Biomedical subjects

Maria Zielenska

Publications and source records attributed to Maria Zielenska.

At least 19 recordsLinked to original sources

PubMed2006

Genomic mechanisms and measurement of structural and numerical instability in cancer cells.

The progression to cancer is often associated with instability and the acquisition of genomic heterogeneity, generating both clonal and non-clonal populations. Chromosomal instability (CIN) describes the excessive rate of numerical and structural genomic change in tumors. Mitotic segregation errors strongly influences copy number, while structural aberrations can occur at unstable genomic regions, or through aberrant DNA repair or methylation. Combined molecular cytogenetic analyses can evaluate cell-to-cell variation, and define the complexity of numerical and structural alterations. Because structural change may occur independently of numerical alteration, we propose the term structural chromosomal instability [(S)-CIN] to distinguish numerical from structural CIN.

Alu Elements↗

PubMed2006

Effects of THBS3, SPARC and SPP1 expression on biological behavior and survival in patients with osteosarcoma.

BACKGROUND: Osteosarcoma is a very aggressive tumor with a propensity to metastasize and invade surrounding tissue. Identification of the molecular determinants of invasion and metastatic potential may guide the development of a rational strategy for devising specific therapies that target the pathways leading to osteosarcoma. METHODS: In this study, we used pathway-focused low density expression cDNA arrays to screen for candidate genes related to tumor progression. Expression patterns of the selected genes were validated by real time PCR on osteosarcoma patient tumor samples and correlated with clinical and pathological data. RESULTS: THBS3, SPARC and SPP1 were identified as genes differentially expressed in osteosarcoma. In particular, THBS3 was expressed at significantly high levels (p = 0.0001) in biopsies from patients with metastasis at diagnosis, which is a predictor of worse overall survival, event-free survival and relapse free survival at diagnosis. After chemotherapy, patients with tumors over-expressing THBS3 have worse relapse free survival. High SPARC expression was found in 51/55 (96.3%) osteosarcoma samples derived from 43 patients, and correlated with the worst event-free survival (p = 0.03) and relapse free survival (p = 0.07). Overexpression of SPP1 was found in 47 of 53 (89%) osteosarcomas correlating with better overall survival, event-free survival and relapse free survival at diagnosis. CONCLUSION: In this study three genes were identified with pattern of differential gene expression associated with a phenotypic role in metastasis and invasion. Interestingly all encode for proteins involved in extracellular remodeling suggesting potential roles in osteosarcoma progression. This is the first report on the THBS3 gene working as a stimulator of tumor progression. Higher levels of THBS3 maintain the capacity of angiogenesis. High levels of SPARC are not required for tumor progression but are necessary for tumor growth and maintenance. SPP1 is not necessary for tumor progression in osteosarcoma and may be associated with inflammatory response and bone remodeling, functioning as a good biomarker.

Adolescent↗

PubMed2006

Metaphase and array comparative genomic hybridization: unique copy number changes and gene amplification of medulloblastomas in South America.

Tumors of the central nervous system are the second most frequent malignancy of childhood, accounting for the majority of cancer-related deaths in this age group. Among these tumors, medulloblastomas (MB) remain in need of further genomic characterization toward understanding of pathogenesis and outcome predictors. Eight pediatric embryonal brain tumors were analyzed: five MB (one being desmoplastic), one PNET, one medulloepithelioma, and one ependymoblastoma. Analyses identified genomic imbalances, including the gain of 16p and the nonsyntenic coamplification of MYCN and TERT loci. More detailed FISH analysis showed that coamplification of MYCN and TERT in one of the MBs manifested as dispersed nuclear speckling, consistent with the presence of double minute chromosomes. There was considerable cell-to-cell copy number heterogeneity present, but it was clear that both genes were amplified concordantly. The amplification of oncogenes seems to play an important role in the pathogenesis of MB, and the association between MYCN and TERT amplifications and poor prognosis has not been well recognized. The uncharacteristic pattern of genomic imbalances detected in MB tumors may be a reflection of the characteristics of these tumors occurring in South America.

