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PubMed2006

Chromogenic in situ hybridization to detect HER-2/neu gene amplification in histological and ThinPrep-processed breast cancer fine-needle aspirates: a sensitive and practical method in the trastuzumab era.

The increasing evidence of trastuzumab efficacy in breast cancer (BC) patients means that an accurate and reproducible evaluation of HER-2 statusis of paramount importance in histological and in cytological samples. Currently, the two main methods used to analyze HER-2 amplification or overexpression are fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Although the two methods are strongly correlated for histological tissue, the evaluation of tumor morphology through FISH may be difficult and fluorescence fades quickly. These limitations can be overcome by chromogenic in situ hybridization (CISH), which can visualize the amplification product along with morphological features. In view of this, in the present study, we analyzed the usefulness of CISH on formalin-fixed, paraffin-embedded (FFPE) BC specimens and investigated whether CISH can be a valid technique in the determination of HER-2 status for fine-needle aspirates (FNAs) processed by liquid-based cytology. The results we obtained in a retrospective series of 111 FFPE BC specimens demonstrated good concordance between CISH and IHC and between CISH and FISH. The former concordance was comparable with that observed between FISH and IHC. When CISH was applied to a prospective series of 53 FNAs, from surgically removed BC, our data showed evidence of a higher concordance of results between liquid-based cytology and the companion FFPE tissues using CISH rather than HercepTesttrade mark. Therefore, CISH analysis, which is avaluable and reproducible alternative to FISH for selecting breast cancer patients for trastuzumab therapy, can lower false-positive immunocytochemistry findings in ThinPrep-processed FNAs.

Antibodies, Monoclonal↗

PubMed2002

Idiopathic hyperammonemia following an unrelated cord blood transplant for mucopolysaccharidosis I.

Bone marrow transplantation (BMT) has been shown to reverse or stabilize some manifestations of mucopolysaccharidosis I (Hurler syndrome). Idiopathic hyperammonemia (IHA) is a rare complication of solid organ and BMT that is characterized by elevated serum ammonia, normal liver enzymes, and abrupt onset of neurologic deterioration. We present the case of a 14-month-old male patient with Hurler syndrome who developed fatal IHA (ammonia = 2297 micromol/L) 31 days after a cord blood transplant. A complete autopsy was performed, with examination of both frozen and formalin-fixed paraffin-embedded (FFPE) tissues using a variety of special stains and electron microscopy. Hyperammonemia was documented by analysis of antemortem serum and postmortem cerebrospinal and vitreous fluid. Other causes of hyperammonemia, including Reye syndrome, were excluded. Histologic changes included centrilobular microvesicular steatosis of the liver and storage product present in multiple organs. The highly water-soluble mucopolysaccharide (MPS) storage product was best identified by colloidal iron staining of FFPE and unfixed air-dried fresh frozen liver sections. Alcian blue stains failed to convincingly demonstrate MPS in any of the liver sections. This is the first published report, to our knowledge, of IHA in a posttransplant patient younger than 18 years old or following transplantation for Hurler syndrome. Demonstration of the hepatic centrilobular microvesicular steatosis characteristic of IHA was complicated by the diffuse storage of MPS within the liver. MPS storage can be best detected in the liver using colloidal iron staining. Oil-red-O staining may be useful to document microvesicular steatosis in cases with a clinical history of hyperammonemia following solid organ or BMT. Determining if certain subsets of children are at increased risk for IHA requires further study.

Ammonia↗

PubMed2024

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article↗

PubMed2024

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Journal Article↗

PubMed2004

PHOTOPROBE biotin: an alternative method for labeling archival DNA for comparative genomic hybridization.

Comparative genomic hybridization (CGH) represents a powerful method for screening the entire genome of solid tumors for chromosomal imbalances. Particularly it enabled the molecular cytogenetic analysis of archival, formalin-fixed, paraffin-embedded (FFPE) tissue. A well-known dilemma, however, is the poor DNA quality of this material with fragment sizes below 1000 bp. Nick translation, the conventionally used enzymatic DNA labeling method in CGH, leads to even shorter fragments often below a critical limit for successful analysis. In this study we report the alternative application of non-enzymatic, PHOTOPROBE biotin labeling for conjugation of the hapten to the DNA prior to in situ hybridization and fluorescence detection. We analyzed 51 FFPE tumor samples mainly from the upper respiratory tract by both labeling methods. In 19 cases, both approaches were successful. The comparison of hybridized metaphases showed a distinct higher fluorescence signal of the PHOTOPROBE samples sometimes with a discrete cytoplasm background which however did not interfere with specificity and sensitivity of the detected chromosomal imbalances. For further 32 cases characterized by an average DNA fragment size below 1000 bp, PHOTOPROBE biotin was the only successful labeling technique thus offering a new option for CGH analysis of highly degraded DNA from archival material.

