Patient with Mosaic Turner Syndrome and a Derivative X Chromosome with a Variant Triple X Diagnosis in Fetus: A Case Report.

Although Turner syndrome is most often sporadic, multigenerational recurrence has been reported more often in the offspring of women with mosaic or variant forms of Turner syndrome. We present a case in which natural conception in a woman with identified 45,X/46,XX mosaicism resulted in a fetus with a gain of a derivative X chromosome. The unexpected fetal finding prompted further cytogenetic evaluation of the patient and subsequent identification of an additional cell line with the same derivative X chromosome, not observed in the initial study. To our knowledge, this is the first case in which further investigation of an abnormal noninvasive prenatal screen resulted in the identification of both maternal and fetal sex chromosome abnormality. We discuss the discordant finding, similar cases, and potential phenotype with respect to skewed X inactivation. We also highlight the use of multiple testing methodologies to characterize the serendipitous identification of a derivative X chromosome.

The recurrent TUBB3 Gly98Ser substitution is the first described to inconsistently result in CFEOM3.

Missense variants in TUBB3 have historically been associated with either congenital fibrosis of the extraocular muscles type 3 (CFEOM3) or malformations of cortical development (MCD). Until a recent report identified two amino acid substitutions in four patients that had clinical features of both disorders, pathogenic variants of TUBB3 were thought distinct to either respective disorder. Three recurrent de novo Gly71Arg TUBB3 substitutions and a single patient with a de novo Gly98Ser substitution blurred the MCD and CFEOM3 phenotypic distinctions. Here we report a second patient with a missense c.292G>A (p.Gly98Ser) substitution, but without CFEOM3, the first reported evidence that even the same TUBB3 substitution can produce a spectrum of TUBB3 syndrome phenotypes. Our patient presented with amblyopia, exotropia, optic disc pallor, and developmental delay. Neuroimaging identified hypoplasia of the corpus callosum, interdigitation of the frontal lobe gyri, and dysplasia or hypoplasia of the optic nerves, basal ganglia, brainstem, and cerebellum. This report identifies the TUBB3 Gly98Ser substitution to be recurrent but inconsistently including CFEOM3, and identifies the absence of joint contractures and the presence of optic disc abnormalities that may be genotype-specific to the TUBB3 Gly98Ser substitution.

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.

A novel t(4;22)(q31;q12) produces an EWSR1-SMARCA5 fusion in extraskeletal Ewing sarcoma/primitive neuroectodermal tumor.

Over 90% of Ewing sarcoma/primitive neuroectodermal tumors (PNETs) feature an 11;22 translocation leading to an EWSR1-FLI1 fusion. Less commonly, a member of the ETS-transcription factor family other than FLI1 is fused with EWSR1. In this study, cytogenetic analysis of an extraskeletal Ewing sarcoma/PNET revealed a novel chromosomal translocation t(4;22)(q31;q12) as the sole anomaly. Following confirmation of an EWSR1 rearrangement by the use of EWSR1 breakpoint flanking probes, a fluorescence in situ hybridization positional cloning strategy was used to further narrow the 4q31 breakpoint. These analyses identified the breakpoint within RP11-481K16, a bacterial artificial chromosome (BAC) clone containing two gene candidates FREM and SMARCA5. Subsequent RACE, RT-PCR, and sequencing studies were conducted to further characterize the fusion transcript. An in-frame fusion of the first 7 exons of EWSR1 to the last 19 exons of SMARCA5 was identified. SMARCA5 encodes for hSNF2H, a chromatin-remodeling protein. Analogous to EWSR1-ETS-expressing NIH3T3 cells, NIH3T3 cells expressing EWSR1-hSNF2H exhibited anchorage-independent growth and formed colonies in soft agar, indicating chimeric protein tumorigenic potential. Conversely, expression of EWSR1-hSNF2H in NIH3T3 cells, unlike EWSR1-ETS fusions, did not induce EAT-2 expression. Mapping analysis demonstrated that deletion of the C-terminus (SLIDE or SANT motives) of hSNF2H impaired, and deletion of the SNF2_N domain fully abrogated NIH3T3 cell transformation by EWSR1-SMARCA5. It is proposed that EWSR1-hSNF2H may act as an oncogenic chromatin-remodeling factor and that its expression contributes to Ewing sarcoma/primitive neuroectodermal tumorigenesis. To the best of our knowledge, this is the first description of a fusion between EWSR1 and a chromatin-reorganizing gene in Ewing sarcoma/PNET and thus expands the EWSR1 functional partnership beyond transcription factor and zinc-finger gene families.

