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Objectives: In precision medicine, where oncologic management is tailored to the individual's clinical and genetic profiles, advanced diagnostic testing provides prognostic information and guides management in a growing number of malignancies. There is a need to capture the work pathologists perform to meet this demand by providing medically relevant, timely, and accurate testing results. This work includes not only direct patient consults (interpretation of results and issuing reports) but the administrative and medical oversight as well as the research needed to provide the necessary quality assurance, quality control, direction, and framework for the laboratory.Methods: An expert panel of Canadian pathologists involved in advanced diagnostics was convened to establish and beta test a model for workload assessment in advanced diagnostics.Results: All aspects of the advanced diagnostics workload were detailed and applied to models based on members' experience, including medical oversight, administration, and the introduction of new testing and platforms. Models for biomarker testing were developed for simple and complex or multiplexed assays, and a detailed model was developed to assess the workload for next-generation sequencing-based assays.Conclusions: This paper provides the first detailed proposal for capturing an advanced diagnostic workload to enable appropriate pathologist allotment for performing all the steps required to run an advanced diagnostic service. [ABSTRACT FROM AUTHOR]

Cancer biomarker studies often require nucleic acid extraction from limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissues, such as histologic sections or needle cores. A major challenge is low quantity and quality of extracted nucleic acids, which can limit our ability to perform genetic analyses, and have a significant influence on overall study design. This study was aimed at identifying the most reliable and reproducible method of obtaining sufficient high-quality nucleic acids from FFPE tissues. We compared the yield and quality of nucleic acids from 0.6-mm FFPE prostate tissue cores across 16 DNA and RNA extraction protocols, using 14 commercially available kits. Nucleic acid yield was determined by fluorometry, and quality was determined by spectrophotometry. All protocols yielded nucleic acids in quantities that are compatible with downstream molecular applications. However, the protocols varied widely in the quality of the extracted RNA and DNA. Four RNA and five DNA extraction protocols, including protocols from two kits for dual-extraction of RNA and DNA from the same tissue source, were prioritized for further quality assessment based on the yield and purity of their products. Specifically, their compatibility with downstream reactions was assessed using both NanoString nCounter gene expression assays and reverse-transcriptase real-time PCR for RNA, and methylation-specific PCR assays for DNA. The kit deemed most suitable for FFPE tissue was the AllPrep kit by Qiagen because of its yield, quality, and ability to purify both RNA and DNA from the same sample, which would be advantageous in biomarker studies. [ABSTRACT FROM AUTHOR]

Abstract: Adult somatic tissue, and the tumours that arise therein, are maintained by a small population of stem cells. In addition to the self-renewal potential and pluripotency, these stem cells express several phenotypic traits that can be used in isolation and enrichment strategies. Since most of the traits are not exclusive to the stem cells however, the resultant populations are typically heterogeneous and variable from one isolation to another. In this article, we review the strategies for isolation of stem cells, and the limitations thereof, with emphasis on mesenchymal tissue and bone tumours. The emerging evidence suggests that stem cell is not a distinct entity, but rather an indefinite state along a spectrum, characterized by phenotypic traits, epigenetic factors and the microenvironment. [Copyright &y& Elsevier]

Abstract: Introduction: Plasmacytoid urothelial carcinoma (PUC) is a rare tumor of the urinary bladder. Its clinical and histopathological features have not been well characterized. In this study we report seven cases of PUC from our institution. Materials and methods: A pilot case of PUC was recently diagnosed at our institution. Cases of urothelial carcinoma (UC) were reviewed for a period of seven years to identify PUC. Representative sections from each case of PUC were submitted for immunohistochemical studies. Clinical charts were reviewed. Results: There were a total of seven cases of PUC out of 260 cases of invasive urothelial carcinoma. The common type of urothelial carcinoma (CUC) was present in focal areas in five cases. Cases with extensive PUC showed coarse and indurated mucosal folds and thickened bladder walls, with no grossly identifiable tumor. Urine cytology showed a scant number of atypical single cells, frequently without tumor diathesis, leading to a shortfall in the positive cytological diagnosis. Histologically, PUC appeared as dyscohesive, plasmacytoid cells with eccentric nuclei, extending widely into the bladder walls and extensively into adjacent pelvic organs. Conclusion: PUC is a distinct clinical and pathological subtype of urothelial carcinoma. The clinical presentation is frequently late due to the frequent absence of hematuria and indurated mucosal surface at cystoscopy. The disease followed an ominous course with recurrence in all the patients, and with patient death. [Copyright &y& Elsevier]

