomic gain and loss events of respective genes, mostly due to the need to utilize a second platform to robustly assess genomic copy number. To further understand the roles of these genomic aberrations in the disease biology of mature B-cell neoplasms, we developed a next-generation sequencing (NGS) panel of 220 genes for profiling of Diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), and secondarily for Chronic Lymphocytic Leukemia (CLL). Methods: The 220 genes represented in the panel included genes based on reported frequency of mutations, targets of therapeutic agents, involvement in disease-en...
175 Background: Post mastectomy radiation (PMR) is usually recommended for T3 or N2 breast cancer... more 175 Background: Post mastectomy radiation (PMR) is usually recommended for T3 or N2 breast cancer (BC). The role of PMR for stage ll BC with T1/T2 lesions remains controversial. The aim of this study was to assess the role of PMR in this subgroup of patients. Methods: A retrospective analysis of a prospectively collected database of all stage ll BC patients treated with mastectomy at our institution between 2005-2008 was performed. Demographics, tumor stage, histology, receptor status, adjuvant therapy, nodal status, failure patterns and disease free survival rates (DFS) were compared between the patients who received PMR vs.those who were not radiated. Results: Eighty-two patients underwent mastectomies for stage II disease with a T1/T2 lesion. Twenty two of those (27%) received PMR. The average follow up time was 47 months. The patients in the PMR group had significantly larger tumors (90% vs. 64%), more advanced stages (55% vs. 17%) and higher grade histology (59% vs. 32%). Menop...
471 Background: Image-guided percutaneous needle biopsies are increasingly utilized for the diagn... more 471 Background: Image-guided percutaneous needle biopsies are increasingly utilized for the diagnosis of renal tumors. Histologic diagnosis of renal mass subtypes, including malignant clear cell (ccRCC), papillary (pRCC), chromophobe (chrRCC) renal cell carcinoma, and benign oncocytomas (OC) can be challenging due to the low cellularity and damaged architecture of needle biopsy specimens. However, each subtype exhibits unique genetic aberrations that can assist in histologic classification and potentially assist in guiding management decisions. We report our initial experience correlating renal mass histology to subtype-associated genomic alterations detected by FISH and a-CGH of percutaneous needle biopsy specimens. Methods: In an ongoing IRB-approved blinded study, 17 samples obtained from 15 patients with known renal masses were submitted to FISH (FReCaD) and targeted a-CGH (UroGenRA-Kidney). Specimens were classified using a subtype classification decision tree algorithm based o...
428 Background: About one-third of patients with clear cell renal cell carcinoma (ccRCC) exhibit ... more 428 Background: About one-third of patients with clear cell renal cell carcinoma (ccRCC) exhibit metastasis at the time of diagnosis and show poor prognosis. The genetic and epigenetic alterations associated with metastasis in ccRCC have variably been studied, and their role in the metastatic process is unclear. The goals of the current study were to identify genomic copy number alterations (CNAs) associated with ccRCC metastasis and examine their clinical utility. Methods: In this IRB-approved study, genome-wide copy number profiling was performed on DNA from 144 ccRCC (81 primary and 63 metastatic lesions). Differential CNAs between primary and metastatic lesions and between different metastatic sites were identified using Fisher’s exact test. Associations between CNAs and overall survival (OS) were tested using the log rank statistic and Kaplan-Meier method. Genomic profiling data of 437 and 240 primary ccRCC (TCGA and PMID: 23797736, respecitively) were used for verification. Re...
Groin pain can be disabling for patients, and complex to diagnose and treat for physicians. Vario... more Groin pain can be disabling for patients, and complex to diagnose and treat for physicians. Various processes can lead to local inflammation, nerve irritation or injury, or referred pain, and present as groin pain. This includes primary processes such as inguinal and femoral hernias, urologic and gynecologic pathology, and hip and lumbar pathology. Secondary groin pain can occur following inguinal hernia repair and lumbar spine surgery.