Biotin↗

PubMed2006

Analysis of ERBB2 and TOP2A gene status using fluorescence in situ hybridization versus immunohistochemistry in localized breast cancer.

The aim of our study was to assess the ERBB2 and TOP2A gene status in breast carcinoma tissue using fluorescence in situ hybridization (FISH) and to compare their amplification with immunohistochemistry assay (IHC) of the ERBB2, resp. topoisomerase IIalpha proteins. TOP2A status is important in tailored treatment as topoisomerase IIalpha is the molecular target for topoisomerase IIalpha inhibitors. This study was conducted to determine whether the methods are equivalent in their assessment of TOP2A status and to correlate the genetic findings with basic tumor and disease characteristics. Locus specific ERBB2, TOP2A genes and chromosome 17 centromeres (CEP17) probes were hybridized to 72 formalin-fixed paraffin-embedded (FFPE) tissue samples from patients with non-metastatic breast carcinoma (M0). The ERBB2, TOP2A and CEP17 signals were counted and gene numbers per nucleus or per CEP17 were calculated, respectively. Sections were also stained with commercial polyclonal antibody (HercepTestTM), anti-topoisomerase IIalpha monoclonal antibody (clone SWT3D1) and scored for the presence of membrane/nuclear staining. ERBB2 amplification was found in 20.3%, ERBB2 and TOP2A co-amplification was detected in 14.5% of cases. Deletion of the ERBB2/TOP2A gene was found in 1.4/2.8% of sections, respectively. Concordance of FISH and IHC techniques in the evaluation of ERBB2 and TOP2A status was found in 88.4% and 66.7%, respectively. The low concordance of FISH versus IHC in the evaluation of TOP2A status was mainly due to the presence of TOP2A amplified tumors in IHC negative or weakly positive specimens. Topoisomerase IIalpha expression was increased in bigger tumors, although direct correlation with tumor grading was not found. ERBB2 amplification was found in more aggressive breast cancers with grades 2 and 3, respectively. Interestingly, chromosome 17 polysomy was more frequently observed among older women (>55 years), suffering usually from less aggressive disease. Our results confirm the high concordance of the ERBB2 and TOP2A gene co-amplification in breast carcinoma. Differences between FISH and IHC in the case of ERBB2 gene status were found only in IHC 2+ sections as reported in the literature. However, our study points to the importance of FISH examination of TOP2A gene status in all tumors with ERBB2 amplification.

Adult↗

PubMed2026

An Instrumental Optimization of a Label-Free Proteomic Method for Trace Protein Input.

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics of trace-level samples, such as tens of cells or spatially resolved tissue regions, offers unique biological insights but is often constrained by the requirement for specialized, costly instrumentation. In this study, we developed a scalable workflow for the deep proteomic analysis of low- to ultralow-input samples by systematically optimizing a widely adopted Orbitrap and UHPLC platform to maximize sensitivity, precision, and throughput. This optimized workflow identified over 5600 proteins from 5 ng of peptides and 3400 proteins from 20 sorted cells, achieving a throughput of 30 analyses per day while maintaining deep proteome coverage and high quantitative reproducibility. Furthermore, by applying this method to spatially resolved proteomics, we identified over 6100 proteins from microscale regions of interest (ROIs) within a formalin-fixed, paraffin-embedded (FFPE) tissue. A data-driven normalization strategy was employed to correct for variable cellularity across tissue regions, effectively revealing intratumor heterogeneity and distinct molecular and functional signatures, including pathway activations not apparent in parallel spatial transcriptomic analysis. Ultimately, this accessible, high-performance method substantially lowers the instrumentation barrier for the deep proteomic profiling of trace-level biological samples.

Proteomics↗

PubMed1992

Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.

AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.

Alleles↗

PubMed2004

Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.

Animals↗

PubMed2026

[State Changes and Stability Grading of Driver Genes in Non-small Cell Lung Cancer Based on Repeated NGS Testing].

