Molecular characterization and clinical impact of t(11;15)(q23;q14–15) MLL-CASC5 rearrangement

The identification of novel translocation partner genes involved in MLL rearrangements increases the chances of identifying novel possibilities for a targeted, molecular therapy. Concerted efforts by many research groups in collaboration with the Diagnostic Center of Acute Leukemia (DCAL) at Frankfurt University have made it possible to establish the MLL recombinome database.1 From these studies it became clear that just a few translocation partner genes account for over 90% of leukemia patients, and that the vast majority of MLL partner genes is relatively infrequent. Nevertheless, these rare but recurrent MLL rearrangements are also of importance, and, therefore, collaborative efforts are required to analyze their function(s) and impact on clinical outcome. In this regard, we recently reported MLL-TET1 rearrangements, which belong to a category of rare MLL rearrangements.2

Table 1.Clinical and laboratory features of three new MLL-CASC5 rearrangement.

Here we present comprehensive analyses of patients with MLL-CASC5 rearrangements resulting from t(11;15)(q23;q14–15) chromosomal translocations of which only 13 cases have been reported so far.93 The CASC5 (alias AF15q14) gene is located at chromosome 15q14, consists of 27 exons, and spans a genomic region of 70.32 kb. The encoded CASC5 protein is known to be associated with cell growth suppression and/or maturation enhancement and thus its disruption could be a key factor for leukemogenesis.4 To date, 3 cases have been molecularly confirmed as MLL-CASC5 positive leukemia (MLL-CASC5).43 However, little is known about the common features of MLL-CASC5 leukemia both in terms of detailed molecular data and clinical aspects. In order to gain further insights into this rare MLL fusion, we have collected t(11;15)(q23;q14–15)/MLL-CASC5 leukemia cases and describe their clinical features (Table 1). By analyzing the molecular and clinical characteristics of these patients along with previously reported t(11;15)(q23;q14–15) cases, we aimed to unravel the distinct features of this cytogenetically described leukemia subtype.

Using the long distance inverse-polymerase chain reaction (LDI-PCR) method, 3 new MLL-CASC5 cases were identified, which have also been included in the recently published MLL recombinome database.1 Basic demographic data, past medical history, and clinical course (if available), as well as cytogenetic, FISH, and LDI-PCR data for the 3 MLL-CASC5 cases were collected and specimens for further analyses were obtained after informed consent. Altogether, now a total of 16 cases with a t(11;15)(q23;q14–15) were analyzed including 10 acute myeloid leukemia (AML) cases (2 M1, 4 M2, 3 M4, and 1 NOS (leukemia not otherwise specified)), 4 acute lymphoblastic leukemia (ALL) cases (1 L1, 2 L2, and 1 unspecified), and 2 cases of myelodysplastic syndrome (MDS). Mean age of the patients was 20.6 years (range 1–54); there were 11 males and 5 females. Although karyotypes were available in all cases, molecular data, including confirmation of MLL-CASC5, and clinical datasets were rather limited.

It is worthy to note that, as previously observed by others,3 abnormalities of chromosome 3 were seen in 10 out of 16 cases (62.5%) with a t(11;15)(q23;q14–15). In an attempt to detect additional copy number alterations, we conducted SNP-microarray analysis on 2 of our 3 cases with chromosome 3 abnormalities (cases 1 and 2) using Affymetrix CytoScan 750K (Affymetrix, Santa Clara, CA, USA). Human Genome Build 37.2 was used for the analyses of copy number variations. These analyses reconfirmed the presence of chromosome 3 abnormalities (case 1: arr 3q12.2q27.3(100,678,757–186,599,696)cth; case 2: arr 3p12.2q26.31(83,272,290–172,476,033)cth). A common deleted or duplicated region on chromosome 3q was identified in both analyzed cases (Table 1). It will be interesting to determine whether the interstitial deletion and 3q gain observed in these 2 cases represent recurrent features in other t(11;15) cases, and whether any commonly deleted or duplicated genes at chromosome 3 cooperate in the leukemogenesis of this rare MLL leukemia. Another notable finding from LDI-PCR analyses of the fusion breakpoints was that our 3 cases showed genomic breakpoints located within exon 11 of the CASC5 gene, suggesting a common breakpoint cluster region (Figure 1). The structures of MLL, CASC5, and the putative fusion protein are presented in Figure 1. Although CASC5 is a known component of the MIS12 complex, which may be fundamental for kinetochore formation and proper chromosome segregation during mitosis (acts in coordination with CENPK to recruit the NDC80 complex to the outer kinetochore),1110 there is not yet enough knowledge on the mechanism of leukemogenesis of MLL-CASC5 fusion protein. According to a recent study by Grossman et al.,12 not only additional cytogenetic aberrations (42.4%) but also RAS pathway mutations including NRAS (22%) and KRAS (20.3%) are frequently found in MLL-rearranged AML (RAS mutation results in our patients are provided in Table 1). Therefore, further studies on the MLL-CASC5 fusion protein itself, and their cooperative mutations such as RAS signaling pathway mutations or chromosome 3q abnormalities, would be required in the near future.

Figure 1.(A) Breakpoint cluster region (BCR) of the CASC5 gene. (B) Size and location of functional domains of the MLL wt, CASC5 wt, and of the MLL-CASC5 (predicted) fusion protein. AT: AT hook, SNL: subnuclear localization; MT: methyltransferase; BD: binding domain; TAD: transcriptional activation domain; PHD: plant homeo domain; SET: Su(var)3e9; Enhancer-of-zeste: Trithorax; CC: coiled coil.

Regarding clinical outcome, out of 8 patients for whom clinical data were available, only 3 are in complete remission, whereas 5 patients died with a mean survival period of 10.4 months. Based on these data it appears that the clinical course and prognosis of MLL-CASC5 leukemia are generally poor and that such patients have relatively short periods of survival. Since MLL fusion genes, particularly in treatment-related or secondary hematopoietic malignancies, are indicative of a rather poor clinical outcome, monitoring for therapy response or an impending relapse using the genomic MLL fusion sequences as patient-specific molecular targets bone marrow transplantation may be indicated.1413 However, due to the small number of MLL-CASC5 leukemia cases, no final conclusion regarding its prognostic relevance can be drawn.

Taken together, to the best of our knowledge, this is the most comprehensive analysis of MLL-CASC5 leukemia to date. Although only a handful of cases have now been confirmed on the molecular level, it is efforts such as the use of LDI-PCR or other new genomic analyses that allowed the advancement of the classification and characterization of MLL-associated leukemias.151 Further studies on MLL-CASC5 patients with regards to the molecular and clinical features will increase our understanding of this specific subtype of MLL-rearranged leukemias.