Cancer Cytogenomics Microarray Consortium Third Annual Meeting, August 6e7, 2012 SESSION1:HEMATOL... more Cancer Cytogenomics Microarray Consortium Third Annual Meeting, August 6e7, 2012 SESSION1:HEMATOLOGICMALIGNANCIES Clinical Laboratory Implementation of the Detection of Genomic Aberrations in Formalin-Fixed ParaffinEmbedded Small Lymphocytic Lymphoma Specimens by Array-CGH Charles Ma, Asha N. Guttapalli, Lizalynn M. Dias, Lynnette M. Cahill, Lan Wang, Jane Houldsworth Cancer Genetics Inc., Rutherford, NJ Due to the highly variable course of the disease, risk stratification is essential for the management of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) patients. For CLL, assessment of prognostic molecular markers including IGHV mutation status and specific genomic aberrations by either FISH and/or, more recently, array-CGH is routinely performed. The same molecular markers have been shown to offer similar prognostic value in SLL. However, the clinical diagnostic evaluation of these markers in SLL is hampered by the solid tissue type. Indeed, formalin-fixed, paraffin-embedded (FFPE) material is often the only available specimen for analysis. Using over 300 FFPE samples, we have established appropriate laboratory procedures and QC matrices for the clinical implementation of array-CGH for FFPE samples. In brief, DNA is extracted from 5 ten micron FFPE sections, and then a minimum of 1ug of DNA is heat fragmented, labeled enzymatically, and hybridized with a similarly fragmented commercial reference DNA to a custom-designed DNA oligonucleotide microarray representing genomic regions that are commonly altered in mature B-cell neoplasms. For ten SLL FFPE samples, adequate DNA was isolated and found to be of varied quality. Using ADM-2, the same genomic aberrations as commonly found in CLL (13q14 loss (MIR15A/16-1), 17p13 loss (TP53), and 11q22 loss (ATM)) were detected in the SLL specimens with high reproducibility and accuracy. DNA dilution studies indicated an analytical sensitivity of 60%70% while FISH in the specimens for detected aberrations revealed sensitivity closer to 30%-40%. All aberrations detected by array-CGH were independently validated by quantitative PCR for genes mapped within the respective regions. Thus, our data indicated that array-CGH is suitable for the detection 2210-7762/$ see front matter a 2012 Elsevier Inc. Elsevier Inc. http://dx.doi.org/10.1016/j.cancergen.2012.07.004 of prognostic genomic aberrations in SLL in a clinical diagnostic setting that could be implemented in patient risk stratification. Conflict of Interest: These authors are employees of Cancer Genetics, Inc., Rutherford, NJ. Array-Based Karyotyping Post Plasma Cell Enrichment for the Detection of Genomic Abnormalities in Multiple Myeloma Barbara K. Zehentner , Luise Hartmann , Krystal Johnson , Christine Stephenson , Douglas Chapman , Monica de Baca , Denise A. Wells , Michael R. Loken , Budi Tirtorahardjo , Shelly R. Gunn , Lony Lim b HematoLogics Inc., Seattle WA; Combimatrix, Irvine, CA Multiple myeloma (MM) is a hematopoietic neoplasm characterized by malignant plasma cells. The discovery of genomic abnormalities present in the monoclonal plasma cells has diagnostic, prognostic, as well as disease monitoring implications. However, technical and disease-related limitations hamper the detection of these abnormalities by metaphase cytogenetic analysis or FISH. In this study, we tested 28 bone marrow specimens with known plasma cell neoplasm for the presence of genomic abnormalities using microarray analysis post plasma cell enrichment and compared the results with other laboratory findings. Metaphase cytogenetic studies were performed on 15 of the 28 samples and identified disease-related genomic aberrations in only 3 of the 15 cases (20%) due to the low proliferative rate of plasma cells; 84.6% of specimens tested positive by MACS-FISH and 89.3% by array comparative genomic hybridization (aCGH). Furthermore, aCGH detected additional abnormalities in 24 cases as compared with FISH alone. We conclude that the combination of plasma cell enrichment and genomic copy number assessment using CGH microarray is a valuable approach for routine clinical use to achieve a more complete genetic characterization of multiple myeloma patients. Conflict of Interest: Employment, stock ownership.
