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HelpTest Code LPLFX Lymphoplasmacytic Lymphoma/Waldenstrom Macroglobulinemia, MYD88 L265P with Reflex to CXCR4, Varies
Reporting Name
Reflex Testing of MYD88 and CXCR4Useful For
Establishing a diagnosis of lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia (LPL/WM)
Helping distinguish LPL/WM low-grade B-cell lymphoma from other subtypes
Aiding in the prognosis and clinical management of lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia
Reflex Tests
| Test ID | Reporting Name | Available Separately | Always Performed |
|---|---|---|---|
| CXCFX | CXCR4, Gene Mutation, Reflex | Yes, (order CXLPL), Bill Only | No |
Testing Algorithm
The algorithm starts with the sensitive MYD88 L265P testing by allele-specific polymerase chain reaction. If a MYD88 L265P variant is detected, additional CXCR4 testing will be performed. If a MYD88 L265P variant is not detected, the algorithm ends, and no further testing is necessary.
Method Name
Allele-Specific Polymerase Chain Reaction (AS-PCR)/Bridged Nucleic Acids (BNA) Clamp Sanger Sequencing/Routine Sanger Sequencing
(BNAClamp is utilized pursuant to a license agreement with BNA Inc.)
Performing Laboratory
Mayo Clinic Laboratories in Rochester
Specimen Type
VariesShipping Instructions
Whole blood or bone marrow specimens must arrive within 10 days of collection.
Necessary Information
The following information is required:
1. Pertinent clinical history
2. Clinical or morphologic suspicion
3. Date and time of collection
4. Specimen source
Specimen Required
Submit only 1 of the following specimens:
Preferred:
Specimen Type: Bone marrow
Container/Tube:
Preferred: Lavender top (EDTA)
Acceptable: Yellow top (ACD)
Specimen Volume: 2 mL
Collection Instructions:
1. Invert several times to mix bone marrow
2. Send bone marrow specimen in original tube. Do not aliquot.
3. Label specimen as bone marrow
Specimen Stability Information: Ambient (preferred)/Refrigerated
Specimen Type: Paraffin-embedded tissue
Container/Tube: Paraffin block
Specimen Stability: Ambient
Specimen Type: Paraffin-embedded bone marrow aspirate clot
Container/Tube: Paraffin block
Specimen Stability: Ambient
Specimen Type: Tissue
Slides: Unstained slides
Specimen Volume: 10 to20 slides
Additional Information: Tissue must demonstrate involvement by a hematologic neoplasm (eg, acute myelocytic leukemia), not solid tumors.
Specimen Stability Information: Ambient
Acceptable:
Specimen Type: Whole blood
Container/Tube:
Preferred: Lavender top (EDTA)
Acceptable: Yellow top (ACD)
Specimen Volume: 3 mL
Collection Instructions:
1. Invert several times to mix blood
2. Send whole blood specimen in original tube. Do not aliquot.
3. Label specimen as blood
Specimen Stability Information: Ambient (preferred)/Refrigerated
Specimen Type: Extracted DNA
Container/Tube: 1.5- to 2-mL tube with indication of volume and concentration of the DNA
Specimen Volume: Entire specimen
Collection Instructions: Label specimen as extracted DNA and list the specimen source. Include indication of volume and concentration of the DNA.
Specimen Stability Information: Frozen (preferred)/Refrigerated/Ambient
Specimen Minimum Volume
Whole blood, Bone marrow: 1 mL
Extracted DNA: 50 mcL with a concentration of at least 20 nanograms per mcL
Other specimen types: See Specimen Required
Specimen Stability Information
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Varies | Varies | 10 days |
Reject Due To
| Gross hemolysis | Reject |
| B5-fixed tissues Decalcified bone marrow biopsies Methanol-acetic acid (MAA)-fixed pellets Paraffin shavings Frozen tissue Moderately to severely clotted |
Reject |
Special Instructions
Reference Values
MYD88 L265P: Mutation present or absent based on expected variant polymerase chain reaction product size for the MYD88 gene (NCBI accession NM_002468.4).
CXCR4: Mutation present or absent in the test region c. 898-1059 (amino acids 300-353) of the CXCR4 gene (NCBI NM_003467.2, GRCh37).
