Testing of t(4;14) in Multiple Myeloma: How to Do It
Cancer Genetics and Therapeutics
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Primary Categories:
- Cancer
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Secondary Categories:
- Cancer
Introduction
Cytogenetics testing is one of the current standards for diagnosis and prognostic follow-up of multiple myeloma (MM). It was recommended to do fluorescent in situ hybridization (FISH) analysis using probes to detect at least t(4;14), t(14;16), t(14;20), trisomies and 17p- on the initial diagnostic bone marrow sample. The presence of 1q gain, 1p-, 17p-, t(4;14), t(14;16) and t(14;20) is considered high-risk factors in MM.
t(4;14) results in placing FGFR3 (fibroblast growth factor receptor 3) and NSD2 (nuclear receptor SET domain protein 2, also known as WHSC1) under the influence of IGH enhancer, hence upregulates FGFR3 and/or NSD2 expression. However, studies indicated that only 30% to 40% of t(4;14) patients were in fact high risk, the remaining patients had intermediate-risk or standard-risk. The high-risk of t(4;14) was neither associated with overexpression of FGFR3 nor NSD2 nor mutations in FGFR3 but associated with truncated fusion transcript of NSD2 (3). The translation start site is located at the exon 2 of NSD2. Patients with the fusion of IGH::NSD2 before the translation start site of NSD2 had significant longer overall survival (OS) than the patients with fusion of IGH::NSD2 after the translation start site. Therefore, identification of the breakpoints of t(4;14) is a critical independent biomarker for separation of high-risk from low-risk patients.
Traditional dual-color, dual-fusion FISH probes for detection of t(4;14) cannot identify the precise breakpoint on FGFR3 or NSD2 and are not suitable for identification of high-risk patients. Therefore, alternative techniques will be needed to identify the precise breakpoints for t(4;14).
Case Presentation
The patient is a 54 y.o. male who presented with generalized pain and fatigue. Serum protein electrophoresis and immunofixation detected IgA Kappa monoclonal protein. Radiology study detected medullary compression and multiple lytic lesions. A bone marrow biopsy revealed extensive marrow involvement by multiple myeloma.
Diagnostic Workup
FISH panel analysis was performed on isolated CD138+ plasma cells from this patient’s bone marrow. Fusions of FGFR3::IGH with 1-2 additional copies of FGFR3 and IGH, trisomy/tetrasomy 9 and 15, 1-2 additional copies of CCND1, MAF, MAFB, trisomy/tetrasomy 17 with 2 copies of TP53 were detected in 86%, 85%, 53%, 81%, 79%, 73%, and 76% of cells, respectively. It was denoted as:
nuc ish(FGFR3,IGH)x3-4(FGFR3 con IGHx2-3)[86/100],(CEP9x3-4)[85/100],(CCND1,IGH)x3-4[81/100], (D13S319,LAMP1)x2[100],(IGHx3)(3’IGH sep 5’IGHx2)[74/100],(IGH,MAF)x3-4[79/100],(IGH,MAFB)x3-4[73/100],(TP53x2,D17Z1x3-4)[76/100]. The overall FISH profile was near-triploidy with t(4;14), which was confirmed by concurrent karyotype analysis.
In order to know the precise breakpoints of t(4;14), the concurrent isolated plasma cells were tested using Saphyr optical genome mapping (OGM) technology (kindly performed by Bionano Genomics, San Diego, CA). The t(4;14) was confirmed by OGM, ogm[GRCh38] t(4;14)(p16.3;q32.33)(1901670;105887565) and the fusion was NSD2::IGH with breakpoint at intron 2 of NSD2. OGM also confirmed the near triploid detected by FISH and karyotype.
Outcome and Follow-Up
Conclusion
OGM detected the precise breakpoints of t(4;14) for our patient. Based on the breakpoints, the expected fusion was IGH::NSD2 exon 3+ and the truncated NSD2 protein will be produced. Thus, our patient has a high-risk MM.
OGM is a powerful technology that can detect whole genome structural and numerical abnormalities. It has much higher resolution than traditional cytogenetics techniques e.g. karyotype and FISH. Our case study demonstrated that karyotype and FISH are not enough to yield precise testing results to meet current clinical needs. High resolution technologies such as OGM are pivotal and needed in diagnosis, prognosis and treatment for MM patients and other hematology cancer patients.