Adolescent↗

PubMed2006

The breakage-fusion-bridge (BFB) cycle as a mechanism for generating genetic heterogeneity in osteosarcoma.

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.

Anaphase↗

PubMed2006

Pediatric undifferentiated sarcoma of the soft tissues: a clinicopathologic study.

Pediatric undifferentiated soft tissue sarcomas represent a major challenge for pathologists and clinicians. The goal of this study was to identify cases that warranted this diagnosis by current standards of analysis and then determine if there are clinicopathological commonalities that may be useful for diagnosis, management, and prognosis. Eighteen potential patients were identified using the institutional pathology database. Three cases were reclassified as specific sarcomas, and 2 cases had insufficient material for molecular analysis, leaving 13 cases for pathological review and 12 patients for radiological and clinical review. There were 7 males and 6 females. The median age at diagnosis was 11 years (1 month to 16 years). Tumors commonly involved the trunk (7 of 13; 54%) and ranged in size from 1.7 to 14.5 cm (mean, 6.7 cm). Eleven patients received ifosfamide/etoposide chemotherapy and 4 received irradiation. Five-year event-free and overall survival (EFS and OS) rates were 54% and 74%, respectively. The predominant histological pattern was round to plump spindled cells forming sheets (9 of 13; 69%) and severe atypia was associated with decreased survival (P = 0.048). Immunohistochemistry showed positivity for vimentin (92%), CD117 (92%), and vascular endothelial growth factor (69%), and 8% to 23% showed focal positivity for epithelial, neural, or myogenic markers. Tumors were uniformly negative for translocations associated with pediatric sarcomas. The presence of certain common morphological and immunohistochemical features in the absence of specific molecular genetic abnormalities allows for a diagnosis of pediatric undifferentiated soft tissue sarcoma; however, whether this group of neoplasms forms a unique category of tumors or a common precursor pathway for a number of different sarcomas awaits further study.

Adolescent↗

PubMed2006

Large scale copy number variation (CNV) at 14q12 is associated with the presence of genomic abnormalities in neoplasia.

BACKGROUND: Advances made in the area of microarray comparative genomic hybridization (aCGH) have enabled the interrogation of the entire genome at a previously unattainable resolution. This has lead to the discovery of a novel class of alternative entities called large-scale copy number variations (CNVs). These CNVs are often found in regions of closely linked sequence homology called duplicons that are thought to facilitate genomic rearrangements in some classes of neoplasia. Recently, it was proposed that duplicons located near the recurrent translocation break points on chromosomes 9 and 22 in chronic myeloid leukemia (CML) may facilitate this tumor-specific translocation. Furthermore, approximately 15-20% of CML patients also carry a microdeletion on the derivative 9 chromosome (der(9)) and these patients have a poor prognosis. It has been hypothesised that der(9) deletion patients have increased levels of chromosomal instability. RESULTS: In this study aCGH was performed and identified a CNV (RP11-125A5, hereafter called CNV14q12) that was present as a genomic gain or loss in 10% of control DNA samples derived from cytogenetically normal individuals. CNV14q12 was the same clone identified by Iafrate et al. as a CNV. Real-time polymerase chain reaction (Q-PCR) was used to determine the relative frequency of this CNV in DNA from a series of 16 CML patients (both with and without a der(9) deletion) together with DNA derived from 36 paediatric solid tumors in comparison to the incidence of CNV in control DNA. CNV14q12 was present in approximately 50% of both tumor and CML DNA, but was found in 72% of CML bearing a der(9) microdeletion. Chi square analysis found a statistically significant difference (p <or= 0.001) between the incidence of this CNV in cancer and normal DNA and a slightly increased incidence in CML with deletions in comparison to those CML without a detectable deletion. CONCLUSION: The increased incidence of CNV14q12 in tumor samples suggests that either acquired or inherited genomic variation of this new class of variation may be associated with onset or progression of neoplasia.

