Luanne M. Wainwright

Spectrum of SMARCB1/INI1 Mutations in Familial and Sporadic Rhabdoid Tumors

Germline mutations and deletions of SMARCB1/INI1 in chromosome band 22q11.2 predispose patients to rhabdoid tumor and schwannomatosis. Previous estimates suggested that 15–20% of rhabdoid tumors were caused by an underlying germline abnormality of SMARCB1. However, these studies were limited by case selection and an inability to detect intragenic deletions and duplications.

Germline INI1 mutation in a patient with a central nervous system atypical teratoid tumor and renal rhabdoid tumor

We describe a four‐month‐old child who presented with an atypical teratoid/rhabdoid tumor of the brain and subsequently developed a renal rhabdoid tumor. Distinct histologic features, immunophenotypic profiles, and deletions of chromosome 22 were supportive of two primary tumors. An identical mutation in exon 7 of the INI1 rhabdoid tumor suppressor gene was identified in both tumors, as well as in normal kidney tissue. We propose that this germline INI1 mutation predisposed the child to the development of both malignancies. These findings lend support to the hypothesis that rhabdoid tumors in all sites have a common genetic etiology. Genes Chromosomes Cancer 28:31–37, 2000.

High-density single nucleotide polymorphism array analysis in patients with germline deletions of 22q11.2 and malignant rhabdoid tumor

Malignant rhabdoid tumors are highly aggressive neoplasms found primarily in infants and young children. The majority of rhabdoid tumors arise as a result of homozygous inactivating deletions or mutations of the INI1 gene located in chromosome band 22q11.2. Germline mutations of INI1 predispose to the development of rhabdoid tumors of the brain, kidney and extra-renal tissues, consistent with its function as a tumor suppressor gene. We now describe five patients with germline deletions in chromosome band 22q11.2 that included the INI1 gene locus, leading to the development of rhabdoid tumors. Two patients had phenotypic findings that were suggestive but not diagnostic for DiGeorge/Velocardiofacial syndrome (DGS/VCFS). The other three infants had highly aggressive disease with multiple tumors at the time of presentation. The extent of the deletions was determined by fluorescence in situ hybridization and high-density oligonucleotide based single nucleotide polymorphism arrays. The deletions in the two patients with features of DGS/VCFS were distal to the region typically deleted in patients with this genetic disorder. The three infants with multiple primary tumors had smaller but overlapping deletions, primarily involving INI1. The data suggest that the mechanisms underlying the deletions in these patients may be similar to those that lead to DGS/VCFS, as they also appear to be mediated by related, low copy repeats (LCRs) in 22q11.2. These are the first reported cases in which an association has been established between recurrent, interstitial deletions mediated by LCRs in 22q11.2 and a predisposition to cancer.

ALK Expression in Rhabdomyosarcomas: Correlation with Histologic Subtype and Fusion Status

Immunohistochemical staining for anaplastic lymphoma kinase (ALK) has been described in rhabdomyosarcomas (RMS), especially the alveolar subtype. Previous studies have yielded conflicting results regarding the pattern of staining (nuclear versus cytoplasmic), and there has been no correlation with PAX3–7/FKHR fusion status. This study was undertaken to evaluate ALK receptor protein expression in a large series of RMS; to correlate these results with fusion status; and to investigate the possibility of 2p23 amplification or translocation using fluorescence in situ hybridization (FISH). Sixty-nine cases of RMS were examined and classified as alveolar RMS (ARMS), embryonal RMS (ERMS), or unclassifiable RMS (URMS) subtypes. Anaplastic lymphoma kinase immunohistochemistry was performed using anti-human CD246 antibody; cases were considered positive when more than 50% of cells had moderate or intense cytoplasmic and/or nuclear staining. There were 30 ARMS, 37 ERMS, and 2 URMS subtypes. Reverse transcription–polymerase chain reaction for PAX3/PAX7-FKHR fusion analysis had been done in all cases of ARMS, in 27 of 37 cases of ERMS, and in both URMS cases. Anaplastic lymphoma kinase staining was positive in 16 of 30 ARMS (53%) and 9 of 39 nonalveolar RMS (23%) cases (P < 0.05). Of the 21 ARMS cases with PAX3-FKHR fusion, 10 of 21 (48%) were positive for ALK staining; of the 6 ARMS cases with PAX7-FKHR fusion, 3 of 6 (50%) were positive for ALK staining; and 3 of 3 (100%) of the fusion-negative ARMS were positive with ALK staining. When comparing each of the ARMS subtypes, statistical significance was not reached. All positive cases showed dot-like cytoplasmic staining; nuclear staining was not seen. Of a subset of 6 ALK-positive ARMS submitted for break-apart FISH for the ALK locus, there was no evidence of a translocation; 1 case had ALK amplification and 2 had low-level gains of the ALK gene. We conclude that there is ALK overexpression in RMS, more commonly in ARMS than in ERMS, most likely independent of fusion status. Amplification or upregulation of ALK may underlie ALK protein overexpression.

