Xianglan Lu

Automated Analysis of Fluorescent in situ Hybridization (FISH) Labeled Genetic Biomarkers in Assisting Cervical Cancer Diagnosis

The numerical and/or structural deviation of some chromosomes (i.e., monosomy and polysomy of chromosomes 3 and X) are routinely used as positive genetic biomarkers to diagnose cervical cancer and predict the disease progression. Among the available diagnostic methods to analyze the aneusomy of chromosomes 3 and X, fluorescence in situ hybridization (FISH) technology has demonstrated significant advantages in assisting clinicians to more accurately detect and diagnose cervical carcinoma at an early stage, in particular for the women at a high risk for progression of low-grade and high-grade squamous intraepithelium lesions (LSIL and HSIL). In order to increase the diagnostic accuracy, consistency, and efficiency from that of manual FISH analysis, this study aims to develop and test an automated FISH analysis method that includes a two-stage scheme. In the first stage, an interactive multiple-threshold algorithm is utilized to segment potential interphase nuclei candidates distributed in different intensity levels and a rule-based classifier is implemented to identify analyzable interphase cells. In the second stage, FISH labeled biomarker spots of chromosomes 3 and X are segmented by a top-hat transform. The independent FISH spots are then detected by a knowledge-based classifier, which enables recognition of the splitting and stringy FISH signals. Finally, the ratio of abnormal interphase cells with numerical changes of chromosomes 3 and X is calculated to detect positive cases. The experimental results of four test cases showed high agreement of FISH analysis results between the automated scheme and the cytogeneticists analysis including 92.7% to 98.7% agreement in cell segmentation and 4.4% to 11.0% difference in cell classification. This preliminary study demonstrates the feasibility of potentially applying the automatic FISH analysis method to expedite the screening and detecting cervical cancer at an early stage.

16p13.3 duplication associated with non-syndromic pierre robin sequence with incomplete penetrance

BackgroundPierre Robin sequence (PRS) is a condition present at birth. It is characterized by micrognathia, cleft palate, upper airway obstruction, and feeding problems. Multiple etiologies including genetic defects have been documented in patients with syndromic, non-syndromic, and isolated PRS.Case presentationWe report a 4-year-old boy with a complex small supernumerary marker chromosome (sSMC) who had non-syndromic Pierre Robin sequence (PRS). The complex marker chromosome, der(14)t(14;16)(q11.2;p13.13), was initially identified by routine chromosomal analysis and subsequently characterized by array-comparative genomic hybridization (array CGH) and confirmed by fluorescence in situ hybridization (FISH). Clinical manifestations included micrognathia, U-type cleft palate, bilateral congenital ptosis, upslanted and small eyes, bilateral inguinal hernias, umbilical hernia, bilateral clubfoot, and short fingers and toes. To our best knowledge, this was the first case diagnosed with non-syndromic PRS associated with a complex sSMC, which involved a 3.8 Mb gain in the 14q11.2 region and an 11.8 Mb gain in the 16p13.13-pter region.ConclusionsWe suggest that the duplicated chromosome segment 16p13.3 possibly may be responsible for the phenotypes of our case and also may be a candidate locus of non-syndromic PRS. The duplicated CREBBP gene within chromosome 16p13.3 is associated with incomplete penetrance regarding the mandible development anomalies. Further studies of similar cases are needed to support our findings.

Deletion of 14q24.1∼q24.3 in a patient with acute lymphoblastic leukemia: a hidden chromosomal anomaly detected by array-based comparative genomic hybridization

A 4-year-old patient, who was newly diagnosed with acute lymphoblastic leukemia (ALL), was referred to us for cytogenetic evaluation. He had a normal karyotype by G-banded chromosome analysis, and a deletion of the ETV6 (alias TEL) gene was determined by fluorescence in situ hybridization analysis using DNA probes specific for t(12;21), which leads to ETV6/RUNX1 (alias TEL/AML1) gene fusion, but no translocation was found between the two genes. To find out if there were possibly additional subtle chromosomal changes undetectable by routine cytogenetics, high-density 385K oligo array comparative genomic hybridization (CGH) assay was performed. Besides the confirmation of the 12p deletion, a deletion of a 12-megabase (Mb) chromosomal segment on 14q24.1 approximately q24.3 was also detected. Within the deleted 12-Mb region, there were a total of 155 genes and at least 28 of these were cancer-related genes. A similar approach by array CGH in patients with ALL, especially in those patients with 12p deletion, could help to determine the significance of this rare event.