Child↗

PubMed2006

Stem cell enrichment approaches.

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.

Animals↗

PubMed2006

Human telomere reverse transcriptase expression predicts progression and survival in pediatric intracranial ependymoma.

PURPOSE: Pediatric intracranial ependymomas are a heterogeneous group of neoplasms with unpredictable clinical and biologic behavior. As part of ongoing studies to identify potential biologic and therapeutic markers, we analyzed the role of human telomere reverse transcriptase (hTERT; the catalytic subunit of telomerase) expression as a prognostic marker for this disease. PATIENTS AND METHODS: Primary intracranial ependymomas that were resected at our institution between 1986 and 2004 were identified through the pathology and oncology databases. A tissue array was constructed from the patient samples and hTERT expression was evaluated by immunohistochemistry. Twenty-one samples were also analyzed for telomerase activity (telomerase repeat amplification protocol assay). RESULTS: Eighty-seven tumors from 65 patients were analyzed. Five-year progression-free survival was 57% (SEM, 12%) and 21% (SEM, 8%) for hTERT-negative and hTERT-positive tumors, respectively (P = .002). Five-year overall survival was 84% (SEM, 7%) and 41% (SEM, 7%) for hTERT-negative and hTERT-positive tumors, respectively (P = .001). There was good correlation between telomerase activity and hTERT expression (kappa = 0.637). Multivariate analysis revealed hTERT expression to be the single most important predictor of survival of all known pathologic, clinical, and treatment factors (hazard ratio, 60.4; 95% CI, 6.4 to 561). All four patients with hTERT-negative tumors at relapse are still alive, with median follow-up of 11.2 years. CONCLUSION: In this study, hTERT expression was the strongest predictor of outcome and was independent of other clinical and pathologic prognostic markers. It represents a simple and reliable biologic prognostic factor for intracranial ependymomas. These results should be confirmed in larger prospective trials.

Adolescent↗

PubMed2006

Interphase FISH analysis of PTEN in histologic sections shows genomic deletions in 68% of primary prostate cancer and 23% of high-grade prostatic intra-epithelial neoplasias.

Prostate cancer (CaP) is characterized by the accumulation of both genetic and epigenetic alterations that transform premalignant lesions to invasive carcinoma. However, the molecular events underlying this critical transition are poorly understood. One of the important genes that might play a role in CaP development is the PTEN gene. At the present time, there has been no systematic analysis of the incidence of genomic PTEN deletion by fluorescence in situ hybridization (FISH) in CaP and associated preneoplastic histologic lesions. This study assesses the frequency of PTEN deletion by interphase FISH analysis in CaP and prostatic intra-epithelial neoplasia (PIN). Dual-color FISH was performed using DNA probes for bands 10q23.3 (PTEN locus) and chromosome 10 centromere using 35 radical prostatectomy specimens. PTEN deletions were not found in 3/3 of stroma, 6/6 samples of benign glandular epithelium, and 12/12 samples of low-grade PIN. However, PTEN deletions were found in 3/13 (23%) of high-grade PIN and 24/35 (68%) of CaP. Concordance was observed between PTEN deletion status and the overall cellular PTEN protein expression levels, as assessed by immunohistochemistry. The high frequency of PTEN deletion observed in CaP versus precursor lesions implicates a pivotal role for PTEN haploinsufficiency in the transition from preneoplastic PIN to CaP. Moreover, this observation is an important consideration for novel therapeutic trials in CaP in which biologic efficacy is influenced by the activity level of PTEN. These findings draw attention to the usefulness of this relatively simple FISH assay for future applications in clinical laboratories.

Gene Deletion↗

PubMed2006

Use of chromogenic in situ hybridization to identify MYCN gene copy number in neuroblastoma using routine tissue sections.