Renal cell carcinoma with novel VCL-ALK fusion: new representative of ALK-associated tumor spectrum.

Renal cell carcinoma represents a model for contemporary classification of solid tumors; however, unusual and unclassifiable cases exist and are not rare in children and young adults. The anaplastic lymphoma kinase (ALK) gene has recently been implicated in subsets of pulmonary, esophageal, breast, and colon cancers. These findings strengthen the importance of molecular classification of carcinomas across different organ sites, especially considering the evolving targeted anticancer therapies with ALK inhibitors. In the current study of six pediatric renal cell carcinomas, two cases exhibited structural karyotypic abnormalities involving the ALK locus on chromosomal band 2p23. Fluorescence in situ hybridization (FISH) studies were positive for an ALK rearrangement in one case, and subsequent 5' rapid amplification of cDNA ends analysis of this tumor revealed that the 3' portion of the ALK transcript encoding for the kinase domain was fused in frame to the 5' portion of vinculin (VCL, NM_003373). The new fusion gene is predicted to have an open reading frame of 4122 bp encoding for a 1374-aa oncoprotein; its expression was shown by immunoblotting with anti-VCL and anti-ALK antibodies in tumor tissue lysates. Immunohistochemistry with the same antibodies demonstrated cytoplasmic and subplasmalemmal localization of the oncoprotein determined by its N-terminal VCL portion. FISH with a custom-designed VCL-ALK dual-fusion probe set confirmed the presence of the fusion in neoplastic cells and demonstrated the potential clinical utility of this approach for detecting VCL-ALK in routinely processed tissue. The five remaining pediatric renal cell carcinomas did not show ALK rearrangement by FISH or ALK expression by immunohistochemistry. The data identify the kidney as a new organ site for ALK-associated carcinomas and VCL as a novel ALK fusion partner. The results should prompt further studies to advance the molecular classification of renal cell carcinoma and help to select patients who would benefit from appropriate targeted therapies.

C11orf95-MKL2 is the resulting fusion oncogene of t(11;16)(q13;p13) in chondroid lipoma.

Chondroid lipoma, a rare benign adipose tissue tumor, may histologically resemble myxoid liposarcoma or extraskeletal myxoid chondrosarcoma, but is genetically distinct. In this study, an identical reciprocal translocation, t(11;16)(q13;p13), was identified in three chondroid lipomas, a finding consistent with previously isolated reports. A fluorescence in situ hybridization (FISH)-based positional cloning strategy using a series of bacterial artificial chromosome (BAC) probe combinations designed to narrow the 16p13 breakpoint revealed MKL2 as the candidate gene. Subsequent 5' RACE studies demonstrated C11orf95 as the MKL2 fusion gene partner. MKL/myocardin-like 2 (MKL2) encodes myocardin-related transcription factor B in a megakaryoblastic leukemia gene family, and C11orf95 (chromosome 11 open reading frame 95) is a hypothetical protein. Sequencing analysis of reverse transcription-polymerse chain reaction (RT-PCR) generated transcripts from all three chondroid lipomas defined the fusion as occurring between exons 5 and 9 of C11orf95 and MKL2, respectively. Dual-color breakpoint spanning probe sets custom-designed for recognition of the translocation event in interphase cells confirmed the anticipated rearrangements of the C11orf95 and MKL2 loci in all cases. The FISH and RT-PCR assays developed in this study can serve as diagnostic adjuncts for the identification of this novel C11orf95-MKL2 fusion oncogene in chondroid lipoma.

An increased frequency of 13q deletions detected by fluorescence in situ hybridization and its impact on survival in children and adolescents with Burkitt lymphoma: results from the Children's Oncology Group study CCG-5961.

Burkitt lymphoma (BL), an aggressive B-cell malignancy, is often curable with short intensive treatment regiments. Nearly all BLs contain rearrangements of the MYC/8q24 region; however, recent cytogenetic studies suggest that certain secondary chromosomal aberrations in BL correlate with an adverse prognosis. In this multi-centre study, the frequency and impact on clinical outcome of del(13q) and +7 in addition to MYC rearrangements as detected by fluorescence in situ hybridization (FISH) in children and adolescents with intermediate and high-risk BL registered on Children's Cancer Group study CCG-5961 were investigated. Analysis with 13q14.3 and 13q34 loci specific probes demonstrated deletions of 13q in 38/90 (42%) cases. The loss of either 13q14.3 or 13q34 alone occurred in 14% and 8% respectively, while 20% exhibited loss of both regions. Gain of chromosome 7 was observed in 7/68 (10%) cases and MYC rearrangements were detected in 84/90 (93%). Prognostic analysis controlling for known risk factors demonstrated that patients exhibiting loss of 13q, particularly 13q14.3, had a significant decrease in 5-year overall survival (77% vs. 95%, P = 0.012). These observations indicate that del(13q) occurs in childhood BL at frequencies higher than previously detected by classical cytogenetics and underscores the importance of molecular cytogenetics in risk stratification.

