RNA-seq NGS to detect KMT2A partial tandem duplication in an AML patient with trisomy 11
Cancer Genetics and Therapeutics
-
Primary Categories:
- Cancer
-
Secondary Categories:
- Cancer
Introduction
Lysine (K)-specific Methyltransferase 2A gene partial tandem duplications (KMT2A-PTDs) are present in 5 - 10% of acute myeloid leukemia (AML) and are associated with adverse outcomes. Traditional cytogenetics techniques, including karyotype and fluorescence in situ hybridization (FISH), cannot detect such aberration, which likely will be missed in diagnosis if only those techniques are used. Few molecular techniques can be used to identify this abnormality, including next-generation sequencing (NGS), Microarray, Polymerase Chain Reaction (PCR), and Multiplex-Ligation Probe Amplification (MLPA). We present a case of therapy-related AML with trisomy 11 and KMT2A-PTD, detected by RNA-seq NGS and RT-PCR.
Case Presentation
A 74-year-old male with a history of colon cancer treated with colectomy and chemotherapy and history of thyroid Hurtle cell carcinoma treated with right lobectomy presented with complaints of fatigue and weakness.
Diagnostic Workup
Complete blood count demonstrated pancytopenia with circulating blasts. Flow cytometry detected 73% myeloid blasts expressing CD34, CD117, HLA-DR, cMPO, CD13 and CD33. Bone marrow biopsy (BMB) showed 30% myeloid blasts with background multilineage dysplasia, suggestive of therapy-related AML. Chromosomal analysis revealed the gain of an intact copy of chromosome 11 in 80% (16/20) of metaphase cells. FISH using KMT2A probes did not detect KMT2A rearrangement but detected gain of one copy of 11q23.3 in 60% (120/200) of the nuclei examined, consistent with trisomy 11. RNA-Seq NGS showed KMT2A-PTD involving exons 2-8 (VAF 20.6%). Primers targeting exons 2 and 8 were designed to run RT-PCR, which confirmed KMT2A-PTD spanning exons 2-8 in the BMB sample.
Treatment and Management
The patient was started on azacitidine and venetoclax. BMB was repeated on day 21 with no morphologic evidence of residual leukemia. Chromosomal analysis revealed gain of an intact copy of chromosome 11 in 32% (8/25) of metaphase cells, consistent with the persistence of the disease. FISH analysis detected a gain of one copy of 11q23.3 in 15.0% (30/200) of the nuclei examined. NGS showed KMT2A-PTD involving exons 2-8 (VAF 6.4%), a hot-spot missense mutation in DNMT3A (VAF 6.5%), and IDH2 R172G (VAF 5.6%). Treatment was continued with no morphologic evidence of leukemia on day 44. Chromosome analysis showed a normal diploid karyotype. NGS showed KMT2A-PTD involving exons 2-8 (VAF 1%), and FISH studies detected a gain of one copy of 11q23.3 in 3.0% (6/200) of nuclei examined, indicative of residual disease. The patient is considered for allogeneic stem cell transplantation.
Discussion
Detection of KMT2A-PTD can guide diagnosis and clinical management, predict outcomes, and serve as a marker of minimal residual disease. However, it is often missed by conventional karyotyping or FISH due to the small size of the rearrangement. Other molecular assays also have limitations in detecting KMT2A-PTD. For example, PCR can only work when the exact breakpoints for the duplication are known, and the sensitivity of MLPA assay is low. On the other hand, RNA-seq NGS is a sensitive assay to detect KMT2A-PTD, and we successfully used it to detect KMT2A-PTD in a patient with therapy-related AML. In addition, KMT2A-PTD has been shown to occur in 25% of patients with concurrent trisomy 11. Therefore, it is essential to correlate cytogenetics findings with NGS results to help establish the diagnosis of the patients.
Conclusion
Accurate detection of KMT2A-PTDs is crucial for diagnosis, treatment, prognosis and disease monitoring. We utilized a comprehensive diagnostic approach including RNA-seq NGS to get a complete and precise assessment for the patient with KMT2A-PTDs, highlighting the essential role of RNA-seq NGS in establish the diagnosis of the patient with this molecular abnormality.
