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Cancer Cytogenetics Laboratory

Diagnostic Test Information

Cytogenetic analysis and preparation of karyotypes

The identification of cytogenetic abnormalities in a bone marrow specimen or tissue sample often plays a central role in the diagnosis and determination of prognosis of a child with cancer. For hematologic disorders, analysis of successive bone marrow or blood samples over time may also be useful for monitoring response to treatment. The types of disorders typically seen in the laboratory include:

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The identification of specific chromosome abnormalities may help classify a malignancy. For example, the t(9;22)(q34;q11.2), also referred to as the Philadelphia (Ph¹) chromosome, is usually indicative of chronic myelogenous leukemia (CML). A cytogenetically identical translocation is also seen in a small percentage of patients with acute lymphocytic leukemia, which is associated with a very poor prognosis. A t(8;21)(q22;q22) defines a subset of patients with acute myelogenous leukemia (AML-M2), whereas a t(8;14)(q24.1;q32) is typically associated with Burkitt’s leukemia/lymphoma. Cytogenetic studies are also used to monitor patients with hematologic disorders and may identify early disease progression, such as the onset of blast crisis in CML, which is often characterized by trisomy 8, isochromosome 17q, and multiple Ph¹ chromosomes. Among the common pediatric solid tumors, a translocation of the Ewings sarcoma (EWS) region in chromosome band 22q12 is characteristic of Ewings sarcoma or peripheral neuroectodermal tumor, whereas a t(2;13) or t(1;13) is diagnostic for alveolar rhabdomyosarcoma. Amplification of the MYCN or MYC oncogene is a poor prognostic feature in neuroblastoma or central nervous system medulloblastoma.


Direct harvest and unstimulated cultures of bone marrow or peripheral blood are prepared to provide metaphases for G-banded analysis. For solid tumors, a direct harvest and cultures of disaggregated tumor tissue are prepared. Cells are cultured until there is sufficient growth to yield metaphase spreads for chromosome analysis and preparation of karyotypes. Analysis of at least 20 metaphases and 4 karyotypes is performed on each patient. Clonal abnormalities are defined as two or more cells having the same gain or structural rearrangement of a chromosome, or loss of a chromosome in at least 3 cells. Abnormalities are characterized according to the International System of Cytogenetic Nomenclature, 2009.


To insure the best interpretation, it is important to provide a preliminary diagnosis and indication for study. On occasion, chromosome studies are unsuccessful. If clinical information has been provided, we may reflex to an interphase FISH assay.

A variety of factors may result in the inability to identify an abnormal clone including:



The following factors are important when interpreting the results

Fluorescence In Situ Hybridization (FISH)

Fluorescence in situ hybridization (FISH) is an important diagnostic tool in cancer cytogenetics. With current FISH techniques, chromosomal deletions, rearrangements or gene amplification can be detected in either metaphase or interphase cells. Complex rearrangements can often be fully characterized using combinations of probes labeled in different colors. We can also monitor the success or failure of a bone marrow transplant if the donor is of the opposite sex, and the remission or relapse of a leukemic clone once an abnormality has been detected. For solid tumors fresh tissue is frequently unavailable or biopsy samples are too small to provide adequate fresh tissue for culture. FISH analysis provides a useful alternative as it can be performed on snap frozen tissue or on formalin fixed paraffin embedded tissue sections.

Available FISH assays

Single Nucleotide Polymorphism Array (SNP Array)

The single nucleotide polymorphism based oligonucleotide array that is currently validated for use in the diagnostic laboratory is the Illumina 610K bead array. Higher resolution platforms are currently in development. This high-resolution platform allows for greater detection of copy number changes (including deletion, single copy gain and amplification) than can be detected at the chromosomal level. The major advantage of a SNP array is that it can also reveal copy number neutral loss of heterozygosity. FISH or MLPA is most useful for alterations that encompass single genes as these assays can be targeted to a specific locus based on the preliminary diagnosis. The arrays are not suitable for ruling out balanced translocations or inversions. DNA is isolated from fresh or frozen tissue, blood or bone marrow and processed by the CHOP Center for Applied Genomics. Interpretation of the resulting data by the laboratory, along with the results of the karyotype and FISH analysis, yields a comprehensive view of the complex changes in the neoplasm. The SNP arrays may reveal underlying constitutional changes that may or may not be related to the malignancy. This may warrant further testing of a peripheral blood sample, and referral to a clinical geneticist for further evaluation and counseling.