Cytogenetics testing is one of the current standards for diagnosis and prognostic follow-up of multiple myeloma (MM). It was recommended to do fluorescent in situ hybridization (FISH) analysis using probes to detect at least t(4;14), t(14;16), t(14;20), trisomies and 17p- on the initial diagnostic bone marrow sample. The presence of 1q gain, 1p-, 17p-, t(4;14), t(14;16) and t(14;20) is considered high-risk factors in MM.
t(4;14) results in placing FGFR3 (fibroblast growth factor receptor 3) and NSD2 (nuclear receptor SET domain protein 2, also known as WHSC1) under the influence of IGH enhancer, hence upregulates FGFR3 and/or NSD2 expression. However, studies indicated that only 30% to 40% of t(4;14) patients were in fact high risk, the remaining patients had intermediate-risk or standard-risk. The high-risk of t(4;14) was neither associated with overexpression of FGFR3 nor NSD2 nor mutations in FGFR3 but associated with truncated fusion transcript of NSD2 (3). The translation start site is located at the exon 2 of NSD2. Patients with the fusion of IGH::NSD2 before the translation start site of NSD2 had significant longer overall survival (OS) than the patients with fusion of IGH::NSD2 after the translation start site. Therefore, identification of the breakpoints of t(4;14) is a critical independent biomarker for separation of high-risk from low-risk patients.
Traditional dual-color, dual-fusion FISH probes for detection of t(4;14) cannot identify the precise breakpoint on FGFR3 or NSD2 and are not suitable for identification of high-risk patients. Therefore, alternative techniques will be needed to identify the precise breakpoints for t(4;14).
Case Presentation
The patient is a 54 y.o. male who presented with generalized pain and fatigue. Serum protein electrophoresis and immunofixation detected IgA Kappa monoclonal protein. Radiology study detected medullary compression and multiple lytic lesions. A bone marrow biopsy revealed extensive marrow involvement by multiple myeloma.
Diagnostic Workup
FISH panel analysis was performed on isolated CD138+ plasma cells from this patient’s bone marrow. Fusions of FGFR3::IGH with 1-2 additional copies of FGFR3 and IGH, trisomy/tetrasomy 9 and 15, 1-2 additional copies of CCND1, MAF, MAFB, trisomy/tetrasomy 17 with 2 copies of TP53 were detected in 86%, 85%, 53%, 81%, 79%, 73%, and 76% of cells, respectively. It was denoted as:
nuc ish(FGFR3,IGH)x3-4(FGFR3 con IGHx2-3)[86/100],(CEP9x3-4)[85/100],(CCND1,IGH)x3-4[81/100], (D13S319,LAMP1)x2[100],(IGHx3)(3’IGH sep 5’IGHx2)[74/100],(IGH,MAF)x3-4[79/100],(IGH,MAFB)x3-4[73/100],(TP53x2,D17Z1x3-4)[76/100]. The overall FISH profile was near-triploidy with t(4;14), which was confirmed by concurrent karyotype analysis.
In order to know the precise breakpoints of t(4;14), the concurrent isolated plasma cells were tested using Saphyr optical genome mapping (OGM) technology (kindly performed by Bionano Genomics, San Diego, CA). The t(4;14) was confirmed by OGM, ogm[GRCh38] t(4;14)(p16.3;q32.33)(1901670;105887565) and the fusion was NSD2::IGH with breakpoint at intron 2 of NSD2. OGM also confirmed the near triploid detected by FISH and karyotype.
Outcome and Follow-Up
Conclusion
OGM detected the precise breakpoints of t(4;14) for our patient. Based on the breakpoints, the expected fusion was IGH::NSD2 exon 3+ and the truncated NSD2 protein will be produced. Thus, our patient has a high-risk MM.
OGM is a powerful technology that can detect whole genome structural and numerical abnormalities. It has much higher resolution than traditional cytogenetics techniques e.g. karyotype and FISH. Our case study demonstrated that karyotype and FISH are not enough to yield precise testing results to meet current clinical needs. High resolution technologies such as OGM are pivotal and needed in diagnosis, prognosis and treatment for MM patients and other hematology cancer patients.