Amplification of MYCN in neuroblastoma is associated with a poor prognosis. However, methods for estimating the number of MYCN genes based on pooled cells do not address copy number heterogeneity at the cell level and can underestimate or even miss amplification. MYCN copy number can be directly assessed by fluorescence in situ hybridization, but evaluation of tissue histology is next to impossible. We have used a chromogenic method for in situ hybridization (CISH) that enables determination of MYCN copy number using routine light microscopy on routinely processed paraffin sections. Of 41 cases studied, CISH identified 100% of the 18 cases that were determined to be amplified by other techniques and was more sensitive than Southern blotting or quantitative DNA polymerase chain reaction. Because the technique evaluates individual tumor cells, heterogeneity of MYCN copy number was apparent from cell to cell. When defined as 50% or greater variation in copy number between cells in amplified tumors, almost 30% of cases were scored as heterogeneous. Heterogeneity reflects different tumor clones and its role has likely been under-recognized and underestimated in neuroblastoma biology. CISH will provide a valuable tool to assess this phenomenon in conjunction with other morphologic parameters in neuroblastoma specimens, to further our understanding of the biology of this childhood tumor.

Blotting, Southern↗

PubMed2006

Pediatric renal cell carcinoma: clinical, pathologic, and molecular abnormalities associated with the members of the mit transcription factor family.

We describe the clinical features, outcome, pathology, cytogenetics, and molecular aspects of 13 pediatric papillary renal cell carcinomas during a 19-year period. Seven cases (54%) had translocations involving Xp11.2 (TFE3). They were identified by cytogenetic, molecular, and/or immunohistochemical analyses. All Xp11.2+ translocations were TFE3+ by immunostaining. Cytogenetic and/or polymerase chain reaction analyses identified 3 cases with t(X17) and 1 case with t(1;17), and all had additional translocations. Histologic features in common in TFE3+ tumors also were present in some TFE3- tumors. One TFE3- tumor had complex cytogenetic abnormalities, 55XY,+2,del(3)(p14),+7,+8,+12,+13,+16,+17,+20[11 ], and 2 cases had normal karyotypes. None had t(6;11)/TFEB+ immunostaining. Five cases had focal, weak MITF tumor immunostaining. The key clinical findings were as follows: (1) The presence of an Xp11.2 (TFE3) translocation frequently is associated with advanced stage at initial examination. (2) All patients who underwent complete, partial nephrectomy with clear margins (adequate only for stage 1) and resection of metastases were alive and relapse-free at last follow-up. (3) The mean +/- SD event-free survival and overall survival rates at 5 years were both 92% +/- 7.4%. (4) One patients with a TFE3+ and MITF+ tumor and 66-87,XXY,der(1)t(1;8)del(4)(q?) der(11)t(11;15)der17t(X;17 abnormalities died 9 months after diagnosis.

Adolescent↗

PubMed2006

The role of telomere maintenance in the spontaneous growth arrest of pediatric low-grade gliomas.

Spontaneous tumor regression of pediatric low-grade gliomas (PLGG). We speculated that lack of telomere maintenance is responsible for this behavior. We first looked for evidence of telomerase activity and alternative lengthening of telomeres (ALT) in 56 PLGG. Telomerase activity was observed in 0 of 11 PLGG, in contrast to 10 of 13 high-grade pediatric brain tumors. There was no ALT in 45 of 45 samples. We then applied Q-FISH to eight patients whose indolent PLGG underwent two metachronous biopsies over a lag of several years. Telomere shortening was observed in the second biopsy in all tumors, but not in normal brain control (P < .0001), indicating that lack of telomere maintenance is associated with continuous telomere erosion. Based on these observations, we found that younger PLGG patients, who exhibit more aggressive and frequently recurrent tumors, had significantly longer telomeres than older ones (P = .00014). Tumors with a terminal restriction fragment length <7.5 did not recur, whereas the presence of longer telomeres (>8.0) conferred a high likelihood of late recurrences in PLGG. Our findings provide a plausible biologic mechanism to explain the tendency of PLGG to exhibit growth arrest and spontaneous regression. Telomere maintenance may therefore represent the first known biologic prognostic marker in PLGG.