T he time has come for lab staff to become more proactive in the management of diabetes by helpin... more T he time has come for lab staff to become more proactive in the management of diabetes by helping generalists diagnose several important related risk factors. Doing so will not only dramatically improve overall care of patients and their quality of life but also reinforce the lab’s reputation as an integral member of the healthcare team. Diabetes is a significant medical problem in this country. Medical costs associated with managing this disease are estimated at $100 billion annually. Moreover, about 30% more US patients die from diabetes than their European counterparts. American diabetic patients also have four times more serious kidney disease than diabetics in the European Union (EU).1,2 In adult patients, diabetes is the leading cause of blindness, amputation, and end-stage renal disease requiring dialysis and renal transplantation.3 About 60% of diabetic patients die of coronary disease, 10% die of stroke, and another 10% suffer from fatal complications related to peripheral vascular disease.4 Diabetic patients who experience a first myocardial infarction have a 25% chance of death within 1 month and a 50% chance within 1 year.5,6
Purpose: Accurate discrimination of benign oncocytoma and malignant renal cell carcinoma is usefu... more Purpose: Accurate discrimination of benign oncocytoma and malignant renal cell carcinoma is useful for planning appropriate treatment strategies for patients with renal masses. Classification of renal neoplasms solely based on histopathology can be challenging, especially the distinction between chromophobe renal cell carcinoma and oncocytoma. In this study we develop and validate an algorithm based on genomic alterations for the classification of common renal neoplasms. Materials and Methods: Using TCGA renal cell carcinoma copy number profiles and the published literature, a classification algorithm was developed and scoring criteria were established for the presence of each genomic marker. As validation, 191 surgically resected formalin fixed paraffin embedded renal neoplasms were blindly submitted to targeted array comparative genomic hybridization and classified according to the algorithm. CCND1 rearrangement was assessed by fluorescence in situ hybridization. Results: The optimal classification algorithm comprised 15 genomic markers, and involved loss of VHL, 3p21 and 8p, and chromosomes 1, 2, 6, 10 and 17, and gain of 5qter, 16p, 17q and 20q, and chromosomes 3, 7 and 12. On histological rereview (leading to the exclusion of 3 specimens) and using histology as the gold standard, 58 of 62 (93%) clear cell, 51 of 56 (91%) papillary and 33 of 34 (97%) chromophobe renal cell carcinomas were classified correctly. Of the 36 oncocytoma specimens 33 were classified as oncocytoma (17 by array comparative genomic hybridization and 10 by array comparative genomic hybridization plus fluorescence in situ hybridization) or benign (6). Overall 93% diagnostic sensitivity and 97% specificity were achieved.
Parasitaemia, the percentage of infected erythrocytes, is used to measure progress of experimenta... more Parasitaemia, the percentage of infected erythrocytes, is used to measure progress of experimental Plasmodium infection in infected hosts. The most widely used technique for parasitaemia determination is manual microscopic enumeration of Giemsa-stained blood films. This process is onerous, time consuming and relies on the expertise of the experimenter giving rise to person-to-person variability. Here the development of image-analysis software, named Plasmodium AutoCount, which can automatically generate parasitaemia values from Plasmodium-infected blood smears, is reported. Giemsa-stained blood smear images were captured with a camera attached to a microscope and analysed using a programme written in the Python programming language. The programme design involved foreground detection, cell and infection detection, and spurious hit filtering. A number of parameters were adjusted by a calibration process using a set of representative images. Another programme, Counting Aid, written in ...
Proceedings of the National Academy of Sciences, 2011
Microtubules are integral to neuronal development and function. They endow cells with polarity, s... more Microtubules are integral to neuronal development and function. They endow cells with polarity, shape, and structure, and their extensive surface area provides substrates for intracellular trafficking and scaffolds for signaling molecules. Consequently, microtubule polymerization dynamics affect not only structural features of the cell but also the subcellular localization of proteins that can trigger intracellular signaling events. In the nematode Caenorhabditis elegans , the processes of touch receptor neurons are filled with a bundle of specialized large-diameter microtubules. We find that conditions that disrupt these microtubules (loss of either the MEC-7 β-tubulin or MEC-12 α-tubulin or growth in 1 mM colchicine) cause a general reduction in touch receptor neuron (TRN) protein levels. This reduction requires a p38 MAPK pathway (DLK-1, MKK-4, and PMK-3) and the transcription factor CEBP-1. Cells may use this feedback pathway that couples microtubule state and MAPK activation to...
