Allele‐specific PCR is a powerful tool for the detection of the MYD88 L265P mutation in diffuse large B cell lymphoma and decalcified bone marrow samples
2015-03-30

The MYD88 L265P mutation can be detected in 90–100% of lymphoplasmacytic lymphomas (LPL) (Treon et al, 2012; Ondrejka et al, 2013). Recognition of mutated MYD88, therefore, represents a highly useful marker in the differential diagnosis separating LPL from other small B-cell lymphoproliferative disorders and plasma cell myeloma (Ondrejka et al, 2013). Moreover, this mutation was detected in around 30% of diffuse large B-cell lymphomas (DLBCL) of the activated B-cell-like (ABC-like) subtype (Ngo et al, 2011) that are characterized by deregulation of the NFκB-signalling pathway (Ngo et al, 2006; Lenz et al, 2008). Targeting the NFκB pathway in MYD88-mutated lymphomas might represent a promising treatment option, and therefore, reliable detection of this mutation may be of increasing importance. Furthermore, the distinction of ABC-like from germinal centre B-cell-like (GCB-like) DLBCL based upon analysis of specific mutations, such as MYD88 L265P, may be of interest. We therefore developed a highly sensitive allele-specific polymerase chain reaction (ASPCR) applicable in daily diagnostics. Although ASPCR approaches for the analysis of formalin-fixed, paraffin-embedded (FFPE) tissue samples have already been described, neither the pre-analytical degradation status of the samples had been tested, nor had a PCR product size guaranteeing an adequate amplification of degraded FFPE samples been considered in previous studies. This ASPCR approach was tested in a cohort of decalcified bone marrow samples and 121 routinely ascertained DLBCL specimens, which, to our knowledge represents the largest series of DLBCL investigated by ASPCR to date. To evaluate the specificity and sensitivity of our ASPCR assay, serial dilution experiments using the Oci-Ly10 cell line heterozygous for MYD88 L265P and peripheral blood mononuclear cells (PBMCs) were performed and evaluated by both ASPCR and subsequent Sanger sequencing. Whereas Sanger sequencing required a minimum tumour cell content of 20% (Fig 1A), ASPCR accurately detected the L265P mutation in samples with a low tumour cell content of up to 1·25% (Fig 1B), well in line with previous findings (Ondrejka et al, 2013). Given that analysis of samples with a tumour cell content lower than 5% occurs only infrequently in daily diagnostic experience, the sensitivity of our ASPCR-assay is highly adequate for this application. Formalin-fixed, paraffin-embedded tissues often yield highly degraded DNA, and therefore the fragmentation status of the DNA was analysed in 121 DLBCL samples using a qualitative PCR approach. DNA quality was estimated upon amplification of three different-sized amplicons of the β-globin gene (HBB; 75 bp, 200 bp, 400 bp). All three amplicons were amplified in 62 samples (51%), indicating a low degree of fragmentation. In contrast, 49 cases (40%) were amplifiable with primers for generation of the 75 bp- and 200 bp-amplicons only. Ten cases (8%) showed high DNA degradation, detecting only the 75 bp-amplicon (Fig 1C). Although degradation of FFPE-derived DNA is a well-known problem in molecular diagnostics, few studies have taken into account this issue when analysing MYD88 mutations (Ondrejka et al, 2013). With establishment of an ASPCR assay that generates small-sized amplicons of 123 bp, this problem was adequately solved. ASPCR yielded results in 119/121 DLBCL (98%). Consequently, only two cases with a high degree of fragmentation and generation of the 75 bp amplicon only, failed in the MYD88 ASPCR. Employing pre-analytical DNA quality testing, therefore, we convincingly showed that our ASPCR assay is usable even when employing highly degraded DNA from FFPE tissues, a prerequisite of pivotal interest in the routine diagnostic setting. Seventy-nine routinely obtained, formalin-fixed, decalcified and paraffin-embedded cases of haematopoietic disorders involving the bone marrow were retrieved from the contributing departments of pathology. All 79 bone marrow samples tested for MYD88 mutation were analysable by ASPCR and 29 L265P mutations were detected. A diagnosis of LPL had been rendered in all cases harbouring the L265P mutation. Three bona fide LPL were negative for the L265P mutation. In contrast, none of the remaining 47 lymphoproliferative disorders used as controls carried the MYD88 L265P mutation (Table 1). These results are well in line with literature data (Treon et al, 2012; Ondrejka et al, 2013). As also shown previously, presence of L265P MYD88 mutation reliably separates LPL from other small B-cell lymphomas, which may have similar morphological features and/or equivocal immunophenotypes, although the mutation has been detected in rare cases of non-LPL. The absence of the MYD88 L265P mutation, however, should not lead to the exclusion of a diagnosis of LPL. As shown in the literature and also in our series, around 10% of LPL are MYD88 L265P-negative (Treon et al, 2012). 121 primary DLBCL were included in the study and classified according to the World Health Organization classification (Swerdlow et al, 2008). Clinical data were not available. Subtyping of DLBCL into GCB and non-GCB DLBCL was performed in 106/121 cases using the immunohistochemical Hans classifier (Hans et al, 2004). MYC translocation data were available in 53/121 cases. MYD88 L265P mutation was detected in 13/119 (11%) informative DLBCL. Subtyping of 106 DLBCL identified 48 GCB DLBCL and 58 non-GCB DLBCL cases. ASPCR analyses were evaluable in 104 of these 106 cases (98%). 10/104 DLBCL (10%) harboured the MYD88 L265P mutation. 7/58 mutation-positive samples (12%) were non-GCB and 3/48 mutation-positive cases (6%) were GCB DLBCL; all the latter had been classified as GCB DLBCL because of their reactivity for CD10. Interestingly, one of these cases showed a triple-hit constellation with simultaneous translocations in MYC, BCL2 and BCL6 (Horn et al, 2013). Testing for MYC translocations was done in all MYD88 L265P mutated and in 40 non-mutated cases. 3/53 (6%) tumours carried the MYC translocation, of which one was also MYD88 L265P mutated. All MYC-positive cases were classified as GCB DLBCL. A second MYD88-positive case was characterized by dual over-expression of MYC and BCL2 proteins. As the MYD88 L265P mutation had been detected almost exclusively in ABC-DLBCL using gene expression profiling for stratification (Ngo et al, 2011; Bohers et al, 2014), our recent observation of CD10 upregulation in MYC-translocated DLBCL of immunoblastic type may be of interest in this context (Horn et al, 2015). Given that ASPCR is a highly sensitive, cost-effective and rapid analysis system, this method represents a promising tool for the detection of MYD88 L265P mutations in the routine diagnostic setting. It remains to be determined whether analysis of MYD88 L265P mutation status in FFPE tissue might be a useful adjunct to differentiate GCB from non-GCB DLBCL subtypes. This work was supported by the Robert-Bosch-Stiftung (Stuttgart, Germany). The authors gratefully acknowledge Petra Hitschke, Stefanie Laukemann, Susanne Gutzeit, Elke Burghart, and Daniela Rauh (Stuttgart) for excellent technical assistance. Conception and design: H.H., A.M.S., G.O., and E.-U.G. Histopathological diagnoses: A.R., G.O., and M.M.O. Provision of study materials: A.R., G.O., M.M.O., and S.P. Collection and assembly of data: A.M.S., G.O., H.H., and E.-U.G. Manuscript writing: A.M.S., H.H., G.O., and M.M.O. All authors read and approved the manuscript. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article.