Karyotype analysis demonstrated trisomy 3 as a sole anomaly in two out of twenty analyzed cells. To confirm clonality of this finding interphase FISH was performed using a chromosome 3 centromere (CEP3) probe, which showed 25 out of 300 cells positive for trisomy 3 (Figure 4)
Figure 4. Conventional cytogenetics (G-banded karyotype) and fluorescence in situ hybridization (FISH) studies were performed on the lymph node involved with PTLD. A. Chromosome analysis revealed 47,XX,+3[2]/46,XX[18]. B. This finding was confirmed by internal FISH studies with a probe for the centromere of chromosome 3, which detected three copies of chromosome 3 in 8.3% (25/300) of the nuclei examined. FISH studies were performed using commercially available Vysis Centromere 3 (CEP 3) probe (Abbott Molecular, Des Plaines, IL), cut off value established for trisomy 3 is 1 percent.
Although bone marrow did not show histologic or immunophenotypic evidence of the involvement by PTLD, molecular studies detected clonal and oligoclonal T-cell gene rearrangements. Patient was treated with Rituximab. Currently patient is seven years old, doing well and is free of the recurrence of PTLD.
4. Discussion
PTLDs represent a heterogeneous, commonly EBV-driven clonal proliferation of B-cells with admixed T-cell and plasma cells3. Although PTLDs are usually diagnosed based on clinical history, morphology, and immunophenotype, clonality studies are used to aid in the diagnosis. Cytogenetics is a powerful technique to determine clonality and, if possible, interpret clinical implications of the particular clone or clones. Although molecular diagnostics dominate the era of the precision medicine, conventional chromosome analysis and FISH studies remain valuable to diagnose gross chromosomal abnormalities in PTLDs. Cytogenetics allows identification of a single cell that bears aneuploidies or large chromosomal aberrations. Furthermore, FISH studies can be utilized to confirm the clonality of findings identified by karyotype. The most common aberrations were trisomies of chromosome 9 and/or 11 associated with EBV positivity, followed by translocations involving 8q24.21 (MYC), 3q27.3 (BCL6), and 14q32.13 (IGH), 14q32.33 (TCL1) 15. The identification of cytogenetic markers that either contribute to the prediction of clinical behavior or response to the treatment are important in managing patients with PTLDs. Although specific mutations have not been reported in PTLDs, aberrant somatic hypermutation was implicated in a pathogenetic process that activates protooncogenes such as such as paired box protein (PAX-5) and avian myelocytomatosis viral oncogene homolog  (C-MYC)16.
Aberrations involving chromosome 3 have been described in a range of low-grade B-cell lymphomas including marginal zone lymphoma, with the incidence reaching 85 percent in splenic marginal zone lymphoma17 18. Trisomy 3 has been reported in other types of B‐cell lymphomas such as follicular lymphoma, small cell lymphocytic lymphoma, and diffuse large cell lymphoma19. Trisomy 3 has also been described in a range of T‐cell lymphomas 20. Chromosome 3 contains proto-oncogenes that were reported in hematologic malignancies including transducin (beta)-like 1 X-linked receptor 1 (TBL1XR1) at 3q26.32, myeloid differentiation primary response gene (88) (MYD88) at 3p22.2, GATA binding protein 2 (GATA2) at 3q21.3, ras homolog family member A (RHOA) at 3p21.31, MDS1 and EVI1 complex locus (MECOM) at 3q26.2, T cell leukemia translocation altered (TCTA) at 3p21.31 and BCL6 transcription repressor (BCL6) at 3q27.3. It is possible that the pathogenesis of PTLDs is mediated by the trisomy 3 through the increased gene dosage of proto-oncogenes, however precise mechanism remains elusive. Recentin vitro studies using spontaneously proliferating EBV-infected B-cells from patients with PTLD demonstrated a higher average growth rate and expression a miR-BHRF1-3, the miRNA that was reported to have transforming abilities 21.
One of the patients presented here (Case 1) also had one cell with a monosomy of chromosome X in addition to trisomy 3. This finding likely represents a subclone, or a secondary finding in addition to trisomy 3. One of the ways that the loss of chromosome X might contribute to the pathogenesis of PTLD is through the loss of the copy of tumor suppressor gene. Several X-linked tumor suppressor genes including APC membrane recruitment protein 1 (AMER1) at Xq11.2 and forkhead box P3 (FOXP3) at Xp11.23 are X-linked tumor suppressor genes involved in prostate cancer22 23.The synchronous trisomy 3 with an extra chromosome X has been previously reported in the pediatric PTLD12. To our knowledge, this is the first report of the pediatric polymorphous PTLD with coexisting trisomy 3 and monosomy of chromosome X.
In the present study, we report two pediatric cases of polymorphous EBV-driven PTLDs with trisomy 3 arising in the setting of cardiac and liver/intestinal allograft. In addition, one of the patients (Case 1) had a monosomy of chromosome X. Both of the patients are doing well, which argues against poor prognostic value of trisomy 3 in PTLDs.
Further studies are necessary to characterize cytogenetic findings in the larger number of pediatric PTLD to understand the significance of trisomy 3. In addition, it is important to understand the significance of trisomy 3 with or without monosomy of chromosome X. Eventually, better understanding of the landscape of cytogenetic abnormalities in pediatric patients with PTLD will contribute to the accurate diagnosis and clinical prognosis.
Author Contributions: Diagnosis, F.Q.R., J.S., S.S.; writing—original draft preparation, A.S, N.G..; writing—review and editing, A.S., F.Q.R.; project conception, F.Q.R.
Funding: This research received no external funding.
Acknowledgments: Authors are thankful to both patients who contributed to this case report.
Conflicts of Interest: The authors declare no conflict of interest.
F.Q.R. ORCID number 0000-0002-6709-6209
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