Increasing numbers of plant-made vaccines and pharmaceuticals are entering the late stage of prod... more Increasing numbers of plant-made vaccines and pharmaceuticals are entering the late stage of product development and commercialization. Despite the theoretical benefits of such production, expression of parasite antigens in plants, particularly those from Plasmodium, the causative parasites for malaria, have achieved only limited success. We have previously shown that stable transformation of tobacco plants with a plant-codon optimized form of the Plasmodium yoelii merozoite surface protein 4 ⁄ 5 (PyMSP4 ⁄ 5) gene resulted in PyMSP4 ⁄ 5 expression of up to 0.25% of total soluble protein. In this report, we describe the rapid expression of PyMSP4 ⁄ 5 in Nicotiana benthamiana leaves using the deconstructed tobacco mosaic virus-based magnICON Ò expression system. PyMSP4 ⁄ 5 yields of up to 10% TSP or 1-2 mg ⁄ g of fresh weight were consistently achieved. Characterization of the recombinant plantmade PyMSP4 ⁄ 5 indicates that it is structurally similar to PyMSP4 ⁄ 5 expressed by Escherichia coli. It is notable that the plant-made PyMSP4 ⁄ 5 protein retained its immunogenicity following long-term storage at ambient temperature within freezedried leaves. With assistance from a mucosal adjuvant the PyMSP4 ⁄ 5-containing leaves induced PyMSP4 ⁄ 5-specific antibodies when delivered orally to naïve mice or mice primed by a DNA vaccine. This study provides evidence that immunogenic Plasmodium antigens can be produced in large quantities in plants using the magnICON Ò viral vector system. Plants have been investigated as an economical expression and delivery system for vaccines targeting a number of infectious diseases (Ma et al., 2005; Spok et al., 2008). However, limited success has been achieved for the expression of parasite antigens in plants, particularly those
The bioinformatics software, Geneious, provides a useful platform for researchers to retrieve and... more The bioinformatics software, Geneious, provides a useful platform for researchers to retrieve and analyse genomic and functional genomics information. However, the main databases that the software is able to access are hosted by NCBI (National Center for Biotechnology Information). The databases of EuPathDB (Eukaryotic Pathogen Database Resources), such as PlasmoDB and PiroplasmaDB, collect more specific and detailed information about eukaryotic pathogens than those kept in NCBI databases. Two plugins for Geneious, one for PlasmaDB and one for PiroplasmaDB were developed. When installed, users can use search facilities to find and import gene and protein sequences from the EuPathDB databases. Users can then use the functions of Geneious to process the sequence information. When information unique to PlasmoDB and PiroplasmaDB is required, the user can access results linked with the gene/protein sequence via the default web browser. The plugins are freely available from the Victorian Bioinformatics Consortium website. The plugins can be modified to access any of the databases of EuPathDB.
Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the ... more Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity, and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments, and proficiency testing on standardized cell line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas below this limit detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false-negatives) were more common than erroneous candidates (false-positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best-practice guidelines and provides a resource for precision oncology. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
1773 Risk stratification in chronic lymphocytic leukemia (CLL) is highly desirable and should com... more 1773 Risk stratification in chronic lymphocytic leukemia (CLL) is highly desirable and should comprise not only evaluation of clinical features but also molecular prognostic markers. Currently such molecular markers include loss of 17p13, 11q22, 13q14, 6q22, and gain of chromosome 12 as assessed by fluorescence in situ hybridization (FISH) and mutation status of the variable region of the IGH gene (IGHV) by sequencing. In recent years, genome-wide scanning technologies such as array-comparative genomic hybridization (array-CGH) have revealed novel and refined known copy number alterations (CNAs) in the CLL genome. In order to evaluate the potential of array-CGH in prognostication in mature B-cell neoplasms, including CLL, and implement array-CGH in a clinical diagnostic laboratory, a targeted oligonucleotide-based microarray was custom designed to represent genomic regions exhibiting gain/loss in these lymphoid neoplasms. The 4 × 44K formatted array included 2 × 17,348 probes for th...
