Conventional Cytogenetic Analysis of Constitutional Abnormalities: A 20-Year Review of Proficiency Test Results From the College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee.

CONTEXT.­: The joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee works to ensure competency and proficiency of clinical cytogenetics testing laboratories through proficiency testing programs for various clinical tests offered by such laboratories, including the evaluation of constitutional abnormalities. OBJECTIVE.­: To review and analyze 20 years of constitutional chromosome analysis proficiency testing results (2003-2022), primarily utilizing G-banded karyograms. DESIGN.­: A retrospective review of results from 2003 through 2022 was performed, identifying challenges addressing constitutional disorders. The chromosomal abnormalities and overall performance were evaluated. RESULTS.­: A total of 184 cases from 161 proficiency testing challenges were administered from 2003 through 2022. Challenges consisted of metaphase images and accompanying clinical history for evaluation of numerical and/or structural abnormalities. Of the 184 cases, only 2 (1%) failed to reach an 80% grading consensus for recognition of the abnormality. Both cases illustrated the limitations of correctly characterizing some chromosomal abnormalities, including recombinant chromosomal abnormalities and isochromosome identification. In addition, 2 cases failed to reach a consensus for nomenclature reporting: 1 with an isochromosome and another with a duplication. CONCLUSIONS.­: This 20-year review illustrates the high rate of competency and proficiency of cytogenetic laboratories in the correct identification of constitutional chromosome abnormalities.

Phagocytosis-initiated tumor hybrid cells acquire a c-Myc-mediated quasi-polarization state for immunoevasion and distant dissemination.

While macrophage phagocytosis is an immune defense mechanism against invading cellular organisms, cancer cells expressing the CD47 ligand send forward signals to repel this engulfment. Here we report that the reverse signaling using CD47 as a receptor additionally enhances a pro-survival function of prostate cancer cells under phagocytic attack. Although low CD47-expressing cancer cells still allow phagocytosis, the reverse signaling delays the process, leading to incomplete digestion of the entrapped cells and subsequent tumor hybrid cell (THC) formation. Viable THCs acquire c-Myc from parental cancer cells to upregulate both M1- and M2-like macrophage polarization genes. Consequently, THCs imitating dual macrophage features can confound immunosurveillance, gaining survival advantage in the host. Furthermore, these cells intrinsically express low levels of androgen receptor and its targets, resembling an adenocarcinoma-immune subtype of metastatic castration-resistant prostate cancer. Therefore, phagocytosis-generated THCs may represent a potential target for treating the disease.

Recurring germline mosaicism in a family due to reversion of an inherited derivative chromosome 8 from an 8;21 translocation with interstitial telomeric sequences.

BACKGROUND: Mosaicism for chromosomal structural abnormalities, other than marker or ring chromosomes, is rarely inherited. METHODS: We performed cytogenetics studies and breakpoint analyses on a family with transmission of mosaicism for a derivative chromosome 8 (der(8)), resulting from an unbalanced translocation between the long arms of chromosomes 8 and 21 over three generations. RESULTS: The proband and his maternal half-sister had mosaicism for a der(8) cell line leading to trisomy of the distal 21q, and both had Down syndrome phenotypic features. Mosaicism for a cell line with the der(8) and a normal cell line was also detected in a maternal half-cousin. The der(8) was inherited from the maternal grandmother who had four abnormal cell lines containing the der(8), in addition to a normal cell line. One maternal half-aunt had the der(8) and an isodicentric chromosome 21 (idic(21)). Sequencing studies revealed microhomologies at the junctures of the der(8) and idic(21) in the half-aunt, suggesting a replicative mechanism in the rearrangement formation. Furthermore, interstitial telomeric sequences (ITS) were identified in the juncture between chromosomes 8 and 21 in the der(8). CONCLUSION: Mosaicism in the proband, his half-sister and half-cousin resulting from loss of chromosome 21 material from the der(8) appears to be a postzygotic event due to the genomic instability of ITS and associated with selective growth advantage of normal cells. The reversion of the inherited der(8) to a normal chromosome 8 in this family resembles revertant mosaicism of point mutations. We propose that ITS could mediate recurring revertant mosaicism for some constitutional chromosomal structural abnormalities.