Cytogenetic abnormalities and clinical correlations in peripheral T-cell lymphoma.

Cytogenetic correlations among most types of peripheral T-cell lymphoma (PTCL) have not been very informative to date. This study aimed to identify recurrent chromosomal abnormalities in angioimmunoblastic T-cell lymphoma (AITL), ALK-negative anaplastic large cell lymphoma (ALK-ALCL) and peripheral T-cell lymphoma, unspecified (PTCL-US), and to evaluate their prognostic value. We reviewed the cytogenetic findings of 90 previously-diagnosed cases of PTCL and correlated the cytogenetic findings with the specific histological subtype. The most common abnormalities for AITL were 5q (55%), 21 (41%) and 3q (36%) gains, concurrent trisomies of 5 and 21 (41%), and loss of 6q (23%). In ALK(-) ALCL, gains of 1q (50%) and 3p (30%), and losses of 16pter (50%), 6q13q21 (30%), 15 (30%), 16qter (30%) and 17p13 (30%) were frequent findings. In PTCL-US, frequent gains involved 7q22q31 (33%), 1q (24%), 3p (20%), 5p (20%), and 8q24qter (22%), and losses of 6q22q24 (26%) and 10p13pter (26%). We did not observe any association between specific chromosomal abnormalities and overall survival (OS). However, cases with complex karyotypes, most frequently observed in ALK(-) ALCL and PTCL-US, had a significantly shorter OS. Although, genetic differences were noted in these subtypes, further studies are needed to determine the key pathogenetic events in PTCL.

Gene targeting in adult rhesus macaque fibroblasts.

BACKGROUND: Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT) has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology. RESULTS: A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer. CONCLUSION: The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.

Loss of the PTCH1 gene locus in cardiac fibroma.

BACKGROUND: Cardiac fibroma (CF) is a rare benign tumor that is poorly characterized genetically. CF is more commonly encountered in patients with Gorlin syndrome (3%) than the general population. Mutations of the tumor suppressor gene PTCH1 are the underlying cause of Gorlin syndrome. METHODS: Conventional cytogenetic analysis was performed on a peripheral blood and a CF sample from a 2-week-old male. In addition, fluorescence in situ hybridization (FISH) studies were performed to assess the copy number of the PTCH1 gene locus (9q22.3) on metaphase and interphase cells from these same specimens using yeast artificial protein (YAC) probe 891G1 and on representative paraffin-embedded tissue sections of two additional CFs (one arising in a 2-month-old female and the other in a 13-week-old male). None of the patients had Gorlin syndrome. RESULTS: Karyotypically, the following abnormal chromosomal complement was detected in the 2-week-old male's CF: 46,XY,del(9)(q22q34)[15]. FISH studies revealed homozygous loss of the PTCH1 locus in the cytogenetically analyzed CF and in the CF arising in the 13-week-old male. Heterozygous loss of this locus was identified in the remaining CF from the 2-month-old female. A mutational mechanism other than deletion may be responsible for PTCH1 inactivation on the other locus in this latter patient. Conventional cytogenetic and FISH studies of the peripheral blood sample from the 2-week-old male were normal. CONCLUSION: These data support a tumor suppressor gene role for PTCH1 in nonsyndromic or sporadic CFs.

Aggressive angiomyxoma with t(12;21) and HMGA2 rearrangement: report of a case and review of the literature.

Conventional cytogenetic analysis of an aggressive angiomyxoma of the rectal wall of a 72-year-old woman revealed a translocation between the long arms of chromosomes 12 and 21, with the karyotype 46,XX,t(12;21)(q15;q21.1). Involvement of the HMGA2 gene locus (12q15) was confirmed by fluorescence in situ hybridization using an HMGA2 breakpoint flanking probe set performed on metaphase and interphase cells from an in situ culture of fresh lesional tissue. Karyotypic rearrangements of 12q13 approximately q15 are considered recurrent in aggressive angiomyxoma, although reported in only five previous cases. Translocation partner chromosome 21 is unique to the present case.