Day(s) Performed
Monday through Friday
CPT Code Information
81305
LOINC Code Information
| Test ID | Test Order Name | Order LOINC Value |
|---|---|---|
| LPLFX | Reflex Testing of MYD88 and CXCR4 | 82140-5 |
| Result ID | Test Result Name | Result LOINC Value |
|---|---|---|
| MP042 | Specimen Type | 31208-2 |
| 601511 | LPLFX Reflex Result | 82140-5 |
| 601510 | Final Diagnosis | 50398-7 |
Forms
1. Hematopathology Patient Information (T676)
2. If not ordering electronically, complete, print, and send a Hematopathology/Cytogenetics Test Request (T726) with the specimen.
Secondary ID
61114Highlights
This test offers highly sensitive detection of the well-characterized hotspot variants c.1013C->G/A, p.S338X and routine Sanger sequencing for other variants in the C-terminus region.
Clinical Information
The MYD88 L265P abnormality is highly associated (>90%) with the pathologic diagnosis of lymphoplasmacytic lymphoma and the clinical syndrome of Waldenstrom macroglobulinemia (LPL/WM), particularly in the setting of an elevated IgM serum monoclonal paraprotein.
CXCR4 mutations are identified in approximately 30% to 40% of LPL/WM patients and are almost always in association with MYD88 L265P, which is highly prevalent in this neoplasm. The status of CXCR4 mutations in the context of MYD88 L265P is clinically relevant as important determinants of clinical presentation, overall survival and therapeutic response to ibrutinib. A MYD88-L265P/CXCR4-WHIM (C-terminus nonsense/frameshift variants) molecular signature is associated with intermediate to high bone marrow disease burden and serum IgM levels, less adenopathy, and intermediate response to ibrutinib in previously treated patients. A MYD88-L265P/CXCR4-WT (wildtype) molecular signature is associated with intermediated bone marrow disease burden and serum IgM levels, more adenopathy, and highest response to ibrutinib in previously treated patients. A MYD88-WT/CXCR4-WT molecular signature is associated with inferior overall survival, lower response to ibrutinib therapy in previously treated patients, and lower bone marrow disease burden in comparison to those harboring a MYD88-L265 variant.
Interpretation
Mutation present or not detected; an interpretive report will be issued.
Cautions
This MYD88 test is a targeted assay and will not detect any alteration at the MYD88 codon 265 that does not result in the L>P (leucine to proline) amino acid change. It will also not detect additional MYD88 variants, including insertion or deletion events. The analytical sensitivity of the assay (1% MYD88 L265P in a wildtype background) can be affected by a variety of factors, including biologic availability (ie, tumor burden), fixation of paraffin-embedded specimens, or nonspecific polymerase chain reaction (PCR) interferences. Rare cases of lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia (LPL/WM) have been reported lacking the MYD88 L265P abnormality, so a negative result would not completely exclude this diagnosis but would make the possibility of LPL/WM more unlikely.
The reflexed test is a targeted assay for the C-terminal end of the CXCR4 gene only. It examines c.898-1059 of the CXCR4 gene (NCBI NM_003467.2 GRCh37) and does not detect variants outside this region. A 1% analytical sensitivity was established at 50 ng DNA input for the hotspot mutations c.1013C>G/A only, which uses bridged nucleic acids (BNA) clamped Sanger sequencing, and DNA that does not meet the established criteria can lead to false-negative results. In the extremely rare event that a rare benign variant (ie, polymorphism), insertion, or deletion occurs at the Sanger sequencing primer binding sites, in cis with a c.1013C>G/A, data can yield a failed result. Routine Sanger sequencing is used to interrogate other mutations in the tested region with a 15% to 20% analytical sensitivity. The analytical sensitivity of the assay can be affected by a variety of factors, including biologic availability (ie, tumor burden), fixation of paraffin-embedded specimens, rare benign variants, insertions, or deletions at the primer binding sites or nonspecific PCR interferences.