Is 5q deletion in de novo Acute Myelogenous Leukemia (AML) with excess blasts a surrogate marker for the cryptic t(7;21)(p22;q22)? A case report and review of literature.

Although the 5q- syndrome is common in both de novo and treatment related myelodysplastic syndrome (MDS) and the World Health Organization defined 5q- syndrome as a specific type of MDS, it is less common in acute myelogenous leukemia (AML). Recently, it was suggested that AML with diploidy/tetraploidy and/or 5q alterations may be associated with the cryptic translocation, t(7;21)(p22;q22) resulting in RUNX1-USP42 gene fusion and this association may have been underestimated. Here, we report another case of de novo AML with cryptic t(7;21)(p22;q22) associated with a 5q deletion.

Oxidatively modified low-density lipoproteins are potential mediators of proteasome inhibitor resistance in multiple myeloma.

Proteasome inhibitor (PI) therapy has improved the survival of multiple myeloma (MM) patients. However, inevitably, primary or acquired resistance to PIs leads to disease progression; resistance mechanisms are unclear. Obesity is a risk factor for MM mortality. Oxidized LDL (OxLDL), a central mediator of atherosclerosis that is elevated in metabolic syndrome (co-occurrence of obesity, insulin resistance, dyslipidemia and hypertension), has been linked to an increased risk of solid cancers and shown to stimulate pro-oncogenic/survival signaling. We hypothesized that OxLDL is a mediator of chemoresistance and evaluated its effects on MM cell killing by PIs. OxLDL potently suppressed the ability of the boronic acid-based PIs bortezomib (BTZ) and ixazomib, but not the epoxyketone-based PI carfilzomib, to kill human MM cell lines and primary cells. OxLDL suppressed BTZ-induced inhibition of proteasome activity and induction of pro-apoptotic signaling. These cytoprotective effects were abrogated when lipid hydroperoxides (LOOHs) associated with OxLDL were enzymatically reduced. We also demonstrated the presence of OxLDL in the MM bone marrow microenvironment as well as numerous granulocytes and monocytes capable of cell-mediated LDL oxidation through myeloperoxidase. Our findings suggest that OxLDL may be a potent mediator of boronic acid-based PI resistance, particularly for MM patients with metabolic syndrome, given their elevated systemic levels of OxLDL. LDL cholesterol-lowering therapy to reduce circulating OxLDL, and pharmacologic targeting of LOOH levels or resistance pathways induced by the modified lipoprotein, could deepen the response to these important agents and offer clinical benefit to MM patients with metabolic syndrome.

Identification of Chromothripsis in Biopsy Using SNP-Based Microarray.

One of the well-known hallmarks of cancer is genomic instability. Although gradualism is a well-established process of cancer evolution, recent studies have shown that chromothripsis or chromoanasynthesis can result in complex genomic rearrangements by a single catastrophic event rather than several incremental steps. These two novel phenomena suggest an evolutionary modality for cancer cells to circumvent individual mutational events with one simultaneous shattering of chromosomes or chromosome regions resulting in the random reassembling of shattered genetic material to form complex derivative chromosomes. Although sequencing methods are ideal for the detection of chromothripsis, single-nucleotide polymorphism (SNP)-based microarray methods are also useful in detecting chromothripsis in biopsy samples. Issues related to sample collection, storage, and transport, especially with tumor biopsies, may limit the options for sequencing studies, and in such cases, SNP-based microarray may be a viable alternative for detecting chromothripsis.

Myelodysplasia and Mast Cell Leukemia with t(9;22).

INTRODUCTION: Mast cell leukemia (MCL) is a rare variant of systemic mastocytosis. Most cases of mast cell leukemia do not have cytogenics performed. Furthermore, there is no consistent chromosomal abnormality identified in MCL. This is the first reported case of MCL with a (9;22) translocation. CASE REPORT: An 80-year-old female presented with pancytopenia and was diagnosed with MDS. Over time, she required hospitalizations for platelet transfusions with increased frequency. She developed fatigue and weakness along with gastrointestinal symptoms. On exam, she had diffuse abdominal tenderness and a maculopapular rash. Her lab results revealed a new basophilia. A bone marrow biopsy showed 100% cellularity with many aggregates of mast cells. Chromosomal analysis showed t(9;22) with confirmed BCR/ABL1 fusion by fluorescence in situ hybridization (FISH). DISCUSSION: MCL has a poor prognosis due to the aggressive nature of the disease and ineffective therapies. Translocation (9;22) is known to be associated with MDS transformations to acute leukemia; however, this translocation has never been reported in MCL. Further research on the relationship between t(9;22) and MCL could lead to development of improved therapeutic options.