Background: Recent advancements in comprehensive sequencing technologies has enabled researchers ... more Background: Recent advancements in comprehensive sequencing technologies has enabled researchers to uncover numerous somatic variants within many disease types, but their respective contributions to disease pathogenesis and potential roles as outcome biomarkers are less well-defined. Even fewer studies have integrated somatic mutation events with genomic gain and loss events of respective genes, mostly due to the need to utilize a second platform to robustly assess genomic copy number. To further understand the roles of these genomic aberrations in the disease biology of mature B-cell neoplasms, we developed a next-generation sequencing (NGS) panel of 220 genes for profiling of Diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), and secondarily for Chronic Lymphocytic Leukemia (CLL). Methods: The 220 genes represented in the panel included genes based on reported frequency of mutations, targets of therapeutic agents, involvement in disease-en...
175 Background: Post mastectomy radiation (PMR) is usually recommended for T3 or N2 breast cancer... more 175 Background: Post mastectomy radiation (PMR) is usually recommended for T3 or N2 breast cancer (BC). The role of PMR for stage ll BC with T1/T2 lesions remains controversial. The aim of this study was to assess the role of PMR in this subgroup of patients. Methods: A retrospective analysis of a prospectively collected database of all stage ll BC patients treated with mastectomy at our institution between 2005-2008 was performed. Demographics, tumor stage, histology, receptor status, adjuvant therapy, nodal status, failure patterns and disease free survival rates (DFS) were compared between the patients who received PMR vs.those who were not radiated. Results: Eighty-two patients underwent mastectomies for stage II disease with a T1/T2 lesion. Twenty two of those (27%) received PMR. The average follow up time was 47 months. The patients in the PMR group had significantly larger tumors (90% vs. 64%), more advanced stages (55% vs. 17%) and higher grade histology (59% vs. 32%). Menop...
471 Background: Image-guided percutaneous needle biopsies are increasingly utilized for the diagn... more 471 Background: Image-guided percutaneous needle biopsies are increasingly utilized for the diagnosis of renal tumors. Histologic diagnosis of renal mass subtypes, including malignant clear cell (ccRCC), papillary (pRCC), chromophobe (chrRCC) renal cell carcinoma, and benign oncocytomas (OC) can be challenging due to the low cellularity and damaged architecture of needle biopsy specimens. However, each subtype exhibits unique genetic aberrations that can assist in histologic classification and potentially assist in guiding management decisions. We report our initial experience correlating renal mass histology to subtype-associated genomic alterations detected by FISH and a-CGH of percutaneous needle biopsy specimens. Methods: In an ongoing IRB-approved blinded study, 17 samples obtained from 15 patients with known renal masses were submitted to FISH (FReCaD) and targeted a-CGH (UroGenRA-Kidney). Specimens were classified using a subtype classification decision tree algorithm based o...
428 Background: About one-third of patients with clear cell renal cell carcinoma (ccRCC) exhibit ... more 428 Background: About one-third of patients with clear cell renal cell carcinoma (ccRCC) exhibit metastasis at the time of diagnosis and show poor prognosis. The genetic and epigenetic alterations associated with metastasis in ccRCC have variably been studied, and their role in the metastatic process is unclear. The goals of the current study were to identify genomic copy number alterations (CNAs) associated with ccRCC metastasis and examine their clinical utility. Methods: In this IRB-approved study, genome-wide copy number profiling was performed on DNA from 144 ccRCC (81 primary and 63 metastatic lesions). Differential CNAs between primary and metastatic lesions and between different metastatic sites were identified using Fisher’s exact test. Associations between CNAs and overall survival (OS) were tested using the log rank statistic and Kaplan-Meier method. Genomic profiling data of 437 and 240 primary ccRCC (TCGA and PMID: 23797736, respecitively) were used for verification. Re...