Acquired genomic copy number changes in CML patients with the Philadelphia chromosome (Ph

Chronic myeloid leukemia (CML) is characterized by the BCR-ABL1 fusion gene; this fusion gene is usually a consequence of the Philadelphia (Ph(+)) chromosome, which results from the t(9;22)(q34;q11.2). Patients newly diagnosed with CML are routinely treated with tyrosine kinase inhibitors; however, the clinical course of the disease can vary, and this variance may be associated with genetic heterogeneity. Array comparative genomic hybridization (CGH) technology is a powerful tool for identifying subtle genomic segmental alterations, which can result from either losses or gains of chromosomal material. These changes may reveal the presence of genes that play important roles in disease initiation or progression or in treatment outcomes. To investigate whether subtle somatic copy number changes (CNCs) are commonly present in CML patients, a pilot study of 19 patients with the Ph(+) chromosome, but who were negative for common secondary chromosomal anomalies [+der(22), +8, i(17q), and +19], was conducted using a high-density whole genomic oligonucleotide array CGH analysis. Four of the 19 cases had somatic segmental CNCs, including the loss of 9q34, 15q25.3, and 15q13 and a gain of 7p21.1-p15.3. The findings demonstrate that subtle genomic changes are relatively common in CML patients with a Ph(+) chromosome and that the clinical significance of these findings, especially the newly discovered regions, must be determined in large patient population studies.

Characterization of a novel t(2;5;11) in a patient with concurrent AML and CLL: a case report and literature review.

Acute myeloid leukemia (AML) that occurs concurrent with a diagnosis of chronic lymphocytic leukemia (CLL) is rare, but the number of cases recognized has recently dramatically increased as a result of the application of flow cytometry. This raises a series of questions regarding the clinical characterization of mixed leukemia, whether this diagnosis possesses unique cytogenetic abnormalities, and the possible association between AML and CLL cell clones. The current study attempts to answer these questions by evaluating an 80-year-old man with concurrent diagnoses of AML-M0 and CLL. Routine G-banded chromosome, array based comparative genomic hybridization, and fluorescence in situ hybridization analyses were used to characterize complex chromosomal rearrangements in the patients bone marrow. Novel complex translocations involving chromosomes 2, 5, and 11, as well as submicroscopic deletions and duplications, were revealed. This case study reports a t(2;5;11) in either AML-M0 or in CLL by using array based comparative genomic hybridization and fluorescence in situ hybridization analyses to facilitate the diagnosis. The study also delineates the clinical characteristics and cytogenetic changes that occur with concurrent AML and CLL.

A novel subtelomeric translocation t(5;9) and a deletion of the RB1 gene in a patient with acute myeloid leukemia (AML-M0)

No chromosomal rearrangements have been identified as specifically associated with minimally differentiated acute myeloid leukemia (AML-M0). Several research groups studied the cytogenetic features of AML-M0 and found that as much as 81% of patients with AML-M0 had chromosomal rearrangements; primarily -5/5q- and/or -7/7q- deletions or translocations involving 12p. A patient, who was diagnosed with AML-M0 eighteen months ago, was referred for cytogenetic evaluation for possible AML relapse. A subtle, cryptic t(5;9)(q35.3;q34.3), plus a deletion of the RB1 gene were detected in 18 out of 20 cells analyzed by FISH utilizing the TelVysion assay kit. To rule out the possibility that these chromosomal changes were related to the relapse of AML in this case, we repeated the same FISH test on the specimen at initial diagnosis before any treatment. The same abnormalities were found. To our knowledge, this is the first case reported with subtelomeric t(5;9)(q35.3;q34.3) and the deletion of the RB1 gene in a patient with AML-M0. Whether the t(5;9) combined with the deletion of the RB1 gene plays an important role in the development of AML-M0 warrants further investigation.