Fetoplacental discrepancy with normal karyotype in amniotic fluid and two different cell lines in placenta.

We present a case of fetoplacental discrepancy in a second-trimester fetus with normal karyotype in amniotic fluid and two different Robertsonian translocations in placenta. A 41-year-old woman of Middle-Eastern origin, gravida 2, para 1, underwent amniocentesis at 16-week gestation because of advanced maternal age. Amniotic fluid karyotype showed a normal 46,XX karyotype with a homozygous inv(9). Parental chromosome analysis showed both parents to be carriers of inv(9) and the parents are not consanguineous. Fetal ultrasound was normal. The mother presented to the clinic 4 weeks later with intrauterine fetal demise. Chromosome analysis from the placenta showed two different cell lines: a balanced (15;21) Roberstonian translocation in 11 cells and an unbalanced (21;21) Robertsonian translocation in 9 cells. The karyotype was interpreted as mos 45,XX,inv(9)(p11q13)x2,der(15;21)(q10;q10)[11]/46,XX,inv(9)(p11q13)x2,+21,der(21;21)(q10;q10). Mother was a carrier for the Cystic Fibrosis (delta F508), Factor V Leiden mutations, HbD-Los Angeles and HbQ-India variants. She also had a sibling with term stillbirth. Her husband's history was unremarkable. Our case appears to be another example of confined placental mosaicism (CPM) with normal fetal karyotype. However, we could not confirm the possibility that CPM contributed to the IUFD in our case given the complex medical history of the mother.

Cytogenetic abnormalities precede morphological abnormalities in developing malignant conditions: report of 2 cases.

We previously hypothesized that cytogenetic abnormalities precede morphological abnormalities in developing malignant conditions. In this context we evaluated additional cases to further confirm that hypothesis. We report on 2 additional cases in which clonal cytogenetic abnormalities were observed in otherwise morphologically normal samples. Case 1 is a bone marrow from a 73 year old male with transformed follicular lymphoma (FL), while case 2 is a lymph node from a 53-year-old with lymphadenopathy, both referred to the cytogenetics laboratory for evaluation. A 73-year-old male presented with an enlarging left inguinal mass surrounding and obliterating the left iliac vein. A tissue core biopsy of the mass revealed recurrent high grade FL with diffuse large B-cell lymphoma (DLBCL). Examination of a random bone marrow biopsy of the adjacent left posterior iliac crest showed only mild hypercellularity (50%) and no evidence of malignancy, and the results were confirmed by flow cytometry. Cytogenetic evaluation revealed an interstitial deletion, del (9)(q13q32). In case 2, morphologically the lymph node showed extensive paracortical hyperplasia with numerous eosinophils and no clear indication of a neoplastic process with no abnormal lymphoid population observed by flow. PCR studies for TCR gamma and IgH gene rearrangements were negative for clonality. Chromosome analysis demonstrated 47,XY,+add(1)(p22),t(3;14)(q27;q11.2)[13]/46,XY[7]. FISH studies showed a BCL6 gene rearrangement but no TCRAD rearrangement. A subsequent inguinal lymph node biopsy showed DLBCL. These cases along with the other cases in the literature provide further evidence of genetic abnormalities preceding morphological abnormalities in developing malignant conditions.

A complex chromosome rearrangement, der(6)ins(6)(p21.1q25.3q27)inv(6)(p25.3q27), in a child with cleidocranial dysplasia.

Complex chromosome rearrangements (CCRs) are structural abnormalities involving >2 chromosomes or >3 breakpoints. It has been suggested that the probability of imbalance increases as the number of breakpoints increase. Here we report a 7-month-old, Hispanic girl presenting with cleidocranial dysplasia (CCD) who was found to have a complex chromosome rearrangement of chromosome 6. Fluorescence in situ hybridization studies with bacterial artificial chromosome (BAC) clones showed that the rearrangement involved insertion of 6q into 6p disrupting the "Runt related transcription factor 2 (RUNX2)" gene at chromosome 6p21.1. In addition, a pericentric inversion of chromosome 6 was identified. Despite the complex nature of the rearrangement, no cryptic deletions or duplications could be detected by array comparative genomic hybridization.