Lysine (K)-specific Methyltransferase 2A gene partial tandem duplications (KMT2A-PTDs) are present in 5 - 10% of acute myeloid leukemia (AML) and are associated with adverse outcomes. Traditional cytogenetics techniques, including karyotype and fluorescence in situ hybridization (FISH), cannot detect such aberration, which likely will be missed in diagnosis if only those techniques are used. Few molecular techniques can be used to identify this abnormality, including next-generation sequencing (NGS), Microarray, Polymerase Chain Reaction (PCR), and Multiplex-Ligation Probe Amplification (MLPA). We present a case of therapy-related AML with trisomy 11 and KMT2A-PTD, detected by RNA-seq NGS and RT-PCR.
Case Presentation
A 74-year-old male with a history of colon cancer treated with colectomy and chemotherapy and history of thyroid Hurtle cell carcinoma treated with right lobectomy presented with complaints of fatigue and weakness.
Diagnostic Workup
Complete blood count demonstrated pancytopenia with circulating blasts. Flow cytometry detected 73% myeloid blasts expressing CD34, CD117, HLA-DR, cMPO, CD13 and CD33. Bone marrow biopsy (BMB) showed 30% myeloid blasts with background multilineage dysplasia, suggestive of therapy-related AML. Chromosomal analysis revealed the gain of an intact copy of chromosome 11 in 80% (16/20) of metaphase cells. FISH using KMT2A probes did not detect KMT2A rearrangement but detected gain of one copy of 11q23.3 in 60% (120/200) of the nuclei examined, consistent with trisomy 11. RNA-Seq NGS showed KMT2A-PTD involving exons 2-8 (VAF 20.6%). Primers targeting exons 2 and 8 were designed to run RT-PCR, which confirmed KMT2A-PTD spanning exons 2-8 in the BMB sample.
Treatment and Management
The patient was started on azacitidine and venetoclax. BMB was repeated on day 21 with no morphologic evidence of residual leukemia. Chromosomal analysis revealed gain of an intact copy of chromosome 11 in 32% (8/25) of metaphase cells, consistent with the persistence of the disease. FISH analysis detected a gain of one copy of 11q23.3 in 15.0% (30/200) of the nuclei examined. NGS showed KMT2A-PTD involving exons 2-8 (VAF 6.4%), a hot-spot missense mutation in DNMT3A (VAF 6.5%), and IDH2 R172G (VAF 5.6%). Treatment was continued with no morphologic evidence of leukemia on day 44. Chromosome analysis showed a normal diploid karyotype. NGS showed KMT2A-PTD involving exons 2-8 (VAF 1%), and FISH studies detected a gain of one copy of 11q23.3 in 3.0% (6/200) of nuclei examined, indicative of residual disease. The patient is considered for allogeneic stem cell transplantation.
Discussion
Detection of KMT2A-PTD can guide diagnosis and clinical management, predict outcomes, and serve as a marker of minimal residual disease. However, it is often missed by conventional karyotyping or FISH due to the small size of the rearrangement. Other molecular assays also have limitations in detecting KMT2A-PTD. For example, PCR can only work when the exact breakpoints for the duplication are known, and the sensitivity of MLPA assay is low. On the other hand, RNA-seq NGS is a sensitive assay to detect KMT2A-PTD, and we successfully used it to detect KMT2A-PTD in a patient with therapy-related AML. In addition, KMT2A-PTD has been shown to occur in 25% of patients with concurrent trisomy 11. Therefore, it is essential to correlate cytogenetics findings with NGS results to help establish the diagnosis of the patients.
Conclusion
Accurate detection of KMT2A-PTDs is crucial for diagnosis, treatment, prognosis and disease monitoring. We utilized a comprehensive diagnostic approach including RNA-seq NGS to get a complete and precise assessment for the patient with KMT2A-PTDs, highlighting the essential role of RNA-seq NGS in establish the diagnosis of the patient with this molecular abnormality.