BACKGROUND: Next-generation sequencing (NGS)-based driver gene testing has become a routine component of molecular subtyping and precision therapy for non-small cell lung cancer (NSCLC). Dynamic genomic monitoring facilitates early detection of resistance-related molecular alterations and informs timely therapeutic adjustments. However, standardized criteria for evaluating the stability of serial NGS testing are currently lacking, and the applicability of NGS using formalin-fixed paraffin-embedded (FFPE) specimens for dynamic monitoring remains poorly defined. This study aims to establish a stability grading system for driver gene status alterations based on repeated NGS testing, and to provide evidence-based support for clinical repeat biopsy strategies. METHODS: Data from 1232 patients with NSCLC who underwent two or more NGS tests on FFPE tissue specimens at Beijing Chest Hospital between June 2019 and April 2026 were collected retrospectively. Patients with an interval of ≥4 months between the initial and last tests were included to ensure the representativeness of temporal analysis, resulting in a main analysis cohort of 942 patients. The Kappa consistency test was used to evaluate the state stability of nine core driver genes [epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1), mesenchymal‑epithelial transition factor (MET), rearranged during transfection (RET), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), erb‑b2 receptor tyrosine kinase 2 (ERBB2), and phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit alpha (PIK3CA)] and to construct a five‑level grading system. Paired variant allele frequency (VAF) differences were compared using the Wilcoxon signed‑rank test. Independent influencing factors for mutation accumulation were identified by binary Logistic regression. RESULTS: The state stability of the nine genes was classified into five levels: EGFR showed high stability (Kappa=0.838), ROS1/ALK/KRAS good stability, BRAF/PIK3CA/RET moderate stability, and ERBB2 low stability, and MET showed high instability. MET exhibited the highest rate of state change (9.3%) with a raw observed agreement of 90.7%. Its Kappa value (0.172) was influenced by the low prevalence (3.7%) compression effect and should therefore be interpreted alongside the observed agreement (90.7%) and the prevalence-adjusted and bias-adjusted Kappa (PABAK). The VAF of PIK3CA increased significantly (P=0.005). T790M positivity increased from 5.8% to 10.8%, and 30 new C797S mutations were detected at the last test (13 with T790M, 17 without). The overall rate of new driver gene variants in the main cohort was 18.0%. Binary Logistic regression showed that a lower number of initial mutated genes was the only independent predictor of new variants [odds ratio (OR)=0.399, P<0.001], while sex and detection interval showed no independent association. CONCLUSIONS: A five level stability grading system for state changes of driver genes in NSCLC based on repeated NGS testing has been established. MET showed the most frequent state changes, which should be interpreted in conjunction with the prevalence effect. The VAF increase of PIK3CA is an observational finding, and its clinical significance requires further prospective validation. A lower initial mutation burden may reflect tumor clonal complexity and was associated with a higher likelihood of subsequent acquisition of new variants. FFPE based NGS is applicable for repeated testing at clinical treatment decision nodes.

Humans↗

PubMed2001

Decreased expression of catenins (alpha and beta), p120 CTN, and E-cadherin cell adhesion proteins and E-cadherin gene promoter methylation in prostatic adenocarcinomas.

BACKGROUND: Catenin/E-cadherin complex proteins play an important role in cell-cell adhesion with decreased expression correlating with adverse prognostic variables in several human malignancies. METHODS: Archival formalin fixed, paraffin embedded (FFPE) sections from 118 prostatic adenocarcinomas (PACs) were immunostained by an automated method (Ventana Medical Systems, Tuscon, AZ) using monoclonal antibodies to catenins alpha and beta, p120 CTN, and E-cadherin proteins. Immunoreactivity was semiquantitatively graded, and results correlated with traditional prognostic parameters. In a subset of 10 randomly selected cases, E-cadherin gene promoter methylation status was determined on FFPE tissues using sodium bisulfite/hydroquinone DNA modification and polymerase chain reaction (PCR) with methylation specific primers (CpG wiz E-cadherin methylation assay; Intergen Co., Purchase, NY). RESULTS: Decreased expression of alpha-catenin (17%), beta catenin (4%), p120 CTN (45%), and E-cadherin (25%) proteins was noted in PACs with downregulation of each protein correlating with high tumor grade (P = 0.01-0.0001). In addition, p120 CTN and E-cadherin expression levels correlated with pathologic stage (P = 0.05; P = 0.02), aneuploidy (P = 0.001; P = 0.0001), and alpha-catenin with aneuploidy (P = 0.0001). p120 CTN loss also correlated with preoperative serum prostate specific antigen (P = 0.05). Two of 10 cases featured no evidence of E-cadherin gene promoter methylation by PCR and both cases retained expression of E-cadherin protein on immunohistochemistry. Of the 8 cases that showed E-cadherin methylation, 5 (68%) featured loss of expression of the protein on immunohistochemistry (P = 0.11). There was no correlation between E-cadherin methylation and adverse prognostic variables. CONCLUSIONS: Decreased expression of catenin/E-cadherin complex cell adhesion proteins is associated with aggressive phenotype in prostatic adenocarcinoma. E-cadherin gene promote methylation is a common event in prostate carcinoma but does not appear to bear prognostic significance in the subset of cases analyzed.