A t(1;9)(q10;q10) translocation with additional 6q23 and 9q22 rearrangements in a case of chondromyxoid fibroma.

Chondromyxoid fibroma (CMF) is a rare cartilaginous tumor of bone. It typically presents in the long tubular bones and to a lesser extent in the small bones of the hands and feet of young adults. To date, several cytogenetic abnormalities have been described in association with CMF. We studied a phalangeal CMF from a 13-year-old female by cytogenetic methods. We found a novel unbalanced translocation between the long arms of chromosomes 1 and 9, resulting in loss of 1p. In addition, rearrangements involving the 6q23 and 9q22 regions were also observed. To our knowledge, this is the first report in the literature describing this novel chromosomal translocation in CMF.

Fusion of HMGA2 to COG5 in uterine leiomyoma.

Uterine leiomyomas are smooth muscle tumors most commonly seen in middle-aged women. Approximately 10% of these tumors contain rearrangements of the chromatin-remodeling gene HMGA2 at the chromosome band 12q14.3. Herein, we report on a uterine leiomyoma with a novel HMGA2 fusion gene. A 44-year-old woman presented with a 20-cm mass uterine leiomyoma. From a histological standpoint, the tumor exhibited extensive hyalinization, very low mitotic activity (<1/10 HPH), and no cytologic atypia. Smooth muscle differentiation was confirmed by the expression of smooth muscle actin and desmin. Standard cytogenetic analysis showed the reciprocal translocation t(7;12)(q31.2;q14.3). Fluorescence in situ hybridization analysis confirmed a balanced rearrangement of the HMGA2 locus in 80% of the cells. 3'RACE reverse-transcription polymerase chain reaction identified the fusion of HMGA2 exon 4 to the COG5 locus on 7q31 (component of oligomeric golgi complex 5 isoform). The fusion sequence is predicted to encode a 96-amino acid chimeric protein that retains all three DNA-binding domains (AT hooks) of HMGA2, but that is shorter than the original HMGA2 protein. Since the general structure of the fusion gene is similar to other previously described HMGA2 fusions, its biologic activity is predicted to be likely similar.

Amplification of the RARA gene in acute myeloid leukemia: significant finding or coincidental observation?

Oncogene amplification resulting in aberrant expression, although common in solid tumors, is rare in acute myeloid leukemia (AML) and is mostly associated with amplification of MYC, RUNX1, and MLL genes. Retinoic acid receptor alpha (RARA) and other target sequences at 17p11.2 often represent the amplicons expressed in breast cancer, not in AML. We present a unique case of a 59-year-old female with a history of breast cancer, now presenting with pancytopenia and bilateral infiltration with effusion in nodules of the right upper lobe of the lung. She was diagnosed with AML-M5. Chromosome analysis demonstrated a hypodiploid clone with complex numerical/structural abnormalities including 5q deletion, monosomy 7, as well as structurally rearranged chromosome 11 and several marker chromosomes. Fluorescence in situ hybridization (FISH) analysis showed amplification of RARA, loss of 7q, monosomy 7, loss of DEK (6p23), and additional copies of NUP214 (9q34) and MLL (11q23). Additional FISH studies showed both ERBB2 and TOP2A genes, which were co-amplified on one of the marker chromosomes. The follow-up bone marrow did not yield any metaphases, but FISH was normal for all probes, including RARA. After a short remission, the patient relapsed and showed clonal evolution. Additional case reports are necessary to assess whether RARA amplification in hematologic malignancies serves as an independent prognostic factor.

Pericentric inversion, inv(14)(p11.2q22.3), in a 9-month old with features of Goldenhar syndrome.

