Section E6.5-6.8 of the ACMG technical standards and guidelines: chromosome studies of lymph node and solid tumor-acquired chromosomal abnormalities.

DISCLAIMER: These ACMG standards and guidelines are developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines is voluntary and does not necessarily ensure a successful medical outcome. These standards and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these standards and guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Cytogenetic analysis of tumor tissue is performed to detect and characterize chromosomal aberrations to aid histopathological and clinical diagnosis and patient management. At the time of diagnosis, known recurrent clonal aberrations may facilitate histopathological diagnosis and subtyping of the tumor. This information may contribute to clinical therapeutic decisions. However, even when tumors have a known recurrent clonal aberration, each tumor is genetically unique and probably heterogeneous. It is important to discover as much about the genetics of a tumor at diagnosis as is possible with the methods available for study of the tumor material. The information gathered at initial study will inform follow-up studies, whether for residual disease detection, determination of relapse and clonal evolution, or identifying a new disease clone.This updated Section E6.5-6.8 has been incorporated into and supersedes the previous Sections E6.4 and E6.5 in Section E: Clinical Cytogenetics of the 2009 Edition (Revised 01/2010), American College of Medical Genetics and Genomics Standards and Guidelines for Clinical Genetics Laboratories. This section deals specifically with the standards and guidelines applicable to lymph node and solid tumor chromosome analysis.Genet Med 18 6, 643-648.

Breast Cancer and Non-Hodgkin Lymphoma in a Young Male with Cowden Syndrome.

Male breast cancer (MBC) is unusual, especially in young adults. Most cases of MBC as a secondary malignancy relate to the previous treatment with ionizing radiation. MBC can be associated with mutations in hereditary cancer predisposition syndrome genes (i.e., BRCA2); however, no such association has been reported in patients with Cowden syndrome (involving the phosphatase and tensin homolog [PTEN] gene). We describe a patient with Cowden syndrome who was initially diagnosed with B-cell lymphoblastic lymphoma at the age of 7 years, then MBC at the age of 31 years, and never received radiation therapy.

Impact of persistent minimal residual disease post-consolidation therapy in children and adolescents with advanced Burkitt leukaemia: a Children's Oncology Group Pilot Study Report.

Patient-specific primers from 10 children/adolescents with Burkitt leukaemia (BL) ± central nervous system disease who were treated with French-British-American/Lymphome Malins de Burkitt 96 C1 plus rituximab were developed from diagnostic blood/bone marrow. Minimal residual disease (MRD) was assessed by real-time polymerase chain reaction at the end of induction (EOI) and consolidation (EOC). Seventy per cent (7/10) and 71% (5/7) were MRD-positive at EOI and EOC, respectively, with no disease recurrences. MRD after induction and consolidation did not predict relapse and subsequent therapy appeared to eliminate MRD. Thus, assessing MRD at a later time point is warranted in future trials to determine its clinical significance.

Aplastic Anemia in Two Patients with Sex Chromosome Aneuploidies.

Sex chromosome aneuploidies range in incidence from rather common to exceedingly rare and have a variable phenotype. We report 2 patients with sex chromosome aneuploidies who developed severe aplastic anemia requiring treatment. The first patient had tetrasomy X (48,XXXX) and presented at 9 years of age, and the second patient had trisomy X (47,XXX) and presented at 5 years of age. Although aplastic anemia has been associated with other chromosomal abnormalities, sex chromosome abnormalities have not been traditionally considered a risk factor for this condition. A review of the literature reveals that at least one other patient with a sex chromosome aneuploidy (45,X) has suffered from aplastic anemia and that other autosomal chromosomal anomalies have been described. Despite the uncommon nature of each condition, it is possible that the apparent association is coincidental. A better understanding of the genetic causes of aplastic anemia remains important.

Assessing the utility of confirmatory studies following identification of large-scale genomic imbalances by microarray.

