Normalized 
CCND1
 expression has prognostic value in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell nonHodgkin lymphoma (NHL; Swerdlow et al, 2008). The molecular hallmark for diagnosis of MCL is the t(11;14)(q13; q32) translocation, resulting in constitutive over-expression of CCND1 (Cyclin D1; Campo et al, 1999; Swerdlow et al, 2008). The current median survival in patients with MCL is only 4–5 years (Herrmann et al, 2009), one of the poorest overall survival rates of all NHLs. Yet, some MCL patients have a more indolent course (Martin et al, 2009; Weigert et al, 2009). While several predictors of outcome for MCL have been proposed, their reliability and reproducibility are not well established (Rosenwald et al, 2003; Martin et al, 2009; Weigert et al, 2009). The chronic lymphocytic leukaemia overlap marker CD23 may be associated with a better prognosis in MCL, but this has not been confirmed (Kelemen et al, 2008). High Ki-67 expression and variant blastoid morphology are useful tissue correlates of poor prognosis, but these measures cannot be applied to patients diagnosed in the leukaemic state (Determann et al, 2008). Thus, at this time, there are no laboratory assays that stratify MCL patients into conservative versus aggressive treatment regimens. The primary aim of this study was to evaluate the prognostic utility of normalized quantitative CCND1 mRNA values in the blood or marrow of patients with newly diagnosed MCL. This novel quantitative CCND1 assay has been validated for establishing a diagnosis of MCL in blood/bone marrow (Hui et al, 2003; Howe et al, 2004), but its use as a prognostic marker has not been evaluated. We further compared the normalized CCND1 assay with CD23 expression and circulating levels of malignant B-cells. All cases tested between October 2002 and May 2010 that demonstrated an elevated normalized CCND1 mRNA value were eligible for inclusion (n = 74). Any cases in which the tissue source was not blood or marrow (n = 6) was subsequently excluded, and inclusion in the final study was restricted to only those patients with a new diagnosis of MCL and no prior treatment (n = 40). The institutional review board approved the review and use of patient information in this study. The normalized CCND1 mRNA assay is a relative fold increase (RFI) determined against the background of B-cells (Howe et al, 2004). The RFI method has been shown to strongly correlate with results obtained using fluorescent insitu hybridization (FISH) with CCND1 probes (Hui et al, 2003). The quantitative assay is briefly described here. RNA was extracted using RNeasy reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. All primers and probes were synthesized as previously described (Howe et al, 2004) and reaction mixtures contained primers for CCND1 and CD19. Reverse transcription and polymerase chain reaction were performed using an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Carlsbad, CA, USA). Critical threshold (Ct) cycle numbers were obtained for amplification of CCND1 and CD19. The DCt values were then obtained by subtracting the Ct value of CD19 from the Ct value of CCND1. The characteristics of our 40 patients with new a diagnosis of MCL in blood (n = 25) or marrow (n = 15) are presented in Table I. All 40 patients met the molecular criteria for diagnosis of MCL as previously described (Howe et al, 2004).