Groin pain can be disabling for patients, and complex to diagnose and treat for physicians. Vario... more Groin pain can be disabling for patients, and complex to diagnose and treat for physicians. Various processes can lead to local inflammation, nerve irritation or injury, or referred pain, and present as groin pain. This includes primary processes such as inguinal and femoral hernias, urologic and gynecologic pathology, and hip and lumbar pathology. Secondary groin pain can occur following inguinal hernia repair and lumbar spine surgery.
Cancer Cytogenomics Microarray Consortium Third Annual Meeting, August 6e7, 2012 SESSION1:HEMATOL... more Cancer Cytogenomics Microarray Consortium Third Annual Meeting, August 6e7, 2012 SESSION1:HEMATOLOGICMALIGNANCIES Clinical Laboratory Implementation of the Detection of Genomic Aberrations in Formalin-Fixed ParaffinEmbedded Small Lymphocytic Lymphoma Specimens by Array-CGH Charles Ma, Asha N. Guttapalli, Lizalynn M. Dias, Lynnette M. Cahill, Lan Wang, Jane Houldsworth Cancer Genetics Inc., Rutherford, NJ Due to the highly variable course of the disease, risk stratification is essential for the management of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) patients. For CLL, assessment of prognostic molecular markers including IGHV mutation status and specific genomic aberrations by either FISH and/or, more recently, array-CGH is routinely performed. The same molecular markers have been shown to offer similar prognostic value in SLL. However, the clinical diagnostic evaluation of these markers in SLL is hampered by the solid tissue type. Indeed, formalin-fixed, paraffin-embedded (FFPE) material is often the only available specimen for analysis. Using over 300 FFPE samples, we have established appropriate laboratory procedures and QC matrices for the clinical implementation of array-CGH for FFPE samples. In brief, DNA is extracted from 5 ten micron FFPE sections, and then a minimum of 1ug of DNA is heat fragmented, labeled enzymatically, and hybridized with a similarly fragmented commercial reference DNA to a custom-designed DNA oligonucleotide microarray representing genomic regions that are commonly altered in mature B-cell neoplasms. For ten SLL FFPE samples, adequate DNA was isolated and found to be of varied quality. Using ADM-2, the same genomic aberrations as commonly found in CLL (13q14 loss (MIR15A/16-1), 17p13 loss (TP53), and 11q22 loss (ATM)) were detected in the SLL specimens with high reproducibility and accuracy. DNA dilution studies indicated an analytical sensitivity of 60%70% while FISH in the specimens for detected aberrations revealed sensitivity closer to 30%-40%. All aberrations detected by array-CGH were independently validated by quantitative PCR for genes mapped within the respective regions. Thus, our data indicated that array-CGH is suitable for the detection 2210-7762/$ see front matter a 2012 Elsevier Inc. Elsevier Inc. http://dx.doi.org/10.1016/j.cancergen.2012.07.004 of prognostic genomic aberrations in SLL in a clinical diagnostic setting that could be implemented in patient risk stratification. Conflict of Interest: These authors are employees of Cancer Genetics, Inc., Rutherford, NJ. Array-Based Karyotyping Post Plasma Cell Enrichment for the Detection of Genomic Abnormalities in Multiple Myeloma Barbara K. Zehentner , Luise Hartmann , Krystal Johnson , Christine Stephenson , Douglas Chapman , Monica de Baca , Denise A. Wells , Michael R. Loken , Budi Tirtorahardjo , Shelly R. Gunn , Lony Lim b HematoLogics Inc., Seattle WA; Combimatrix, Irvine, CA Multiple myeloma (MM) is a hematopoietic neoplasm characterized by malignant plasma cells. The discovery of genomic abnormalities present in the monoclonal plasma cells has diagnostic, prognostic, as well as disease monitoring implications. However, technical and disease-related limitations hamper the detection of these abnormalities by metaphase cytogenetic analysis or FISH. In this study, we tested 28 bone marrow specimens with known plasma cell neoplasm for the presence of genomic abnormalities using microarray analysis post plasma cell enrichment and compared the results with other laboratory findings. Metaphase cytogenetic studies were performed on 15 of the 28 samples and identified disease-related genomic aberrations in only 3 of the 15 cases (20%) due to the low proliferative rate of plasma cells; 84.6% of specimens tested positive by MACS-FISH and 89.3% by array comparative genomic hybridization (aCGH). Furthermore, aCGH detected additional abnormalities in 24 cases as compared with FISH alone. We conclude that the combination of plasma cell enrichment and genomic copy number assessment using CGH microarray is a valuable approach for routine clinical use to achieve a more complete genetic characterization of multiple myeloma patients. Conflict of Interest: Employment, stock ownership.