PURPOSE: The identification of clinically relevant genomic dosage anomalies assists in accurate diagnosis, prognosis, and medical management of affected individuals. Technological advancements within the field, such as the advent of microarray, have markedly increased the resolution of detection; however, clinical laboratories have maintained conventional techniques for confirmation of genomic imbalances identified by microarray to ensure diagnostic accuracy. In recent years the utility of this confirmatory testing of large-scale aberrations has been questioned but has not been scientifically addressed. METHODS: We retrospectively reviewed 519 laboratory cases with genomic imbalances meeting reportable criteria by microarray and subsequently confirmed with a second technology, primarily fluorescence in situ hybridization. RESULTS: All genomic imbalances meeting reportable criteria detected by microarray were confirmed with a second technology. Microarray analysis generated no false-positive results. CONCLUSION: Confirmatory testing of large-scale genomic imbalances (deletion of ≥150 kb, duplication of ≥500 kb) solely for the purpose of microarray verification may be unwarranted. In some cases, however, adjunct testing is necessary to overcome limitations inherent to microarray. A recommended clinical strategy for adjunct testing following identified genomic imbalances using microarray is detailed.

Early onset, EBV(-) PTLD in pediatric liver-small bowel transplantation recipients: a spectrum of plasma cell neoplasms with favorable prognosis.

EBV(-) posttransplantation lymphoproliferative disorders (PTLDs) are rare compared with EBV(+) PTLDs, occur later after transplantation, and have a poor response to treatment. Few studies have reported EBV(-) PTLD in pediatric solid-organ transplantation recipients. We describe 5 cases of EBV(-) PTLD in recipients of combined liver and small bowel allografts ranging in age from 16 months to 7 years. EBV(-) PTLD developed 9-22 months (median, 15) after transplantation. Morphologically, the lesions ranged from atypical plasma cell hyperplasia (a term not currently included in the World Health Organization classification) to plasmacytoma like. In all cases, in situ hybridization for EBV was negative, and molecular studies demonstrated clonal IgH gene rearrangements. Protein electrophoresis showed multiple clonal paraproteins in 4 of 5 cases. In 2 cases with a donor-recipient sex mismatch, FISH cytogenetics demonstrated that the plasma cells were of mixed donor/recipient origin. One patient died before therapy. Four patients were treated with high-dose dexamethasone, and 1 patient subsequently required thalidomide. All 4 remain in remission 75-128 months (median, 86) after diagnosis. In contrast to reports of EBV(-) PTLD in adults, these plasma cell lesions occurred early after transplantation and resolved completely after minimal treatment.

Rituximab with chemotherapy in children and adolescents with central nervous system and/or bone marrow-positive Burkitt lymphoma/leukaemia: a Children's Oncology Group Report.

Children and adolescents with Burkitt Lymphoma (BL) and combined central nervous system (CNS) and bone marrow involvement still have a poor prognosis with chemotherapy alone. We therefore investigated in children and adolescents with bone marrow (≥25% blasts) and/or CNS-positive Burkitt lymphoma the chemoimmunotherapy combination of rituximab (375 mg/m(2) ) and the standard chemotherapy arm of our previously reported French-American-British (FAB) Lymphome Malins de Burkitt (LMB) 96 trial. Central pathological and cytogenetic characterization was also performed. There were 40 evaluable patients with Burkitt histology (25 with leukaemia and 15 with CNS disease ± leukaemia). The chemoimmunotherapy regimen was well tolerated. The incidence of grade III/IV mucositis during induction cycles with combined chemotherapy and rituximab was 31% and 26%, respectively. The 3-year event-free survival (EFS)/overall survival (OS) was 90% (95% confidence interval [CI], 76-96%) in the entire cohort and 93% (95% CI, 61-99%) in patients with CNS disease. Based on the results of this trial, an international randomized study of FAB/LMB 96 chemotherapy ± rituximab for high-risk patients is currently under investigation.

Molecular distinctions between pediatric and adult mature B-cell non-Hodgkin lymphomas identified through genomic profiling.

Burkitt lymphoma (BL) predominates in pediatric patients, whereas diffuse large B-cell lymphoma (DLBCL) is uncommon. In contrast to adults, BL and DLBCL are treated similarly in children and both entities have superior outcomes in children compared with adults. Gene expression profiling (GEP) and miRNA expression profiling clearly differentiated pediatric DLBCL from BL, forming distinct clusters regardless of patient age. However, pathway analysis of GEP data identified minor differences between corresponding pediatric and adult tumors. Predominance (6:1) of the germinal center B-cell subtype to activated B-cell subtype was found among pediatric DLBCL. Two cases were molecularly classified as primary mediastinal B-cell lymphoma. We observed frequent abnormalities in 8q24 in pediatric DLBCL, including MYC rearrangement in 31% (5 of 16) and gain or amplification in 50% (6 of 12) nonrearranged cases. MYC rearrangement was present in 96% (23 of 24) BL cases. Array-based CGH analysis identified abnormalities that are shared between adult and pediatric DLBCL (+12q15, +19q13, -6q), and abnormalities unique to the pediatric cases (-4p14, -19q13.32, +16p11.2), suggesting distinct pathogenetic mechanisms relative to age. Elucidation of the underlying target genes may provide insight into factors that modulate outcome and could provide potential novel therapeutic targets with less toxicity for pediatric patients with B-cell non-Hodgkin lymphoma.

