MOLECULAR DIAGNOSTIC ON BONE MARROW BIOPSY SPECIMEN: AN INNOVATIVE CLINICAL TOOL WITH MAJOR IMPACT ON CLINICAL DECISIONS.
(Abstract release date: 05/19/16)
EHA Library. Gavillet M. 06/09/16; 134573; PB1673
Dr. Mathilde Gavillet
Contributions
Contributions
Abstract
Abstract: PB1673
Type: Publication Only
Background
Genetics is currently central to diagnosis, prognosis and measure of response to therapy (minimal residual disease, MRD) for patients with acute leukemia, myeloid or lymphoid. These tests are usually conducted on bone marrow aspirate by various techniques (karyotype, fluorescence in situ hybridization, reverse transcription polymerase chain reaction, or DNA sequencing). In case of a dry tap these most valuable information are often lost.
Aims
Since January 2014, we implemented a method for DNA and RNA extraction from bone marrow biopsy specimen in case of a dry tap at diagnosis or during follow up. Here we present the method, results and relevance of the analysis conducted so far.
Methods
Bone marrow biopsy is immediately sunken in either RNAlater (QIAGEN, as for other products listed below) for transcript expression analysis such as acute myeloid leukemia (AML) recurrent genetic anomaly or MRD determination. In case DNA-based analysis is warranted, such as analysis of BCR rearrangements and NOTCH mutations, the biopsy specimen is put into phosphate buffer saline (PBS). Biopsy material is then manually crushed and solved in Qiazol for RNA and PBS for DNA. Manual extraction according to the phenol/chloroform is performed prior to RNA purification, quantification and reverse transcription according to manufacturer’s instruction. DNA purification and quantification is performed similarly. Analyses are performed according to established standards for bone marrow aspirates.
Results
We retrospectively identified 32 analysis from 20 patients with either acute lymphoid leukemia (ALL, n=6), acute myeloid leukemia (AML, n=12) or high risk myelodysplastic syndromes (MDS, n=2). Dry tap occurred either at diagnosis (n=11) or during follow up (n=21), regardless of diagnosis (lymphoid or myeloid neoplasia), blast count (median 5%, range: 1-95%) or marrow fibrosis (median 2, range 0-4). In seven samples, this technique allowed to identify a mutation in NMP1, with impact on diagnosis and prognosis, which is not amendable to other techniques (karyotype or FISH). Seventeen more samples allowed to quantify minimal residual disease (levels of WT1 or BCR-ABL transcripts), which is either not possible or less refined by alternative cytogenetic methods. Seven patients had more than one sample, which allowed correlation with and refinement of response assessment by histology. On 11 occasions (37.5%) the result of molecular analysis had a major impact on therapy (mostly distinction of Philadelphia positive vs. negative ALL n=5, targeted therapy for FLT3-ITD transcript n=1, identification of relapse/refractory disease n=2, decision between autologous vs. allogeneic transplantation for consolidation n=2).
Conclusion
The method presented here is a readily accessible variant of the commonly used molecular diagnostic techniques. Its use can confirm, expand and usually refine the analysis available on the bone marrow biopsy specimen, and in one third of the cases, have a significant impact on therapy options.
Session topic: E-poster
Keyword(s): Acute leukemia, Bone marrow biopsy, Molecular markers, Quantitative molecular analysis
Type: Publication Only
Background
Genetics is currently central to diagnosis, prognosis and measure of response to therapy (minimal residual disease, MRD) for patients with acute leukemia, myeloid or lymphoid. These tests are usually conducted on bone marrow aspirate by various techniques (karyotype, fluorescence in situ hybridization, reverse transcription polymerase chain reaction, or DNA sequencing). In case of a dry tap these most valuable information are often lost.
Aims
Since January 2014, we implemented a method for DNA and RNA extraction from bone marrow biopsy specimen in case of a dry tap at diagnosis or during follow up. Here we present the method, results and relevance of the analysis conducted so far.