Mechanisms underlying prostate cancer (CaP) initiation and progression are poorly understood. A chromosomal instability mechanism leading to the generation of numerical and structural chromosomal changes has been implicated in the preneoplastic and neoplastic stages of CaP. Telomere dysfunction is one potential mechanism associated with the onset of such instability. To determine whether there was alteration in telomere length and chromosome number, 15 paraffin-embedded prostatectomy specimens were investigated using quantitative peptide nucleic acid (PNA) FISH analysis of representative foci of carcinoma, putative precancerous lesions (high-grade prostatic intraepithelial neoplasia, HPIN) and nondysplastic prostate epithelium. A significant decrease in telomere length was shown in both HPIN and CaP in comparison with normal epithelium. In addition, elevated rates of aneusomy suggested that increased levels of chromosomal aberrations were associated with decreased telomere length. Moreover, multiple foci of HPIN were shown to have a heterogeneous overall reduction of telomere length. This reduction was more evident in the histologic regions of the prostate containing CaP. Such observations lend support to the hypothesis that telomere erosion may be a consistent feature of CaP oncogenesis and may also be associated with the generation of chromosomal instability that characterizes this malignancy.Oncogene (2003) 22, 1978-1987. doi:10.1038/sj.onc.1206227 [ABSTRACT FROM AUTHOR]

The way in which cytogenetic aberrations develop in prostate cancer (CaP) is poorly understood. Spectral karyotype (SKY) analysis of CaP cell lines has shown that they have unstable karyotypes and also have features associated with chromosomal instability (CIN). To accurately determine the incidence of de novo structural and numerical aberrations in vitro in CaP, we performed SKY analysis of three independent clones derived from one representative cell line, DU145. The frequent generation of new chromosomal rearrangements and a wide variation in the number of structural aberrations within two to five passages suggested that this cell line exhibited some of the features associated with a CIN phenotype. To study numerical cell-to-cell variation, chromosome 8 aneusomy was assessed in the LNCaP, DU145, and PC-3 cell lines and a patient cohort of 15 CaP primary tumors by interphase fluorescence in situ hybridization (FISH). This analysis showed that a high frequency of numerical alteration affecting chromosome 8 was present in both in vitro and in CaP tissues. In comparison to normal controls, the patient cohort had a statistically significant (P<.05), greater frequency of cells with one and three centromere 8 copies. These data suggest that a CIN-like process may be contributing towards the generation of de novo numerical and structural chromosome abnormalities in CaP. Neoplasia (2001) 3, 62–69. [ABSTRACT FROM AUTHOR]

Examines the relative, spatial relationship of small nuclear ribonucleoproteins (snRNPs) and of DNase I hypersensitive chromatin (DHC) in rat pheochromocytoma cells. Testing of the hypothesis that the nonrandom organization of the contents of the interphase nuclei represents a compartmentalization of function; Nerve growth factor-induced differentiation.

OBJECTIVE To investigate the predictive value (PV) for all prostate cancers and for clinically significant cancer undiagnosed after a 10-core biopsy protocol, as the 10-core transrectal ultrasonography-guided biopsy is considered the standard technique of prostatic biopsy due to its high rate of detection of prostatic adenocarcinoma. PATIENTS AND METHODS In all, 132 consecutive radical prostatectomy (RP) specimens, with their corresponding 10-core biopsies, were reviewed. Cases with unilateral core involvement by prostate cancer were retained for study. Morphometric analysis was conducted on the biopsy-negative hemi-prostates to determine the PV of the biopsy protocol with respect to the size, position and clinical significance of the lesion. RESULTS In all, 70 resected prostates (RP) had unilateral core involvement by prostate cancer. In 38 cases, there was cancer in the biopsy-negative hemi-prostates (group 1); in the remaining 32 the hemi-prostates were free of cancer (group 2). Group 1 was categorized by morphometric criteria. Specifically, 23 cases had one to eight foci of prostate cancer in the posterior nontransitional zone (NTZ) (group 1a), while 15 had two to six foci of prostate cancer in the transitional zone (TZ), or the anterior horn (AH) of the peripheral zone or the TZ and AH (group 1b). There were two cases with clinically significant prostate cancer in group 1a, and six in group 1b. CONCLUSIONS The PV of a negative five-core biopsy protocol on a hemi-prostate is 54% for prostate cancer and 11% for clinically significant prostate cancer. Most clinically significant prostate cancers were in the AH/TZ of the prostate. [ABSTRACT FROM AUTHOR]

The detection of gene fusions by RNA-based next-generation sequencing (NGS) is an emerging method in clinical genetic laboratories for oncology biomarker testing to direct targeted therapy selections. A recent Canadian study (CANTRK study) comparing the detection of NTRK gene fusions on different NGS assays to determine subjects' eligibility for tyrosine kinase TRK inhibitor therapy identified the need for recommendations for best practices for laboratory testing to optimize RNA-based NGS gene fusion detection. To develop consensus recommendations, representatives from 17 Canadian genetic laboratories participated in working group discussions and the completion of survey questions about RNA-based NGS. Consensus recommendations are presented for pre-analytic, analytic and reporting aspects of gene fusion detection by RNA-based NGS. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is characterized by chromosomal instability and high copy number gene amplification. The breakage–fusion–bridge (BFB) cycle is a well-established mechanism of genome instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. To determine whether the BFB cycle could be increasing the de novo rate of formation of cytogenetic aberrations in OS, the frequency of anaphase bridge configurations and dicentric chromosomes in four OS cell lines was quantified. An increased level of anaphase bridges and dicentrics was observed in all the OS cell lines. There was also a strong association between the frequencies of anaphase bridges, dicentrics, centrosomal anomalies, and multipolar mitotic figures in all the OS cell lines, indicating a possible link in the mechanisms that led to the structural and numerical instabilities observed in OS. In summary, this study has provided strong support for the role of the BFB cycle in generating the extensive structural chromosome aberrations, as well as cell-to-cell cytogenetic variation observed in OS, thus conferring the genetic diversity for OS tumor progression. [ABSTRACT FROM AUTHOR]