Occurrence of nephroblastomatosis with dup(18)(q11.2-q23) implicates trisomy 18 tumor screening protocol in select patients with 18q duplication.

Duplications of the long arm of chromosome 18 have been previously reported in patients with phenotypic findings similar to full trisomy 18. Trisomy 18 increases the risk for Wilms tumor and it is currently recommended that these patients undergo abdominal ultrasonography screening every 6 months. We report on nephroblastomatosis in a 27-month-old male with a 55 Mb duplication of chromosome 18q11.2-q23 (chr18:22693370-77982126, hg 19) and propose that the trisomy 18 tumor screening protocol could also benefit patients with large 18q duplications.

Duplication of 20qter and deletion of 20pter due to paternal pericentric inversion: patient report and review of 20qter duplications.

Duplications of the terminal long arm of chromosome 20 are rare chromosomal anomalies. We report a male infant found on array comparative genomic hybridization analysis to have a 19.5 Mb duplication of chromosome 20q13.12-13.33, as well as an 886 kb deletion of 20p13 at 18,580-904,299 bp. This anomaly occurred as the recombinant product of a paternal pericentric inversion. There have been 23 reported clinical cases involving 20qter duplications; however, to our knowledge this is only the second reported patient with a paternal pericentric inversion resulting in 46,XY,rec(20)dup(20q). This patient shares many characteristics with previously described patients with 20qter duplications, including microphthalmia, anteverted nares, long ears, cleft palate, small chin, dimpled chin, cardiac malformations, and normal intrauterine growth. While there is variable morbidity in patients with terminal duplications of 20q, a review of previously reported patients and comparison to our patient's findings shows significant phenotypic similarity.

Allogeneic stem cell transplantation for Philadelphia chromosome-positive acute myeloid leukemia.

Philadelphia chromosome-positive acute myeloid leukemia (Ph(+)-AML) has a poor response to anthracycline- and cytarabine-containing regimens, high relapse rate, and dismal prognosis. Although therapy with imatinib and allogeneic stem cell transplantation (allo-SCT) is promising, relatively short follow-up limits understanding of long-term results of these therapies. This report describes the outcomes of 3 cases of Ph(+)-AML diagnosed and transplanted at the University of Nebraska Medical Center between 2004 and 2011. These patients, young and without major comorbidities, received induction therapy with 7 days of cytarabine and 3 days of idarubicin along with imatinib and consolidation therapy with high-dose cytarabine (with or without imatinib). All patients underwent 10/10 HLA-matched peripheral blood allo-SCT (sibling donor for first and third patients and unrelated donor for the second patient; all had acute graft-versus-host disease (GVHD), and the first and third patients had chronic GVHD. All patients are currently alive and experiencing complete remission at 116, 113, and 28 months after diagnosis, respectively. This report shows that the use of allo-SCT with resultant graft-versus-leukemia effect and the addition of imatinib can result in long-term remission and possible cure in some patients with Ph(+)-AML.

Cytosine arabinoside and mitoxantrone followed by second allogeneic transplant for the treatment of children with refractory juvenile myelomonocytic leukemia.

Hematopoietic stem cell transplantation (HSCT) remains the only curative option for most patients with juvenile myelomonocytic leukemia (JMML). However, persistent disease and relapse rates after transplant range from 26% to 58%. We report the successful use of second HSCT after preparation with mitoxantrone and cytosine arabinoside (Ara-C) for patients with refractory or recurrent disease. Between 1993 and 2006, 5 children who underwent HSCT at our institution as initial therapy for JMML had persistent disease or relapsed. Pre-HSCT conditioning varied and donors were either HLA-matched siblings (n=2) or matched unrelated donors (n=3). After initial HSCT, they subsequently received high-dose Ara-C (3 g/m IV) every 12 hours on days -8 through -3 and mitoxantrone (10 mg/m/d IV) on days -8, -7, -6 followed by second HSCT from their original donors. All 5 patients are alive at 88, 179, 199, 234, and 246 months with no evidence of JMML, no significant toxicity, and 100% donor chimera as determined by PCR short-tandem repeat analysis. Our experience supports second transplant utilizing high-dose Ara-C and mitoxantrone in children with JMML who do not respond or relapse after first transplant.