Methods
Bone marrow biopsy is immediately sunken in either RNAlater (QIAGEN, as for other products listed below) for transcript expression analysis such as acute myeloid leukemia (AML) recurrent genetic anomaly or MRD determination. In case DNA-based analysis is warranted, such as analysis of BCR rearrangements and NOTCH mutations, the biopsy specimen is put into phosphate buffer saline (PBS). Biopsy material is then manually crushed and solved in Qiazol for RNA and PBS for DNA. Manual extraction according to the phenol/chloroform is performed prior to RNA purification, quantification and reverse transcription according to manufacturer’s instruction. DNA purification and quantification is performed similarly. Analyses are performed according to established standards for bone marrow aspirates.
Results
We retrospectively identified 32 analysis from 20 patients with either acute lymphoid leukemia (ALL, n=6), acute myeloid leukemia (AML, n=12) or high risk myelodysplastic syndromes (MDS, n=2). Dry tap occurred either at diagnosis (n=11) or during follow up (n=21), regardless of diagnosis (lymphoid or myeloid neoplasia), blast count (median 5%, range: 1-95%) or marrow fibrosis (median 2, range 0-4). In seven samples, this technique allowed to identify a mutation in NMP1, with impact on diagnosis and prognosis, which is not amendable to other techniques (karyotype or FISH). Seventeen more samples allowed to quantify minimal residual disease (levels of WT1 or BCR-ABL transcripts), which is either not possible or less refined by alternative cytogenetic methods. Seven patients had more than one sample, which allowed correlation with and refinement of response assessment by histology. On 11 occasions (37.5%) the result of molecular analysis had a major impact on therapy (mostly distinction of Philadelphia positive vs. negative ALL n=5, targeted therapy for FLT3-ITD transcript n=1, identification of relapse/refractory disease n=2, decision between autologous vs. allogeneic transplantation for consolidation n=2).
Conclusion
The method presented here is a readily accessible variant of the commonly used molecular diagnostic techniques. Its use can confirm, expand and usually refine the analysis available on the bone marrow biopsy specimen, and in one third of the cases, have a significant impact on therapy options.
Session topic: E-poster
Keyword(s): Acute leukemia, Bone marrow biopsy, Molecular markers, Quantitative molecular analysis
Abstract: PB1673
Type: Publication Only
Background
Genetics is currently central to diagnosis, prognosis and measure of response to therapy (minimal residual disease, MRD) for patients with acute leukemia, myeloid or lymphoid. These tests are usually conducted on bone marrow aspirate by various techniques (karyotype, fluorescence in situ hybridization, reverse transcription polymerase chain reaction, or DNA sequencing). In case of a dry tap these most valuable information are often lost.
Aims
Since January 2014, we implemented a method for DNA and RNA extraction from bone marrow biopsy specimen in case of a dry tap at diagnosis or during follow up. Here we present the method, results and relevance of the analysis conducted so far.
Methods
Bone marrow biopsy is immediately sunken in either RNAlater (QIAGEN, as for other products listed below) for transcript expression analysis such as acute myeloid leukemia (AML) recurrent genetic anomaly or MRD determination. In case DNA-based analysis is warranted, such as analysis of BCR rearrangements and NOTCH mutations, the biopsy specimen is put into phosphate buffer saline (PBS). Biopsy material is then manually crushed and solved in Qiazol for RNA and PBS for DNA. Manual extraction according to the phenol/chloroform is performed prior to RNA purification, quantification and reverse transcription according to manufacturer’s instruction. DNA purification and quantification is performed similarly. Analyses are performed according to established standards for bone marrow aspirates.