Clinical Reference
1. Treon SP, Xu L, Yang G, et al: MYD88 L265P somatic mutation in Waldenstrom's macroglobulinemia. N Engl J Med. 2012 Aug 30;367(9):826-833. doi: 10.1056/NEJMoa1200710
2. Varettoni M, Arcaini L, Zibellini S, et al: Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom's macroglobulinemia and related lymphoid neoplasms. Blood. 2013 Mar 28;121(13):2522-2528. doi: 10.1182/blood-2012-09-457101
3. Xu L, Hunter ZR, Yang G, et al: MYD88 L265P in Waldenstrom macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction. Blood. 2013 Mar 14;121(11):2051-2058. doi: 10.1182/blood-2012-09-454355
4. Poulain S, Roumier C, Decambron A, et al: MYD88 L265P mutation in Waldenstrom macroglobulinemia. Blood. 2013 May 30;121(22):4504-4511. doi: 10.1182/blood-2012-06-436329
5. Gachard N, Parrens M, Soubeyran I, et al: IGHV gene features and MYD88 L265P mutation separate the three marginal zone lymphoma entities and Waldenstrom macroglobulinemia/lymphoplasmacytic lymphomas. Leukemia. 2013 Jan;27(1):183-189. doi: 10.1038/leu.2012.257
6. Ondrejka SL, Lin JJ, Warden DW, et al: MYD88 L265P somatic mutation: its usefulness in the differential diagnosis of bone marrow involvement by B-cell lymphoproliferative disorders. Am J Clin Pathol. 2013 Sept;140(3):387-394. doi: 10.1309/AJCP10ZCLFZGYZIP
7. Hunter Z, Xu L, Yang G, et al: The genomic landscape of Waldenstrom macroglobulinemia is characterized by highly recurring MYD88 and WHIM-like CXCR4 mutations, and small somatic deletions associated with B-cell lymphomagenesis. Blood. 2014 Mar 13;123(11):1637-1646. doi: 10.1182/blood-2013-09-525808
9. Poulain S, Roumier C, Venet-Caillault A, et al: Genomic landscape of CXCR4 mutations in Waldenstrom macroglobulinemia. Clin Cancer Res. 2016 Mar 15;22(6):1480-1488. doi: 10.1158/1078-0432.CCR-15-0646
10. Roccaro A, Sacco A, Jimenez C, et al: C1013G/CXCR4 acts as a driver mutation of tumor progression and modulator of drug resistance in lymphoplasmacytic lymphoma. Blood. 2014 Jun 26;123(26):4120-4131. doi: 10.1182/blood-2014-03-564583
11. Schmidt J, Federmann B, Schindler N, et al: MYD88 L265P and CXCR4 mutations in lymphoplasmacytic lymphoma identify cases with high disease activity. Br J Haematol. 2015 Jun;169(6):795-803. doi: 10.1111/bjh.13361
12. Treon SP, Cao Y, Xu L, et al: Somatic mutations in MYD88 and CXCR4 are determinants of clinical presentation and overall survival in Waldenstrom macroglobulinemia. Blood. 2014 May 1;123(18):2791-2796. doi: 10.1182/blood-2014-01-550905
13. Treon SP, Tripsas CK, Meid K, et al. Ibrutinib in previously treated Waldenstrom's macroglobulinemia. N Engl J Med. 2015 Apr 9;372(15):1430-1440. doi: 10.1056/NEJMoa1501548
Method Description
Extracted DNA from the clinical specimen is subjected to allele-specific polymerase chain reaction (PCR) using MYD88 exon 5 primers that simultaneously amplify both a wild-type sequence fragment and a fragment containing the specific nucleotide change resulting in L265P if present. PCR products are visualized by capillary electrophoresis and the presence of mutated and wildtype amplicons is determined according to the expected specific PCR product sizes.(Unpublished Mayo method)
The C-terminal end of CXCR4 (NM_003467.2, c. 898-1059) is amplified from extracted genomic DNA by PCR, followed by Sanger sequencing and capillary electrophoresis analysis. Review of the sequence data is performed using a combination of automated calls and manual inspection. (Unpublished Mayo method)
The hotspot mutations c.1013C>G/A (p.S338X) are examined using bridged nucleic acids clamped Sanger sequencing with an analytic sensitivity of 1%. All other genetic mutations in the test region are examined by routine Sanger sequencing with an analytic sensitivity of 15% to 20%.(Unpublished Mayo method)