Aberrant immunoglobulin class switch recombination and switch translocations in activated B cell-like diffuse large B cell lymphoma.

To elucidate the mechanisms underlying chromosomal translocations in diffuse large B cell lymphoma (DLBCL), we investigated the nature and extent of immunoglobulin class switch recombination (CSR) in these tumors. We used Southern blotting to detect legitimate and illegitimate CSR events in tumor samples of the activated B cell-like (ABC), germinal center B cell-like (GCB), and primary mediastinal B cell lymphoma (PMBL) subgroups of DLBCL. The frequency of legitimate CSR was lower in ABC DLBCL than in GCB DLBCL and PMBL. In contrast, ABC DLBCL had a higher frequency of internal deletions within the switch mu (Smu) region compared with GCB DLBCL and PMBL. ABC DLBCLs also had frequent deletions within Sgamma and other illegitimate switch recombinations. Sequence analysis revealed ongoing Smu deletions within ABC DLBCL tumor clones, which were accompanied by ongoing duplications and activation-induced cytidine deaminase-dependent somatic mutations. Unexpectedly, short fragments derived from multiple chromosomes were interspersed within Smu in one case. These findings suggest that ABC DLBCLs have abnormalities in the regulation of CSR that could predispose to chromosomal translocations. Accordingly, aberrant switch recombination was responsible for translocations in ABC DLBCLs involving BCL6, MYC, and a novel translocation partner, SPIB.

Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia.

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻5). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.

Rituximab pharmacokinetics in children and adolescents with de novo intermediate and advanced mature B-cell lymphoma/leukaemia: a Children's Oncology Group report.

The ANHL01P1 trial was undertaken to determine pharmacokinetics and safety following the addition of rituximab to French-American-British/Lymphome Malins de Burkitt (FAB/LMB96) chemotherapy in 41 children and adolescents with Stage III/IV mature B-cell lymphoma/leukaemia. Patients received rituximab (375 mg/m(2) ) days -2 and 0 of two induction cycles and day 0 of two consolidation cycles. Highest peak levels were achieved following the second dose of each induction cycle [299 ± 19 and 384 ± 25 µg/ml (Group-B); 245 ± 31 and 321 ± 32 µg/ml (Group-C)] with sustained troughs and t½ of 26-29 d. Rituximab can be safely added to FAB chemotherapy with high early rituximab peak/trough levels and a long t½.

Report of a patient with developmental delay, hearing loss, growth retardation, and cleft lip and palate and a deletion of 7q34-36.1: review of distal 7q deletions.

The use of aCGH has improved our ability to find subtle cytogenetic abnormalities as well as to find more precise information in patients with previously known abnormalities. In addition, it has allowed more specific genotype-phenotype correlation. In this report we describe a patient with a chromosomal deletion initially diagnosed with conventional cytogenetic analysis which was redemonstrated and more specifically described upon aCGH analysis. Our patient is a 12-year-old female born to a 26-year-old G1P0 mother. She was noted as a neonate to have a bilateral cleft lip and cleft palate, abnormal external ears, dysmorphic facies, and moderate to severe hearing loss. She has subsequently shown developmental delay, hyperreflexia, seizures, hyperactivity, and absence of speech. Chromosomal analysis showed deletion of 7q34q36.1. FISH studies confirmed the deletion was interstitial. Parental chromosomes were performed and did not show any cytogenetic abnormalities. aCGH was recently performed for the patient to further characterize the breakpoints of the deletion and confirmed the deletion was interstitial and of 13.2 Mb in size. Both proximal and terminal 7q deletion show a different phenotype than that of our patient. A number of patients with similar deletions have been found and while significant variability is observed, a number of findings appear to be common to deletions in this region. Therefore, we feel that distal interstitial deletions of chromosome 7q represent a recognizable phenotype and could be considered a separate deletion syndrome.