No evidence for hypermethylation of the hSNF5/INI1 promoter in pediatric rhabdoid tumors

The hSNF5/INI1 gene on chromosome 22 has been implicated as a tumor suppressor gene in pediatric rhabdoid tumor, an aggressive malignancy that generally occurs in the first two years of life. The most common sites for tumor development are the brain and kidney. We and other investigators have identified deletions and mutations of the INI1 gene in the majority of rhabdoid tumors of the central nervous system, kidney, and extrarenal tissues. At least 20% of cases do not have genomic alterations of INI1, although expression at the RNA or protein level may be decreased. The aim of this study was to determine whether hypermethylation or mutation of the 5′ promoter region of INI1, or hypermethylation of CpG dinucleotides in a GC‐rich repeat region within the first intron, could account for the decreased expression of INI1 observed in these tumors. We employed bisulfite modification, polymerase chain reaction, and sequence analysis to determine the methylation status of the cytosine nucleotides in the predicted promoter region of the INI1 gene, and two GC repeat regions in intron 1. DNA from 24 tumors with or without coding‐sequence mutations was analyzed. None of the tumors demonstrated methylation of the promoter or intron 1 regions. This mechanism is unlikely to account for the inactivation of INI1 in rhabdoid tumors without coding‐sequence mutations. One tumor demonstrated a potential mutation in the promoter region, but further studies are required for determining its functional significance.

Clinical utilization of high-resolution single nucleotide polymorphism based oligonucleotide arrays in diagnostic studies of pediatric patients with solid tumors

High-resolution single nucleotide polymorphism (SNP) arrays have been effectively implemented as a first tier test in clinical cytogenetics laboratories for the detection of constitutional chromosomal abnormalities in patients with suspected genomic disorders. We recently published our experience utilizing SNP array analysis of bone marrow aspirates as a clinical test for patients with suspected leukemia or lymphoma in the Clinical Cancer Cytogenetics Laboratory at The Childrens Hospital of Philadelphia. In the present report we summarize our clinical experience using the Illumina HumanHap610 BeadChip array (Illumina, San Diego, CA) for whole genome analysis of pediatric solid tumors. A total of 168 DNA samples isolated from a variety of solid tumors, including brain tumors, sarcomas, neuroblastomas, and Wilms tumors, as well as benign neoplasms and reactive processes, were analyzed over a 2 1/2 year period. One hundred thirty-seven of 168 (82%) specimens had at least one copy number alteration or region of loss of heterozygosity detected by the SNP array. Thirty-three of 168 (20%) of cases had a normal karyotype or targeted fluorescence in situ hybridization (FISH) study, but had an abnormal finding by the array analysis. Sixty-three of 168 (37%) samples for which cytogenetic studies were unsuccessful or not performed demonstrated an abnormal array result. In 44 of 168 cases (26%) the array and karyotype or FISH were abnormal, but each demonstrated alterations not detected by the other methodology. Based on our experience in the last 2 1/2 years, we suggest that SNP array analysis can be used as a first tier clinical test for the majority of pediatric solid tumors.

Atypical teratoid/rhabdoid tumor in a patient with Beckwith–Wiedemann syndrome†

Beckwith–Wiedemann syndrome (BWS) is a genetic disorder associated with an increased risk of childhood tumors. Here we describe a patient with BWS who developed a central nervous system atypical teratoid/rhabdoid tumor (AT/RT). To our knowledge, despite the known cancer predisposition, this patient is the first described with BWS to develop an AT/RT. Due to the high propensity of these patients to develop childhood tumors, in addition to routine diagnostic tests, analysis of the tumor DNA using the Illumina Infinium whole‐genome genotyping 550K Beadchip was performed to investigate a possible common underlying mechanism for his BWS and AT/RT. The only alteration detected was monosomy 22, which was accompanied by a somatic mutation in the INI1 rhabdoid tumor gene. These results suggest that, despite an underlying cancer predisposition, the occurrence of BWS and AT/RT in this patient may be unrelated.