Goldenhar syndrome, also called hemifacial microsomia or oculo-auriculo-verterbal dysplasia (OAVS) (MIM 164210), is a birth defect involving the first and second branchial arch derivatives with an incidence of 1/5000. The variable phenotype includes mostly unilateral deformity of the external ear and small ipsilateral half of the face with epibulbar dermoid and vertebral anomalies. A genome-wide search in one family suggested linkage to a region of 10.7 cM on chromosome 14q32; however, no candidate genes have been identified. We report on a 9-month old with OAVS and a pericentric inversion of chromosome 14 which he inherited from his phenotypically normal mother. Fluorescence in-situ hybridization analysis with bacterial artificial chromosome clones from chromosome 14 showed the breakpoint on 14q maps distal to 14q21.2, thus confirming the cytogenetic breakpoints. In light of previous linkage studies mapping OAVS to 14q, we propose that the long arm breakpoint in our proband disrupted a potential candidate gene for OAVS resulting in his clinical phenotype.

The phenotypic spectrum of trisomy 2: report of two new cases.

We describe two cases of trisomy 2. The first case is a child with mosaic trisomy 2 who presented with mental retardation, multiple congenital anomalies, and dysmorphic findings similar to Pallister-Killian syndrome. The second case was an acardiac and acranial fetus with complete trisomy 2. We review the phenotypic spectrum associated with trisomy 2.

Molecular cytogenetic characterization of an interstitial de novo 13q deletion in a 3-month-old with severe pediatric gastroesophageal reflux.

Gastroesophageal reflux (GER) occurs when gastric contents travel back into the esophagus through the esophageal sphincter. GER is very common in infants with most growing out of it, but some continue to have chronic symptoms throughout childhood and adulthood. A gene for severe pediatric gastroesophageal reflux disease (GERD) was identified by linkage analysis and was mapped to chromosome 13. We report here a de novo interstitial deletion of chromosome 13 in a 3-month-old biracial male who presented to the emergency room with severe GER and failure to thrive. Chromosome analysis showed an interstitial deletion of chromosome 13, with the karyotype reported as 46, XY, del(13)(q12.3q14.1). BAC-FISH analysis demonstrated that the deletion encompasses 12.3 Mb and does involve the GERD1 locus. The GERD1 locus has been mapped to a 9-cM interval between the markers CAGR1 and D13S263, both of which are deleted in our patient. We propose that the GER phenotype in our patient is due to a haploinsufficiency of GERD1.

Fine mapping of breakpoints in two unrelated patients with rare overlapping interstitial deletions of 9q with mild dysmorphic features.

Approximately, 20 cases of interstitial deletions of 9q have been reported in the literature spanning the breakpoints from 9q21 to 9q34. Unlike the 9q subtelomeric deletions, the interstitial deletions do not demonstrate a specific recognizable phenotype, although the majority of patients had microcephaly. Lack of precise molecular delineation of the extent of deletions in the published cases makes it difficult to develop an accurate genotype-phenotype correlation. We report on fine mapping of breakpoints using the Affymetrix Human Mapping 500K Array Set in two unrelated female patients with overlapping de novo deletion in 9q. SNP oligonucleotide microarray analysis (SOMA) indicated these to be relatively large deletions with Patient 1 having a 6.47 Mb deletion (>60 genes) spanning 9q32-q33.2 and Patient 2 having a 9.68 Mb deletion (>20 genes) localized to 9q31.1-q33.1. FISH analysis with BAC clones localized to the breakpoints showed discrepant results in Patient 1. Based on the review of previously reported interstitial 9q deletion patients and our patients, the minimal region of overlap (MRO) appears to encompass the 9q32 region and a phenotype characterized by microcephaly, neurological dysfunction and facial dysmorphism can be deduced. Our study shows the investigative nature of the latest array technology and the limitations of this technology in the accurate delineation of breakpoints.

Partial trisomy and partial monosomy resulting from a reciprocal segregating in a large family.

Partial trisomy 7p with partial monosomy 9p is a rare disorder with only 3 cases reported. Both these abnormalities i.e., partial trisomy 7p and partial monosomy 9p result in distinct clinical phenotypes. However, patients with combined 7p trisomy/9p monosomy present with a phenotype consistent with trisomy 7p. We present a fourth case of trisomy 7p/monosomy 9p with long term follow-up and document the medical complications associated with this disorder. Long term follow-up of patients with chromosome abnormalities provides a unique opportunity to document the medical history and complications associated with such abnormalities.