Characterization of six novel patients with MECP2 duplications due to unbalanced rearrangements of the X chromosome.

Males with duplication of the Xq28 region, including methyl CpG-binding protein 2 (MECP2), exhibit a characteristic phenotype, including developmental delay, intellectual disability, limited or absent speech, limited or absent ambulation, and recurrent respiratory infections. We report six males with MECP2 duplications identified using array comparative genomic hybridization. The minimal sizes of these duplications range from ∼0.08 to 14.13 Mb, which, to the best of our knowledge, are respectively the smallest and largest minimal size duplications molecularly characterized to date. Adjunct metaphase fluorescence in situ hybridization analysis further classified these duplications as tandem or as products of complex chromosomal rearrangements. Specifically, one complex rearrangement was described as a der(12)t(X;12)(q28;q24.33), which is the first report of a translocation involving MECP2 on Xq and chromosome 12. The other complex rearrangement was described as a rec(X)dup(Xq)inv(X)(p22.32q28)mat. Synthesis of the dysmorphic features identified in individuals with rec(X) chromosomes, including deletions in the pseudoautosomal region 1 (PAR1) at Xp22.33/Yp11.3 and duplications of the distal Xq region including MECP2, revealed a high prevalence of undescended testes (7/8) and micropenis (3/8) in this cohort. Given that micropenis is rare in the general population, but present in 38% of individuals in this cohort, a dosage anomaly at one or both loci may be a significant risk factor for this condition. Therefore, we recommend microarray testing for patients with unexplained micropenis, particularly when accompanied by other phenotypic anomalies.

The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma.

We used gene expression profiling to establish a molecular diagnosis of mantle cell lymphoma (MCL), to elucidate its pathogenesis, and to predict the length of survival of these patients. An MCL gene expression signature defined a large subset of MCLs that expressed cyclin D1 and a novel subset that lacked cyclin D1 expression. A precise measurement of tumor cell proliferation, provided by the expression of proliferation signature genes, identified patient subsets that differed by more than 5 years in median survival. Differences in cyclin D1 mRNA abundance synergized with INK4a/ARF locus deletions to dictate tumor proliferation rate and survival. We propose a quantitative model of the aberrant cell cycle regulation in MCL that provides a rationale for the design of cell cycle inhibitor therapy in this malignancy.

Primary follicular lymphoma of the testis in children and adolescents.

This study reports 6 cases of primary follicular lymphoma of the testis (PFLT) in children and adolescents correlated with clinical presentation, pathologic features, treatment, and outcome. All 6 patients (age, 3 to 16 y; median, 4 y) had PFLT grade 3 with disease limited to the testis, completely resected and treated with 2 courses of chemotherapy (cyclophosphamide, vincristine, prednisone, doxorubicin). Event-free survival was 100% (follow-up: median, 73 mo; mean, 53 mo; range, 6 to 96 mo). In conclusion, clinical outcome in children and adolescents with PFLT is excellent with treatment including complete surgical resection and 2 courses of cyclophosphamide, vincristine, prednisone, doxorubicin.

Genome wide copy number analysis of paediatric Burkitt lymphoma using formalin-fixed tissues reveals a subset with gain of chromosome 13q and corresponding miRNA over expression.

The majority of paediatric Burkitt lymphoma (pBL) patients that relapse will die of disease, but markers for this high-risk subset are unknown. MYC translocations characterize pBL, but additional genetic changes may relate to prognosis and serve as potential biomarkers. We utilized a molecular inversion probe single nucleotide polymorphism assay to perform high resolution, genome-wide copy number analysis on archival formalin-fixed, paraffin-embedded pBL and germline tissues. We identified copy number abnormalities (CNAs) in 18/28 patients (64%) with a total of 62 CNAs that included 32 gains and 30 copy number losses. We identified seven recurrent CNAs including 1q gain (7/28, 25%), 13q gain (3/28, 11%), and 17p loss (4/28, 14%). The minimum common amplified region on 13q was at 13q31 and included the MIR17HG (MIR17-92) locus. Samples with this gain had higher levels of MIR17 RNA and showed a tendency for early relapse. Tumour-specific uniparental disomy was identified in 32% of cases and usually was recurrent. These results demonstrate that high-resolution copy number analysis can be performed on archival lymphoma tissue specimens, which has significance for the study of rare diseases.