Copy number alterations determined by single nucleotide polymorphism array testing in the clinical laboratory are indicative of gene fusions in pediatric cancer patients

Gene fusions resulting from structural rearrangements are an established mechanism of tumorigenesis in pediatric cancer. In this clinical cohort, 1,350 single nucleotide polymorphism (SNP)‐based chromosomal microarrays from 1,211 pediatric cancer patients were evaluated for copy number alterations (CNAs) associated with gene fusions. Karyotype or fluorescence in situ hybridization studies were performed in 42% of the patients. Ten percent of the bone marrow or solid tumor specimens had SNP array‐associated CNAs suggestive of a gene fusion. Alterations involving ETV6, ABL1‐NUP214, EBF1‐PDGFRB, KMT2A(MLL), LMO2‐RAG, MYH11‐CBFB, NSD1‐NUP98, PBX1, STIL‐TAL1, ZNF384‐TCF3, P2RY8‐CRLF2, and RUNX1T1‐RUNX1 fusions were detected in the bone marrow samples. The most common alteration among the low‐grade gliomas was a 7q34 tandem duplication resulting in a KIAA1549‐BRAF fusion. Additional fusions identified in the pediatric brain tumors included FAM131B‐BRAF and RAF1‐QKI. COL1A1‐PDGFB, CRTC1‐MAML2, EWSR1, HEY1, PAX3‐ and PAX7‐FOXO1, and PLAG1 fusions were determined in a variety of solid tumors and a novel potential gene fusion, FGFR1‐USP6, was detected in an aneurysmal bone cyst. The identification of these gene fusions was instrumental in tumor diagnosis. In contrast to hematologic and solid tumors in adults that are predominantly driven by mutations, the majority of hematologic and solid tumors in children are characterized by CNAs and gene fusions. Chromosomal microarray analysis is therefore a robust platform to identify diagnostic and prognostic markers in the clinical setting.

Dramatic response of acute monoblastic leukemia to a single dose of docetaxel.

The development of secondary acute myeloid leukemia following chemotherapy and/or radiation therapy for other malignancies is well documented. Indeed, the World Health Organization has categorized these leukemias separately from the de novo cases, with emphasis upon those specifically related to alkylating agents and those related to topoisomerase II inhibitors [1]. Although the leukemias secondary to alkylating agents tend to arise 5 – 6 years following exposure to the toxic agent and are commonly preceded by a myelodysplastic syndrome, the leukemias induced by treatment with topoisomerase II inhibitors often present earlier (2 – 3 years after exposure) and without a preceding myelodysplastic syndrome. Although the former leukemias are associated with deletions of chromosome 5 and/or 7, the latter often carry translocations involving the MLL gene in chromosome band 11q23. Unfortunately, most secondary leukemias, especially those related to alkylating therapy, are notoriously refractory to treatment with short overall survival. To date, allogeneic bone marrow transplantation remains the only hope for long-term survival. Here, we present a case report of a patient with a dramatic response to unconventional therapy for a secondary myeloid leukemia with monoblastic morphology. The patient is a 52-year-old woman with a history of T2N2M0 breast cancer diagnosed and treated in 2005 with lumpectomy, breast radiation and adjuvant chemotherapy, who presented in March 2007 with pancytopenia (WBC 0.86 10/L with an ANC of 300 cells/mL, hemoglobin 8.3 g/dL, and platelet count 80,000/mL). The prior adjuvant chemotherapy for her breast cancer had consisted of a topoisomerase II inhibitor (Adriamycin 60 mg/m) and an alkylating agent (Cytoxan 600 mg/m) in combination for four cycles, followed by paclitaxel (175 mg/m) for four cycles. Subsequently, she was receiving anastrazole (1 mg/day). A bone marrow biopsy was performed at presentation at an outside hospital in March 2007. With an immunohistochemical analysis pending, a preliminary morphologic diagnosis of extensive bone marrow involvement by metastatic breast cancer was rendered, and the patient was treated with a single dose of docetaxel (75 mg/m) and then transferred to the Hospital of the University of Pennsylvania (HUP) for further treatment of her presumed metastatic disease. However, the results of an initial limited immunohistochemical analysis of the neoplasm revealed thatthe tumor was negative for an epithelial cell marker AE1/3 and positive for a hematopoietic cell antigen CD45, a phenotype incompatible with a carcinoma. The bone marrow biopsy H&E stained slides reviewed at HUP demonstrated marked