Coexistence of t(2;14;11)(p16.1;q32;q23) and t(14;19)(q32;q13.3) chromosome translocations in a patient with chronic lymphocytic leukemia: A case report

Rationale: With combination of multiple techniques, we have successfully characterized unique, complex chromosomal changes in a patient with chronic lymphocytic leukemia (CLL), a lymphoproliferative disorder. Diagnoses: The diagnosis was based on white blood cell, flow cytometry, and immunophenotypes and confirmed by karyotype, fluorescence in situ hybridization, and array comparative genomic hybridization from the patients blood culture. Interventions: The patient was given fludarabine, cyclophosphamide and rituximab (FCR) for 6 cycles. Outcomes: After completion of 6 cycles of FCR, the computed tomography scans of the neck/chest/abdomen/pelvic showed that the patient in CR. During the 10-month follow-up, the patients clinical course remained uneventful. Lessons: The translocation t(14;19) identified in this patient is a recurrent translocation found in patients with chronic B-cell lymphoproliferative disorders and the 3-way translocation involving chromosomes 2, 14, and 11 may play a role as an enhancer.

Clinical report: variable phenotypic expression in a large sibling cohort with a deletion of 4p16.1

We report a half‐sibling cohort with deletion of 4p16.1, astigmatism, gross and fine motor delay, variable intellectual disability, and variable behavioral concerns. However, two siblings without the deletion also had learning delays and psychological concerns. Thus, variable phenotypic expression was seen and the significance of deletion of 4p16.1 remains unclear.

Two patients with small chromosome 22q11.21 alterations and central nervous system abnormalities

BackgroundCentral nervous system features have been rarely described to be associated with the small deletion or duplication of chromosome 22q11.21.Case presentationWe report two patients with chromosome 22q11.21 alterations and central nervous system abnormalities. Features described include semilobar holoprosencephaly in the patient with the small deletion and Chiari I malformation in the patient with the small duplication.ConclusionsThis report will aid in the characterization of the clinical significance of interstitial duplications and deletions on the long-arm of chromosome 22. Areas of future research would benefit from additional analysis of the described regions with inclusion of the phenotypic findings described in this case report to provide additional insight into the pathogenicity of the described alterations.

Abstract 3913: Establishing a genomic profile of four gastric carcinoma cell lines using array CGH, FISH and cytogenetic analyses

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Four gastric carcinoma cell lines (CRL-1739TM, CRL-5822TM, CRL-5971TM and CRL-5973TM), established in the 1970s and 1980s, were purchased from ATCC. These cell lines have been distributed throughout the world and are regularly used for biomedical research. Cytogenetic information is available for three of these cell lines, and two of the three are known to have complex chromosomal rearrangements, though these have not been well characterized. In part, this is due to the complexity of chromosomal changes and the limited ability of G-banded chromosome analysis. Establishing a genomic profile of these cell lines is vitally important, so that their genotypes and phenotypes can be compared. In the current study, G-banded chromosome analysis, array CGH and FISH analyses were performed on these 4 cell lines. The G-banded chromosome analysis was performed and followed by array CGH using 720k oligonucleotide based array chip. The imbalanced regions and chromosome rearrangements, detected by G-banded chromosome and array CGH analyses, were confirmed by FISH using the appropriate DNA probes. Based on the combined data generated by these assays, we established genomic profiles of each cell line; deletions, duplications, translocations and insertions and a chromosomal homogenous staining region. Deletions of 3p and 18q, duplication of 8q, 19q and extra chromosome 20 were found to be common. These findings are consistent with array CGH analyses of fresh gastric tumor samples (data to be presented separately). The results of this study are important as they will provide important information on the status of genomic profiles, allow the expression patterns of gene(s) to be compared, and provide a mechanism for monitoring the genomic changes in these cell lines. This information, we believe, will allow laboratories to make more informed decisions about the need for replacing existing cell lines due to changes in their biological characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3913. doi:10.1158/1538-7445.AM2011-3913