The use of formalin‐fixed, paraffin‐embedded lymph node samples for the detection of minimal residual disease in mantle cell lymphoma (2014)

There is a growing evidence regarding the usefulness of minimal residual disease (MRD) monitoring in mantle cell lymphoma (MCL) (Pott, 2011). MRD assessment in MCL can be performed either at the gene expression level using CCND1-IGH transcript (Pott, 2011), via CCND1 overexpression (Brizova et al, 2008) or at the DNA level. Quantitative polymerase chain reaction (qPCR) for clone-specific immunoglobulin (IGH) gene rearrangement is currently the most sensitive and robust tool for MRD detection and has been thoroughly standardized for use in acute lymphoblastic leukaemia (ALL) (van der Velden et al, 2007). Circulating lymphoma cells are present in most MCL cases at diagnosis, at a median 6% in the peripheral blood (PB) and 7% in bone marrow (BM) (Pott et al, 2010). Therefore, a clone-specific target (IGH or CCND1-IGH) can be derived from diagnostic BM or PB. This material can be further used as a dilution standard for MRD quantification at later time points. Using this approach, MRD assessment is possible in 80–85% of MCL cases (Pott, 2011). In the remaining cases, there is either no BM/PB infiltration or it is too low for the reliable identification of a clonal IGH target on the background of normal B lymphocytes. In those cases, a fresh diagnostic lymph node sample could serve for target identification and dilution series construction, but this is rarely available, especially in a multi-centre setting. Unlike fresh samples, formalin-fixed, paraffin-embedded (FFPE) tissues are used routinely for histopathological diagnosis and are usually archived in diagnostic centres. However concerns regarding the use DNA from FFPE samples include the poor integrity and quantity of isolated DNA and the impossibility of quantifying the initial lymphoma infiltration for setting the dilution curve. Here, we describe a method for MRD quantification based on FFPE tissue samples than can reliably detect MRD in MCL patients (detailed in the Supporting Information). The DNA was isolated from FFPE sections and checked for integrity via amplification of differently sized fragments (van Dongen et al, 2003) and for amplificability via the ALB control gene (Pongers-Willemse et al, 1998). The lymphoma infiltration in the lymphoma section was estimated using CCND1 staining. Further steps of the method are identical with the setting of patient-specific MRD assay from BM/PB and include screening for clonal IGH or CCND1-IGH rearrangements, sequencing of junctional regions, design of patient-specific primers and optimization of qPCR assays (Verhagen et al, 2000; van Dongen et al, 2003; Pott et al, 2013). The 17 diagnostic FFPE tissues analysed included 14 lymph nodes, one tonsil, one intestine with mesenteric node and one parotid gland. Figure 1 shows lymph nodes sections with a high (A) and lower (B) infiltration of MCL cells as assessed via CCND1 expression. A median 70% (range 50–90%) infiltration was determined in the tissue sections. Dilution series into polyclonal DNA from healthy donors (‘buffy coat’) were prepared for each patient. Table SI shows the parameters of the standard curves. All but two systems reached a sensitivity of 10−5; all assays achieved a quantitative range (QR) of at least 5 × 10−4 according to the EuroMRD guidelines (van der Velden et al, 2007). In six patients with BM or PB infiltration higher than 10%, the dilution curve was constructed from the BM (4×) or PB (2×) in parallel to FFPE-derived systems. In total, 28 MRD samples were evaluated on both systems. Both gold standard and FFPE-derived assays gave strongly concordant results (Fig 2A; Spearman's correlation coefficient 0·975, P < 0·0001). We then evaluated the applicability of FFPE-based MRD detection in 11 patients with low or no lymphoma infiltration at the time of diagnosis or relapse. As it was not possible to directly compare the two PCR approaches, we compared the FFPE results with 8-colour flow cytometry (FC) (van Dongen et al, 2012) (Fig 2B). We only evaluated the diagnostic samples because, in the follow-up samples, the majority of MRD results were negative for both PCR and FC. The sensitivity of 8-colour FC depends on the individual MCL phenotype. Generally, FC gave unambiguous quantitative results in values higher than approximately 10−3 (0·1%) and could be used to define negative results in concordance with FFPE-based PCR. FFPE-based PCR MRD correlated well with quantitative FC values above this value and gave numerical values within the quantitative range of each respective PCR assay below the 0·1% FC sensitivity threshold (Fig 2B). MRD quantification using qPCR based on patient-specific IGH rearrangement has been standardized by the EuroMRD group for use in ALL and multiple rounds of quality controls and standardization has also been achieved for MCL. The lack of initial lymphoma material has been a drawback in a substantial proportion of patients with low or no lymphoma infiltration in BM or PB. Gimenez et al (2012) described a method based on cloning individual rearrangements and quantification of MRD on plasmid standards in patients with lack of initial lymphoma material. However, this method is laborious and has a substantial risk of PCR contamination due to plasmid use. The amount of FFPE-isolated DNA is frequently lower than that of standard DNA isolated from BM/PB samples because of the small size of the sample. To save the material for multiple examinations, a standard curve dilution can start at 10−2 or even lower, providing that the standard curve has at least three points and would cover at least 2 logs, as required by the EuroMRD criteria (van der Velden et al, 2007). We admit that there can be an inter-observer difference in the exact percentage of CCND1-expressing cells reported. However, even a two-fold difference in infiltration percentage would only correspond to one qPCR cycle and therefore would not grossly affect the overall accuracy of qPCR MRD, which is often rounded to whole logs in clinical protocols. Taken together, this study demonstrated that it is possible to establish reliable MRD assays even with small, archived FFPE biopsy samples. This can be clinically relevant in approximately 20% of MCL patients without a MRD target because it can clearly determine the presence or absence of lymphoma dissemination both at diagnosis and during the clinical course of the disease. Supported by the Ministry of Health, Czech Republic - Conceptual Development of Research Organization, Motol University Hospital, Prague, Czech Republic 00064203, IGA-MZ NT/13072-4, PRVOUK-27/LF1/1 and OPPK CZ.2.16/3.1.00/24022. MK, performed research, wrote the paper; EF, performed research, wrote the paper; PK, performed and designed research, KF, ML, EM, BB and HM, performed research; MT and RK designed research, wrote the paper; JT proposed and designed research, wrote the paper. The authors have no conflicts of interest to disclose. Data S1. Materials and methods. Table SI. Parameters of MRD systems generated from FFPE MCL samples. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.