T he time has come for lab staff to become more proactive in the management of diabetes by helpin... more T he time has come for lab staff to become more proactive in the management of diabetes by helping generalists diagnose several important related risk factors. Doing so will not only dramatically improve overall care of patients and their quality of life but also reinforce the lab’s reputation as an integral member of the healthcare team. Diabetes is a significant medical problem in this country. Medical costs associated with managing this disease are estimated at $100 billion annually. Moreover, about 30% more US patients die from diabetes than their European counterparts. American diabetic patients also have four times more serious kidney disease than diabetics in the European Union (EU).1,2 In adult patients, diabetes is the leading cause of blindness, amputation, and end-stage renal disease requiring dialysis and renal transplantation.3 About 60% of diabetic patients die of coronary disease, 10% die of stroke, and another 10% suffer from fatal complications related to peripheral vascular disease.4 Diabetic patients who experience a first myocardial infarction have a 25% chance of death within 1 month and a 50% chance within 1 year.5,6
Purpose: Accurate discrimination of benign oncocytoma and malignant renal cell carcinoma is usefu... more Purpose: Accurate discrimination of benign oncocytoma and malignant renal cell carcinoma is useful for planning appropriate treatment strategies for patients with renal masses. Classification of renal neoplasms solely based on histopathology can be challenging, especially the distinction between chromophobe renal cell carcinoma and oncocytoma. In this study we develop and validate an algorithm based on genomic alterations for the classification of common renal neoplasms. Materials and Methods: Using TCGA renal cell carcinoma copy number profiles and the published literature, a classification algorithm was developed and scoring criteria were established for the presence of each genomic marker. As validation, 191 surgically resected formalin fixed paraffin embedded renal neoplasms were blindly submitted to targeted array comparative genomic hybridization and classified according to the algorithm. CCND1 rearrangement was assessed by fluorescence in situ hybridization. Results: The optimal classification algorithm comprised 15 genomic markers, and involved loss of VHL, 3p21 and 8p, and chromosomes 1, 2, 6, 10 and 17, and gain of 5qter, 16p, 17q and 20q, and chromosomes 3, 7 and 12. On histological rereview (leading to the exclusion of 3 specimens) and using histology as the gold standard, 58 of 62 (93%) clear cell, 51 of 56 (91%) papillary and 33 of 34 (97%) chromophobe renal cell carcinomas were classified correctly. Of the 36 oncocytoma specimens 33 were classified as oncocytoma (17 by array comparative genomic hybridization and 10 by array comparative genomic hybridization plus fluorescence in situ hybridization) or benign (6). Overall 93% diagnostic sensitivity and 97% specificity were achieved.
Parasitaemia, the percentage of infected erythrocytes, is used to measure progress of experimenta... more Parasitaemia, the percentage of infected erythrocytes, is used to measure progress of experimental Plasmodium infection in infected hosts. The most widely used technique for parasitaemia determination is manual microscopic enumeration of Giemsa-stained blood films. This process is onerous, time consuming and relies on the expertise of the experimenter giving rise to person-to-person variability. Here the development of image-analysis software, named Plasmodium AutoCount, which can automatically generate parasitaemia values from Plasmodium-infected blood smears, is reported. Giemsa-stained blood smear images were captured with a camera attached to a microscope and analysed using a programme written in the Python programming language. The programme design involved foreground detection, cell and infection detection, and spurious hit filtering. A number of parameters were adjusted by a calibration process using a set of representative images. Another programme, Counting Aid, written in ...