Results
We retrospectively identified 32 analysis from 20 patients with either acute lymphoid leukemia (ALL, n=6), acute myeloid leukemia (AML, n=12) or high risk myelodysplastic syndromes (MDS, n=2). Dry tap occurred either at diagnosis (n=11) or during follow up (n=21), regardless of diagnosis (lymphoid or myeloid neoplasia), blast count (median 5%, range: 1-95%) or marrow fibrosis (median 2, range 0-4). In seven samples, this technique allowed to identify a mutation in NMP1, with impact on diagnosis and prognosis, which is not amendable to other techniques (karyotype or FISH). Seventeen more samples allowed to quantify minimal residual disease (levels of WT1 or BCR-ABL transcripts), which is either not possible or less refined by alternative cytogenetic methods. Seven patients had more than one sample, which allowed correlation with and refinement of response assessment by histology. On 11 occasions (37.5%) the result of molecular analysis had a major impact on therapy (mostly distinction of Philadelphia positive vs. negative ALL n=5, targeted therapy for FLT3-ITD transcript n=1, identification of relapse/refractory disease n=2, decision between autologous vs. allogeneic transplantation for consolidation n=2).
Conclusion
The method presented here is a readily accessible variant of the commonly used molecular diagnostic techniques. Its use can confirm, expand and usually refine the analysis available on the bone marrow biopsy specimen, and in one third of the cases, have a significant impact on therapy options.
Session topic: E-poster
Keyword(s): Acute leukemia, Bone marrow biopsy, Molecular markers, Quantitative molecular analysis
Type: Publication Only
Background
Genetics is currently central to diagnosis, prognosis and measure of response to therapy (minimal residual disease, MRD) for patients with acute leukemia, myeloid or lymphoid. These tests are usually conducted on bone marrow aspirate by various techniques (karyotype, fluorescence in situ hybridization, reverse transcription polymerase chain reaction, or DNA sequencing). In case of a dry tap these most valuable information are often lost.
Aims
Since January 2014, we implemented a method for DNA and RNA extraction from bone marrow biopsy specimen in case of a dry tap at diagnosis or during follow up. Here we present the method, results and relevance of the analysis conducted so far.
Methods
Bone marrow biopsy is immediately sunken in either RNAlater (QIAGEN, as for other products listed below) for transcript expression analysis such as acute myeloid leukemia (AML) recurrent genetic anomaly or MRD determination. In case DNA-based analysis is warranted, such as analysis of BCR rearrangements and NOTCH mutations, the biopsy specimen is put into phosphate buffer saline (PBS). Biopsy material is then manually crushed and solved in Qiazol for RNA and PBS for DNA. Manual extraction according to the phenol/chloroform is performed prior to RNA purification, quantification and reverse transcription according to manufacturer’s instruction. DNA purification and quantification is performed similarly. Analyses are performed according to established standards for bone marrow aspirates.
Results
We retrospectively identified 32 analysis from 20 patients with either acute lymphoid leukemia (ALL, n=6), acute myeloid leukemia (AML, n=12) or high risk myelodysplastic syndromes (MDS, n=2). Dry tap occurred either at diagnosis (n=11) or during follow up (n=21), regardless of diagnosis (lymphoid or myeloid neoplasia), blast count (median 5%, range: 1-95%) or marrow fibrosis (median 2, range 0-4). In seven samples, this technique allowed to identify a mutation in NMP1, with impact on diagnosis and prognosis, which is not amendable to other techniques (karyotype or FISH). Seventeen more samples allowed to quantify minimal residual disease (levels of WT1 or BCR-ABL transcripts), which is either not possible or less refined by alternative cytogenetic methods. Seven patients had more than one sample, which allowed correlation with and refinement of response assessment by histology. On 11 occasions (37.5%) the result of molecular analysis had a major impact on therapy (mostly distinction of Philadelphia positive vs. negative ALL n=5, targeted therapy for FLT3-ITD transcript n=1, identification of relapse/refractory disease n=2, decision between autologous vs. allogeneic transplantation for consolidation n=2).
Conclusion
The method presented here is a readily accessible variant of the commonly used molecular diagnostic techniques. Its use can confirm, expand and usually refine the analysis available on the bone marrow biopsy specimen, and in one third of the cases, have a significant impact on therapy options.
Session topic: E-poster
Keyword(s): Acute leukemia, Bone marrow biopsy, Molecular markers, Quantitative molecular analysis
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