Adenocarcinoma↗

PubMed1990

In situ hybridization for the detection of human parvovirus B19 nucleic acid sequences in paraffin-embedded specimens.

Parvovirus infection of pregnant women leading to a transplacentar infection of the fetus may result in hydrops fetalis, and ultimately in intrauterine death of the fetus. In situ hybridization with a biotinylated as well as with a 35S-labeled probe for human parvovirus B19 was performed on formalin-fixed paraffin-embedded (FFPE) tissue from a fetus suffering from non-immunologic hydrops fetalis. Histology was suggestive of viral infection probably with human parvovirus. Parvovirus DNA could be detected and precisely localized mainly in the nuclei of erythroid precursors cells within fetal blood vessels of all organs examined. There was no detection of B19 nucleic acid in parenchymal cells of the placenta or the fetal organs, nor within maternal blood cells. These findings are in agreement with the well-known properties of animal parvoviruses to replicate exclusively in proliferating cells. Taking into consideration the problems in diagnosing human parvovirus infection by light microscopy, we conclude that in situ hybridization with an appropriate non-radioactive probe is a valuable, rapid and safe complementary detection method for the diagnosis and study of human parvovirus infections. The 35S-labeled probe is more sensitive than the biotinylated probe, but has the disadvantages of lower resolution of the signal, longer duration of the assay, the hazard of radioactivity and the shorter shelf-life of the probe.

Adult↗

PubMed2004

Upregulation of focal adhesion kinase (FAK) expression in ductal carcinoma in situ (DCIS) is an early event in breast tumorigenesis.

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in a subset of invasive breast cancers. FAK transmits signals that mediate several functions including tumor cell proliferation, migration, adhesion and survival. We used immunohistochemical techniques to assess FAK expression in patients with fibrocystic disease (FCD), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC). Formalin-fixed, paraffin-embedded (FFPE) tissue sections were obtained from 119 patients (12 FCD, 38 ADH, 51 DCIS and 18 IDC). The anti-FAK 4.47 monoclonal antibody was used to detect FAK expression. FAK expression was scored as high (3 or 4 intensity and > or =90% positive cells) or low. The DCIS tissue sections demonstrated high FAK expression in 34/51 (66%) of the sections. High FAK expression was demonstrated in 6/18 (33%) of the IDC tissue sections and 8/38 (21%) of the ADH tissue sections. None (0/12) of the FCD tissues sections stained high for FAK. The pattern of FAK expression in DCIS was significantly higher than ADH (p < 0.0001) and IDC (p = 0.02). We conclude that FAK overexpression in preinvasive, DCIS tumors precedes tumor cell invasion or metastasis, suggesting that FAK may function as a survival signal and be an early event in breast tumorigenesis.

Breast Diseases↗

PubMed1999

Enhanced sensitivity with a novel TCRgamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases.

BACKGROUND: Clonal rearrangement of genes encoding the immunoglobulins (Ig) and T-cell antigen receptors (TCR) are considered to be useful markers for the diagnosis of lymphoma and for determining the clonal origins of B- and T-cell populations in lymphoid neoplasms. METHODS AND RESULTS: Polymerase chain reaction-based clonality assays for TCRgamma, TCRbeta, and immunoglobulin (Ig) heavy chain (IgH) gene rearrangements were evaluated for diagnostic sensitivity and specificity in 569 formalin-fixed, paraffin-embedded (FFPE) tissues. Combined TCRbeta and TCRgamma assays enhanced the routine detection of TCR clonality to 90% of all peripheral T-cell lymphoma (PTCL) cases. IgH clonality was detected in 59% of 241 peripheral B-cell lymphoma (BCL) cases and 6% of 169 PTCL cases. Of 452 lymphomas, 5% could not be classified phenotypically as B or T lineage after immunohistochemical and clonality studies. Of all BCL cases analyzed, 24% had detectable TCRbeta and/or TCRgamma clonality. Of these BCL with biclonal results, 47% were extranodal lymphomas from skin and various tissues. CONCLUSIONS: Clonality assays were useful for distinguishing reactive or benign lymph nodes from neoplastic lymphoid infiltrates in most cases. The inclusion of TCRb and TCRg assays in the assessment of lymphomas results in a significant increase in the sensitivity of clonality detection, but is of limited utility in assessing the T- or B-cell phenotype of the tumor.

B-Lymphocytes↗

PubMed2006

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.

Base Sequence↗

PubMed2005

An expression-based site of origin diagnostic method designed for clinical application to cancer of unknown origin.