INI1/SMARCB1 Deletion and Mutation Analysis

Deletions and mutations of the INI1/SMARCB1/hSNF5/BAF47 locus in chromosome band 22q11.2 have been demonstrated in rhabdoid tumors of the kidney, CNS, and extra-renal sites. INI1 is a member of the ATP-dependent SWI/SNF chromatin-remodeling complex. Although the specific function of INI1 in rhabdoid tumor development is unknown, it is hypothesized that INI1 exerts its tumor-suppressor function by modulating the transcription of a variety of cellular genes. The types of deletions and the location of mutations within the coding sequence for renal, extra-renal, and CNS rhabdoid tumors may ultimately yield clues regarding the function of INI1 in the development of rhabdoid tumors in young children. The laboratory uses a variety of approaches to detect these abnormalities.

Fresh or frozen tumor tissue is required for DNA isolation and analysis. If frozen tissue is not available formalin fixed tissue may be submitted, however the success rate will be lower. Approximately 30% of children with rhabdoid tumors may have an underlying (germline) deletion or mutation of the INI1 gene that predisposed them to cancer. Peripheral blood samples are therefore requested to rule out any abnormality that is identified in the tumor tissue. Genetic testing of the parents is also recommended to determine if a germline mutation or deletion is inherited or de novo. Blood from both parents is requested at the time of submission to provide a more rapid turn-around time. Three to 5 cc of blood should be collected in both a purple (EDTA) and green (sodium heparin) tube. Blood should be kept at room temperature and not frozen. A lymphoblastoid cell line will be set up from the green top, and DNA will be isolated from the purple top.

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After DNA is isolated from the tumor and blood, polymerase chain reaction (PCR) analysis and DNA sequencing is performed. The DNA sequence analysis will reveal any mutations within the nine coding exons of the INI1 gene. If a mutation is found in the tumor DNA, the blood will be screened. If the mutation is found to be germline (present in the blood), the parents will be screened to determine if the germline mutation is inherited or de novo. 

Representative sequencing output for mutation detection:


If a mutation is inherited from one of the parents, additional family members can be screened.

If needed, RNA can be extracted from frozen tissue for reverse transcription PCR (RT-PCR). The cDNA will then be sequenced and analyzed for mutations. cDNA based analysis is particularly useful for determining the possible effects of splice site mutations, and to determine whether the gene is expressed at the mRNA level.  

In addition to the sequence analysis, a determination of gene copy number is completed. This may reveal the presence of a deletion that affects one or both copies of all or part of the gene. We utilize a Multiplex Ligation-dependent Probe Amplification (MLPA) assay using the SMARCB1 kit from MRC-Holland to detect the copy number of the nine exons of the INI1 gene, as well as probes proximal and distal to INI1 on chromosome 22. Duplications may also be detected within the gene. The MLPA assay cannot detect copy number neutral loss of heterozygosity (CN LOH). If necessary, SNP array analysis is performed. This array can confirm a heterozygous or homozygous deletion, and will also reveal CN LOH of the INI1 region. The MLPA assay and the SNP array are not available for formalin fixed samples.

Representative results from the SNP array demonstrating a homozygous deletion that includes the INI1 rhabdoid tumor gene in chromosome band 22q11.23.

In addition to the SNP array and MLPA, fluorescence in situ hybridization (FISH) is performed on frozen and paraffin samples to determine copy number of the INI1 gene. Deletions may be present in one (heterozygous) or both (homozygous) copies of chromosome 22. 

Turn around time is typically 3 weeks, although may be longer if additional family members need to be tested.