Astrocytoma↗

PubMed2006

Three-color FISH analysis of TMPRSS2/ERG fusions in prostate cancer indicates that genomic microdeletion of chromosome 21 is associated with rearrangement.

The recent description of novel recurrent gene fusions in approximately 80% of prostate cancer (PCa) cases has generated increased interest in the search for new translocations in other epithelial cancers and emphasizes the importance of understanding the origins and biologic implications of these genomic rearrangements. Analysis of 15 PCa cases by reverse transcription-polymerase chain reaction was used to detect six ERG-related gene fusion transcripts with TMPRSS2. No TMPRSS2/ETV1 chimeric fusion was detected in this series. Three-color fluorescence in situ hybridization confirms that TMPRSS2/ERG fusion may be accompanied by a small hemizygous sequence deletion on chromosome 21 between ERG and TMPRSS2 genes. Analysis of genomic architecture in the region of genomic rearrangement suggests that tracts of microhomology could facilitate TMPRSS2/ERG fusion events.

Chromosome Aberrations↗

PubMed2005

HER2 amplification and overexpression is not present in pediatric osteosarcoma: a tissue microarray study.

The HER2 gene, located on 17q, encodes a 185-kD transmembrane tyrosine kinase receptor. Amplification of this gene with overexpression of the gene product occurs in about 30% of cases of breast cancer and is considered to be a poor prognostic indicator for this tumor. Results for HER2 expression in osteosarcoma are controversial, with some studies reporting up to 61% of positive cases and others reporting only negative results. Further, expression of HER2 is reported to be a favorable prognostic indicator by some groups and unfavorable by others. The present study used tissue microarrays containing 34 samples of osteosarcoma from 18 patients to analyze HER2 expression by immunohistochemistry and gene copy number by chromogenic in situ hybridization. The microarray included 13 pretreatment biopsies, 11 posttreatment resection specimens, and 10 resected metastases and comprised 18 osteoblastic, 6 chondroblastic, 5 fibroblastic, and 5 mixed subtypes. HER2 protein expression was seen in 4 of 34 (12%) tumor samples that originated from 2 of 18 patients (11%). The staining pattern was consistently weak and focal, and immunohistochemical overexpression of the HER2 protein, defined as complete membrane positivity, was never observed. Further, the presence of HER2 gene amplification was not detected in any osteosarcoma by chromogenic in situ hybridization. Therefore, therapies based on antibodies directed against the HER2 protein are unlikely to have much value in the treatment of pediatric osteosarcomas. From a technical standpoint, this study also demonstrates the value of tissue microarrays in screening tumors at the protein and gene levels using conventional light microscopy.

Adolescent↗

PubMed2005

An integrated mBAND and submegabase resolution tiling set (SMRT) CGH array analysis of focal amplification, microdeletions, and ladder structures consistent with breakage-fusion-bridge cycle events in osteosarcoma.

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 genomic instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. However, until now, there have been no high-resolution cytogenetic or genomic array studies of BFB events in OS. In the present study, multicolor banding (mBAND) FISH and submegabase resolution tiling set (SMRT) array comparative genomic hybridization (CGH) were used to identify and map genomic signatures of BFB events in four OS cell lines and one patient tumor. The expected intermediates associated with BFB-dicentric chromosomes, inverted duplications, and intra- and interchromosomal amplifications-were identified. mBAND analysis provided detailed mapping of rearrangements in 1p, 6p, and 8q and showed that translocation junctions were often in close proximity to fragile sites. More detailed mBAND studies of OS cell line MG-63 revealed ladderlike FISH signals of equally spaced interchromosomal coamplifications of 6p21, 8q24, and 9p21-p22 in a homogeneously staining region (hsr). Focal amplifications that concordantly mapped to the hsr were localized to discrete genomic intervals by SMRT array CGH. The complex amplicon structure in this hsr suggests focal amplifications immediately adjacent to microdeletions. Moreover, the genomic regions in which there was deletion/amplification had a preponderance of fragile sites. In summary, this study has provided further support for the role of the BFB mechanism and fragile sites in facilitating gene amplification and chromosomal rearrangement in OS.