Lack of a common or characteristic cytogenetic anomaly in solitary fibrous tumor.

Solitary fibrous tumor is a mesenchymal tumor that was initially described as a pleural-based lesion, but later was discovered in many other locations. The light-microscopic appearance of solitary fibrous tumor may overlap with other diagnostic entities; however, consistent tumor cell CD34 immunoreactivity is useful in establishing the diagnosis. Limited data suggest that solitary fibrous tumors are karyotypically diverse, and no common or characteristic anomaly has yet emerged for this entity. Cytogenetic analysis of two solitary fibrous tumors, one peritoneal and the other arising in the liver, revealed predominantly structural abnormalities in the former and numerical imbalances in the latter. Clonal karyotypic abnormalities were lacking in three additional solitary fibrous tumors.

Characterization of childhood precursor T-lymphoblastic lymphoma by immunophenotyping and fluorescent in situ hybridization: a report from the Children's Oncology Group.

BACKGROUND: T-lymphoblastic lymphoma (T-LBL) accounts for 25-30% of childhood non-Hodgkin's lymphoma and is closely related to T-lymphoblastic leukemia (T-ALL). Recently, we demonstrated distinct differences in gene expression between childhood T-LBL and T-ALL, but molecular pathogenesis and relevant protein expression patterns in T-LBL remain poorly understood. PROCEDURE: Children with T-LBL with disseminated disease were registered and treated on COG protocol 5971. Paraffin-embedded tumor tissue was obtained at diagnosis for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) studies. We determined the pattern and intensity of staining for c-Myc, Skp2, Mib-1, p53, TCL-1, bcl-2, and bcl-6 proteins by IHC and c-Myc, p53, bcl-2, bcl-6, and TCR alpha/delta molecular alterations by FISH in 22 pediatric T-LBL cases. RESULTS: The majority of T-LBL samples expressed Mib-1 (59%) and c-Myc (77%) proteins in greater than 50% of the cells, but Skp2 (14%), p53 (14%), and bcl-2 (23%) expression was less common. FISH studies demonstrated 18% gains and 10% losses in c-Myc, 16% gains in p53, 12% gains and 6% losses in bcl-2, and 6% gains and 19% losses in bcl-6 with little direct correlation between the IHC and FISH studies. CONCLUSIONS: Childhood T-LBL is a highly proliferative tumor associated with enhanced expression of c-Myc protein, but without detectable c-Myc molecular alterations. FISH studies did not identify consistent etiologies of molecular dysregulation, and future studies with other molecular approaches may be required to elucidate the molecular pathogenesis of childhood T-LBL.

Imbalances of chromosomes 4, 9, and 12 are recurrent in the thecoma-fibroma group of ovarian stromal tumors.

Traditional cytogenetic studies of ovarian stromal tumors are few, although trisomy 12 has been frequently documented with fluorescence in situ hybridization (FISH). In the current study, karyotypic analysis of four ovarian stromal tumors and a review of the literature suggest that numerical abnormalities of chromosomes 4 and 9 might also be important, possibly as secondary changes. To determine the frequency of 4, 9, and 12 aneuploidy in a larger group of ovarian tumors, FISH studies were performed on eight fibromas, three thecomas, one fibrothecoma, and five cellular fibromas. Trisomy 12 was identified in all five cellular fibromas as well as in two fibromas and the fibrothecoma. Gain of chromosome 9 was confined to the cellular fibromas. Loss of chromosomes 4 and/or 9 was prominent in the fibromas. These findings confirm the presence of trisomy 12 as a nonrandom chromosomal abnormality in ovarian stromal tumors. Moreover, these conventional and molecular cytogenetic data indicate that gain of chromosome 9 in addition to gain of chromosome 12 is prominent in cellular fibroma. In contrast, loss of chromosomes 4 and/or 9 are recurrent in fibroma. In summary, imbalances of chromosomes 4 and 9 appear to represent important secondary abnormalities in the thecoma-fibroma ovarian tumor group.

Monoallelic deletion of the p53 gene through chromosomal translocation in a small cell osteosarcoma.