BRAF Analysis

Fusions of the 5’ region of KIAA1549 with the distal region of BRAF have been identified in approximately 80% of pilocytic astrocytomas. Additionally, a specific mutation in exon 15 of BRAF has been detected in 50% of gangliogliomas, as well as a variety of other grade II gliomas. These abnormalities result in constitutive activation of BRAF, which is a member of the RAF family of serine/threonine protein kinases and is a key intermediary in the RAS-RAF-MEK-ERK-MAP kinase signaling pathway. This pathway is implicated in a wide variety of cellular functions, including cell proliferation, cell-cycle arrest, terminal differentiation, and apoptosis. The laboratory uses a variety of approaches to detect these abnormalities.

Fresh or frozen tumor tissue is required for DNA and RNA isolation and analysis. For suspected pilocytic astrocytomas, RNA is isolated from the tumor and reverse transcription polymerase chain reaction (RT-PCR) for the KIAA1549-BRAF fusion is performed. This testing will detect all five fusion variants that have been described. For suspected gangliogliomas DNA is isolated from the tumor and PCR analysis and DNA sequencing of exons 11 and 15 of BRAF is performed. The DNA sequence analysis will reveal any mutations within these two exons. If no fusion product is detected in a pilocytic astrocytoma, DNA will be isolated and screening for BRAF mutations will be performed; if no BRAF mutation is detected in a ganglioglioma, RNA will be isolated and screening for a KIAA1549-BRAF fusion will be performed. In the event that no BRAF abnormality is detected a SNP array analysis is available as a reflex test.


  1. Biegel JA, Zhou J-Y, Rorke LB, Stenstrom C, Wainwright LM, Fogelgren B. Germline and acquired mutations of INI1 in atypical teratoid and rhabdoid tumors. Cancer Res. 59:74-79, 1999. Read the abstract »
  2. Jackson EM, Shaikh TH, Gururangan S, Jones MC, Malkin D, Nikkel SM, Zuppan CW, Wainwright LM, Zhang F, Biegel JA. High-density single nucleotide polymorphism array analysis in patients with germline deletions of 22q11.2 and malignant rhabdoid tumor, Human Genet. DOI 10.1007/s00439-007-0386-3, 2007. Read the abstract »
  3. Janson K, Nedzi LA, David O, Schorin M, Walsh JW, Battacharjee M, Pridjian G, Tan L, Judkins AR, Biegel, JA. Predisposition to atypical teratoid/rhabdoid tumor due to an inherited hSNF5/INI1mutation. Ped Blood & Cancer. Oct 2005, epub ahead of print. Read the abstract »
  4. Roberts CW, Biegel JA. The role of SMARCB1/INI1 in development of rhabdoid tumor. Cancer Biology & Therapy. 8:412-414, 2009.
  5. Jackson EM, Sievert AJ, Gai X, Hakonarson H, Judkins AR, Tooke L, Perin JC, Xie H, Shaikh TH, Biegel JA. Genomic analysis using high-density single nucleotide polymorphism-based oligonucleotide arrays and multiplex ligation-dependent probe amplification provides a comprehensive analysis of INI1/SMARCB1 in malignant rhabdoid tumors. Clin Cancer Res. 2009 Mar 10. [Epub ahead of print] Read the abstract »
  6. Sievert AJ, Jackson EM, Gai X, Hakonarson H, Judkins AR, Resnick AC, Sutton LN, Storm PB, Shaikh TH, Biegel JA. Duplication of 7q34 in pediatric low-grade astrocytomas detected by high-density single-nucleotide polymorphism-based genotype arrays results in a novel BRAF fusion gene. Brain Pathol. 2009 Jul;19(3):449-58. Epub 2008 Oct 21. Read the article »
  7. Dougherty MJ, Santi M, Brose MS, Ma C, Resnick AC, Sievert AJ, Storm PB, Biegel JA. Activating mutations in BRAF characterize a spectrum of pediatric low-grade gliomas. Neuro Oncol. 2010 Feb 14. [Epub ahead of print] Read the abstract »



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