Cell Line, Tumor↗

PubMed2005

Imatinib mesylate: an attractive alternative in young children with large, surgically challenging dermatofibrosarcoma protuberans.

To document the clinical activity of imatinib mesyalte in a child with a dermatofibrosarcoma protuberans (DFSP). An 18-month-old girl presented with a large extremity DFSP. As surgical resection would have caused unacceptable functional defects, imatinib mesylate was administered to induce tumor reduction and or stabilization. After 23 weeks of therapy, magnetic resonance imaging (MRI) of the tumor showed a reduction in the subcutaneous thickness in the transverse plane. The drug was tolerated well without any adverse reactions. Imatinib mesylate offers a non-surgical alternative for the treatment of large DFSP in children.

Benzamides↗

PubMed2005

Clinical and molecular characteristics of pediatric gastrointestinal stromal tumors (GISTs).

BACKGROUND: To describe the clinical characteristics, molecular features, treatment, and outcome of six pediatric patients with gastrointestinal stromal tumors (GISTs). PATIENTS AND METHODS: Retrospective clinical review of GISTs, seen at The Hospital for Sick Children (HSC) Toronto, over an 11-year period. All specimens were stained for the CD 117 and CD 34 antigens. Three specimens were sequenced for mutations in exons 9, 11, and 13 of the c-kit gene. RESULTS: Five patients were evaluated and treated at HSC and one was referred for histopathological consultation only. The median patient age at diagnosis was 13.6 years, (6.9-14.8 years); four were female. All patients presented with anemia secondary to gastrointestinal (GI) bleeding. The disease was localized in five patients and two had other malignancies consistent with the diagnoses of Carney's triad. Immunohistochemical staining for CD 117 and CD 34 showed heavy cytoplasmic localization in all of the tumor cells. A novel point mutation of KIT in codon 456 of exon 9 was found in one case. Complete surgical resection was achieved in the five patients managed at our center and none received adjuvant therapies. Disease recurred locally in one patient. Four patients are alive and one is lost to follow-up. CONCLUSIONS: In children and adolescents, GISTs should be considered in the differential diagnosis of anemia secondary to GI hemorrhage. The absence of an exon 11 mutation and the identification of a novel mutation in exon 9 suggest that pediatric GISTs may respond differently to currently available targeted therapies and therefore should be studied within the context of collaborative group trials.

Adolescent↗

PubMed2005

Association of the t(12;22)(q13;q12) EWS/ATF1 rearrangement with polyphenotypic round cell sarcoma of bone: a case report.

The t(12;22)(q13;q12) chromosomal rearrangement results in an EWS/ATF1 fusion transcript and is associated with clear cell sarcoma (CCS). CCS is an uncommon tumor arising in tendons and aponeuroses of the extremities and shows evidence of melanocytic differentiation at the light microscopic, immunohistochemical, and/or ultrastructural level. Only 5 cases have been reported to arise in bone, none of which had molecular confirmation of the diagnosis. The current report describes a 7-year-old girl with a primary round cell sarcoma of the left humerus showing polyphenotypic differentiation on immunohistochemical analysis. Antibodies directed at melanocytic antigens were negative, and there was no evidence of melanocytic differentiation by light microscopy or ultrastructural analysis. Cytogenetic analysis revealed rearrangement of the EWS locus within 22q12. RT-PCR and sequence analysis revealed the presence of a fusion transcript bringing together exon 7 of EWS with exon 5 of ATF1, consistent with a type 2 transcript reported in association with CCS. However, given the lack of morphologic features usually present in CCS, a diagnosis of polyphenotypic round cell sarcoma was made. This tumor thus expands the spectrum of neoplasms associated with the t(12;22)(q13;q12) rearrangement.

Antineoplastic Combined Chemotherapy Protocols↗