Background: Resistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression profiling to delineate major deregulated pathways and biomarkers associated with the development of intrinsic chemotherapy resistance upon exposure to standard first-line therapy for ovarian cancer.Methods: The study cohort comprised 28 patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. All 28 patients had advanced stage, high-grade serous ovarian cancer, and were treated with standard platinum-based chemotherapy. Twelve patient tumours demonstrating relative resistance to platinum chemotherapy corresponding to shorter PFS (< eight months) were compared to sixteen tumours from platinum-sensitive patients (PFS > eighteen months). Whole transcriptome profiling was performed using an Affymetrix high-resolution microarray platform to permit global comparisons of gene expression profiles between tumours from the resistant group and the sensitive group.Results: Microarray data analysis revealed a set of 204 discriminating genes possessing expression levels which could influence differential chemotherapy response between the two groups. Robust statistical testing was then performed which eliminated a dependence on the normalization algorithm employed, producing a restricted list of differentially regulated genes, and which found IGF1 to be the most strongly differentially expressed gene. Pathway analysis, based on the list of 204 genes, revealed enrichment in genes primarily involved in the IGF1/PI3K/NF κB/ERK gene signalling networks.Conclusions: This study has identified pathway specific prognostic biomarkers possibly underlying a differential chemotherapy response in patients undergoing standard platinum-based treatment of serous epithelial ovarian cancer. In addition, our results provide a pathway context for further experimental validations, and the findings are a significant step towards future therapeutic interventions. [ABSTRACT FROM AUTHOR]

Abnormal cell division leading to the gain or loss of entire chromosomes and consequent genetic instability is a hallmark of cancer. Centromere protein -A (CENPA) is a centromere-specific histone-H3-like variant gene involved in regulating chromosome segregation during cell division. CENPA is one of the genes included in some of the commercially available RNA based prognostic assays for breast cancer (BCa)—the 70 gene signature MammaPrint® and the five gene Molecular Grade Index (MGISM). Our aim was to assess the immunohistochemical (IHC) expression of CENPA in normal and malignant breast tissue. Clinically annotated triplicate core tissue microarrays of 63 invasive BCa and 20 normal breast samples were stained with a monoclonal antibody against CENPA and scored for percentage of visibly stained nuclei. Survival analyses with Kaplan-Meier (KM) estimate and Cox proportional hazards regression models were applied to assess the associations between CENPA expression and disease free survival (DFS). Average percentage of nuclei visibly stained with CENPA antibody was significantly higher (p = 0.02) in BCa than normal tissue. The 3-year DFS in tumors over-expressing CENPA (>50% stained nuclei) was 79% compared to 85% in low expression tumors (<50% stained nuclei). On multivariate analysis, IHC expression of CENPA showed weak association with DFS (HR > 60.07; p = 0.06) within our small cohort. To the best of our knowledge, this is the first published report evaluating the implications of increased IHC expression of CENPA in paraffin embedded breast tissue samples. Our finding that increased CENPA expression may be associated with shorter DFS in BCa supports its exploration as a potential prognostic biomarker. [ABSTRACT FROM AUTHOR]

Prostate cancer (CaP) is a multifocal heterogenous disease. A major challenge in CaP research is to identify genetic biomarkers that herald aggressive transformation. To investigate the effect of tumor heterogeneity on the analysis of genomic aberration, we compared the results of comparative genomic hybridization (CGH) analysis of DNA extracted from tumor bulk against that of DNA amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from homogeneous cell population obtained by laser capture microdissection of discrete tumor foci. Sampling by microdissection, aberrations were observed in three of three foci of carcinoma involved with prostatic capsule, and in two of three prostatic intraepithelial neoplasia (PIN) foci examined. Carcinoma foci consistently exhibited more extensive aberrations than the PIN samples obtained from the same tumor. Within these samples, the different tumor foci exhibited gain of 8q, whereas PIN showed no consistent aberration. Using bulk extracted DNA, CGH detected aberrations in only 3 of 21 samples investigated, despite the known trisomy 8 status, as revealed by fluorescence in situ hybridization. The results of this study demonstrate that CGH analysis using bulk dissected fresh tissue is insufficiently sensitive to fully detect the chromosomal numerical aberrations in CaP. Given the considerable intratumor genomic heterogeneity, CGH with microdissection and DOP-PCR amplification provides a more complete repertoire of aberrations as well as a better phenotype-genotype correlation in prostate tumors. [Copyright &y& Elsevier]