Proceedings of the National Academy of Sciences, 2011
Microtubules are integral to neuronal development and function. They endow cells with polarity, s... more Microtubules are integral to neuronal development and function. They endow cells with polarity, shape, and structure, and their extensive surface area provides substrates for intracellular trafficking and scaffolds for signaling molecules. Consequently, microtubule polymerization dynamics affect not only structural features of the cell but also the subcellular localization of proteins that can trigger intracellular signaling events. In the nematode Caenorhabditis elegans , the processes of touch receptor neurons are filled with a bundle of specialized large-diameter microtubules. We find that conditions that disrupt these microtubules (loss of either the MEC-7 β-tubulin or MEC-12 α-tubulin or growth in 1 mM colchicine) cause a general reduction in touch receptor neuron (TRN) protein levels. This reduction requires a p38 MAPK pathway (DLK-1, MKK-4, and PMK-3) and the transcription factor CEBP-1. Cells may use this feedback pathway that couples microtubule state and MAPK activation to...
Increasing numbers of plant-made vaccines and pharmaceuticals are entering the late stage of prod... more Increasing numbers of plant-made vaccines and pharmaceuticals are entering the late stage of product development and commercialization. Despite the theoretical benefits of such production, expression of parasite antigens in plants, particularly those from Plasmodium, the causative parasites for malaria, have achieved only limited success. We have previously shown that stable transformation of tobacco plants with a plant-codon optimized form of the Plasmodium yoelii merozoite surface protein 4 ⁄ 5 (PyMSP4 ⁄ 5) gene resulted in PyMSP4 ⁄ 5 expression of up to 0.25% of total soluble protein. In this report, we describe the rapid expression of PyMSP4 ⁄ 5 in Nicotiana benthamiana leaves using the deconstructed tobacco mosaic virus-based magnICON Ò expression system. PyMSP4 ⁄ 5 yields of up to 10% TSP or 1-2 mg ⁄ g of fresh weight were consistently achieved. Characterization of the recombinant plantmade PyMSP4 ⁄ 5 indicates that it is structurally similar to PyMSP4 ⁄ 5 expressed by Escherichia coli. It is notable that the plant-made PyMSP4 ⁄ 5 protein retained its immunogenicity following long-term storage at ambient temperature within freezedried leaves. With assistance from a mucosal adjuvant the PyMSP4 ⁄ 5-containing leaves induced PyMSP4 ⁄ 5-specific antibodies when delivered orally to naïve mice or mice primed by a DNA vaccine. This study provides evidence that immunogenic Plasmodium antigens can be produced in large quantities in plants using the magnICON Ò viral vector system. Plants have been investigated as an economical expression and delivery system for vaccines targeting a number of infectious diseases (Ma et al., 2005; Spok et al., 2008). However, limited success has been achieved for the expression of parasite antigens in plants, particularly those
The bioinformatics software, Geneious, provides a useful platform for researchers to retrieve and... more The bioinformatics software, Geneious, provides a useful platform for researchers to retrieve and analyse genomic and functional genomics information. However, the main databases that the software is able to access are hosted by NCBI (National Center for Biotechnology Information). The databases of EuPathDB (Eukaryotic Pathogen Database Resources), such as PlasmoDB and PiroplasmaDB, collect more specific and detailed information about eukaryotic pathogens than those kept in NCBI databases. Two plugins for Geneious, one for PlasmaDB and one for PiroplasmaDB were developed. When installed, users can use search facilities to find and import gene and protein sequences from the EuPathDB databases. Users can then use the functions of Geneious to process the sequence information. When information unique to PlasmoDB and PiroplasmaDB is required, the user can access results linked with the gene/protein sequence via the default web browser. The plugins are freely available from the Victorian Bioinformatics Consortium website. The plugins can be modified to access any of the databases of EuPathDB.
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Papers by Charles Ma