Small cell osteosarcoma is a rare bone tumor of high-grade malignancy that most often arises in the metaphysis of long bones in the second decade of life. Cytogenetic and molecular genetic findings in small cell osteosarcoma are poorly defined. Conventional cytogenetic analysis of a small cell osteosarcoma arising in the proximal tibia of a 9-year-old male revealed a diploid chromosomal complement with complex structural rearrangements involving chromosomes 6, 16, and 17. Immunohistochemical assessment of p53 protein expression revealed nuclear p53 immunoreactivity in approximately 15% of the neoplastic cells. Subsequent fluorescence in situ hybridization (FISH) analyses confirmed loss of the p53 gene locus on the derivative chromosome 17 homolog and were negative for amplification of the MDM2, CDK4, c-MYC, HER-2/neu, CCND1, and COPS3 gene loci. To the best of our knowledge, this represents the first demonstration of monoallelic deletion of p53 in small cell osteosarcoma, suggesting that p53 alterations may play an important role in the development of small cell osteosarcoma as well as conventional osteosarcoma.

A (9;11)(q34;q13) translocation in a hibernoma.

The diagnosis of hibernoma has historically been made by histopathologic examination and finding of characteristic brown fat cells with granular multivacuolated cytoplasm. The diagnosis of hibernoma may be complicated, however, because seemingly diagnostic cells could be mistakenly identified as lipoblasts, leading to the erroneous diagnosis of well-differentiated liposarcoma. Cytogenetic alterations in lipomatous tumors are well established and could be used for diagnostic purposes. Previous cytogenetic abnormalities reported in hibernomas have included alteration of 11q13 region. Here, we present a case of a hibernoma with a novel cytogenetic alteration involving a reciprocal translocation between 9q and 11q that was useful in establishing the final diagnosis.

Monosomy 22 as a diagnostic aid in a case of late recurrence of adult granulosa cell tumor of the ovary.

Cytogenetic analysis of a case of metastatic granulosa cell tumor recurring 21 years after oophorectomy revealed monosomy 22. This anomaly, typical of granulosa cell tumor, coupled with the pathologic and immunophenotypic findings assisted in establishing the proper diagnosis of this lesion in the absence of the original histopathologic slides.

Papillary tumor of the pineal region with synchronous suprasellar focus and novel cytogenetic features.

Papillary tumor of the pineal region (PTPR) is an uncommon neoplasm with variable biologic behavior. Cytogenetic and molecular diagnostic studies are rare, yielding no definitive genetic signature. We report a case of PTPR with a multicentric presentation and unusual cytogenetic features. A 25-year-old man presented with headache, disconjugate gaze, and confusion. Mass lesions in the pineal and suprasellar regions, with identical imaging characteristics, were identified. The former extended partially into the fourth ventricle. It showed typical PTPR histology and losses of chromosomes 3, 7, 10, 14, 18, and Y with gains of chromosomes 3, 8, and 9. Seventeen months postsurgery, the patient is stable without disease progression after radiation therapy. Synchronous mass lesions at presentation and losses of chromosomes 3, 7, 14, 18, and Y are unusual features, adding to the available data and underscoring the biologic and genetic variability associated with these neoplasms.

Inconspicuous insertion 22;12 in myxoid/round cell liposarcoma accompanied by the secondary structural abnormality der(16)t(1;16).

In myxoid/round cell liposarcoma, the t(12;16)(q13;p11) and its associated fusion transcript, FUS-CHOP, characterize greater than 95% of cases. The variant translocation t(12;22)(q13;q12) and associated EWS-CHOP fusion transcript are rare. A second non-random aberration observed in roughly 20% of Ewing's sarcomas, and to a lesser extent other select sarcomas, is the unbalanced 1;16 translocation. Recognition of this secondary aberration in the absence of an obvious primary karyotypic abnormality strongly suggests that the use of other genetic approaches will be informative in uncovering a clinically suspected primary anomaly. The following case illustrates the utility of molecular cytogenetic and reverse transcriptase-polymerase chain reaction techniques in diagnosing an ins(22;12)(q12;q13q14) and associated EWS-CHOP fusion transcript in a myxoid/round cell liposarcoma exhibiting a der(16)t(1;16)(q11;q11).

Translocation (1;4)(p31;q34) in nonossifying fibroma.

Nonossifying fibroma is a benign, lytic lesion of fibrous origin most often observed in the metaphyseal region of the long bones in children and adolescents. It is frequently asymptomatic and is often characterized by a history of spontaneous resolution. Herein, we report a clinicohistopathologically typical case of nonossifying fibroma arising in the tibia of an 18-year-old skeletally mature female. Conventional cytogenetic analysis revealed a reciprocal translocation involving bands 1p31 and 4q34 [t(1;4)(p31;q34)]. To the best of our knowledge, this is only the second reported case of a clonally aberrant nonossifying fibroma.