Gene expression profiling offers a promising new technique for the diagnosis and prognosis of cancer. We have applied this technology to build a clinically robust site of origin classifier with the ultimate aim of applying it to determine the origin of cancer of unknown primary (CUP). A single cDNA microarray platform was used to profile 229 primary and metastatic tumors representing 14 tumor types and multiple histologic subtypes. This data set was subsequently used for training and validation of a support vector machine (SVM) classifier, demonstrating 89% accuracy using a 13-class model. Further, we show the translation of a five-class classifier to a quantitative PCR-based platform. Selecting 79 optimal gene markers, we generated a quantitative-PCR low-density array, allowing the assay of both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue. Data generated using both quantitative PCR and microarray were subsequently used to train and validate a cross-platform SVM model with high prediction accuracy. Finally, we applied our SVM classifiers to 13 cases of CUP. We show that the microarray SVM classifier was capable of making high confidence predictions in 11 of 13 cases. These predictions were supported by comprehensive review of the patients' clinical histories.

Gene Expression Profiling↗

PubMed2006

Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules.

BACKGROUND: Fine needle aspiration (FNA) is widely utilized for evaluation of patients with thyroid nodules. However, approximately 30% are indeterminate for malignancy. Recently, a mutation in the BRAF gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC). In this retrospective study, we assessed the utility of BRAF V600E mutation detection for refining indeterminate preoperative cytologic diagnoses in patients with PTC. METHODS: Archival indeterminate thyroid FNAs and corresponding formalin-fixed, paraffin-embedded (FFPE) surgical samples with PTC were identified in our patient files. DNA extracted from slide scape lysates and 5 mum FFPE sections were evaluated for the BRAF V600E mutation using LightCycler PCR and fluorescent melting curve analysis (LCPCR). Amplification products that showed deviation from the wild-type genomic DNA melting peak, discordant FNA and FFPE matched pairs, and all benign control samples, underwent direct DNA sequencing. RESULTS: A total of 19 indeterminate thyroid FNAs demonstrating PTC on FFPE surgical samples were included in the study. Using BRAF mutation analysis, the preoperative diagnosis of PTC was confirmed in 3/19 (15.8%) FNA samples that could not be conclusively diagnosed on cytology alone. However, 9/19 (47.4%) FFPE tissue samples were positive for the V600E mutation. Of the discordant pairs, 5/6 FNAs contained less than 50% tumor cells. CONCLUSION: When used with indeterminate FNA samples, BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. However, overall tumor cell content of some archival FNA smear slides is a limiting factor for mutation detection.

Journal Article↗

PubMed1997

Clinical relevance of DNA ploidy and proliferative activity in childhood rhabdomyosarcoma: a retrospective analysis of patients enrolled onto the Italian Cooperative Rhabdomyosarcoma Study RMS88.

PURPOSE: Evaluation of the possible clinical relevance of DNA ploidy and proliferative activity assessed as S-phase fraction (SPF) in childhood rhabdomyosarcoma (RMS). PATIENTS AND METHODS: We conducted a retrospective study on 59 RMS patients enrolled onto the ICS-RMS88 protocol (seven botryoid, 35 embryonal, and 17 alveolar RMS), for which formalin-fixed paraffin-embedded (FFPE) tissue was available. Nuclear suspensions for cytometric investigation were obtained using a mechanical disaggregation. Tumors were distinguished according to their DNA index (DI) value as follows: diploid (0.9 < DI < 1.1), hyperdiploid (1.1 < or = DI < 1.8 or DI > or = 2.2), and tetraploid (1.8 < or = DI < 2.2); for analysis of SPF, a cutoff value of 14% was used. RESULTS: DNA histograms were diploid in 19 (33%) cases, hyperdiploid in 29 (49%), and tetraploid in 10 (32%). One patient showed both a hyperdiploid and a tetraploid peak. The 5-year overall survival (OS) rate by ploidy status was 73% in hyperdiploid patients as compared with 33% and 25% in diploid and tetraploid patients, respectively (P = .0012). A striking difference emerged when the 5-year OS for the combined diploid and tetraploid RMS groups was compared with survival of the hyperdiploid RMS group: 30% versus 73%, respectively (P = .0006). In addition, the SPF was prognostically relevant: 5-year OS by SPF less than or greater than 14% was 70% and 36%, respectively (P = .009). Multivariate analysis confirmed the importance of DNA content (P = .0006) and SPF (P = .034) in predicting survival. CONCLUSION: These findings confirm that ploidy and SPF are important new prognostic factors that are able to identify selected groups of patients at high risk of treatment failure, even if the tumor's presentation is favorable according to standard criteria.

Adolescent↗