NRIP3: a novel translocation partner of MLL detected in a pediatric acute myeloid leukemia with complex chromosome 11 rearrangements

MLL-rearranged acute myeloid leukemia (AML) is associated with an adverse outcome in most treatment protocols. The MLL-gene exhibits an 8-kb breakpoint cluster region, which behaves as a hotspot for chromosomal translocations. So far more than 50 different fusion partners of the MLL-gene have been identified.1 In pediatric AML, the main translocations are: t(9;11), t(11;19); t(6;11) and t(10;11) accounting for almost 15–20% of the cases(2). Here, we report on a novel translocation partner of MLL on chromosome 11 in a pediatric AML case.

A 5-month old boy presented with AML FAB-M5 and a white blood cell count of 9.8×10/L. CSF analysis showed CNS involvement with a cell count of 1.4×10 cells/mm and with 93% blasts. Immunophenotyping of bone marrow and peripheral blood further confirmed a monoclonal population in at least 50% of which showed the following aberrant phenotype: CD34, CD117, CD13, CD33, CD15s, CD14, CD4, CD45 and MPO. The child responded well to chemotherapy according to the DCOG-AML 97 protocol. The patient is in continuous complete remission seven years after diagnosis.

RBA and QFQ-banded karyotyping and fluorescence in situ hybridization (FISH) showed a double inversion on chromosome 11 in combination with a rearrangement involving chromosome 3 (Figure 1).

At the time of diagnosis the karyotype was 46,XY,der(3)t(3;11)(p21;q23)ins(3;11)(q23;p12p15),der(11)d el(11)(p12p15)inv(11)(p1?2q1?1)inv(11)(q1?1q23)t(3;11)(p2 1;q23). FISH using the MLL Dual Color, Break Apart Rearrangement Probe (Vysis/Abbott, Des Plaines, IL, USA) confirmed the MLL gene rearrangement showing the 3’ probe on the short arm of the der(3) and the 5’ probe high on the long arm of the der(11).

Long distance inverse (LDI)-PCR was performed as previously described.1 In this case this technique revealed that 5’MLL (intron 9) was fused to NRIP3 (intron 1) located on chromosome 11p15. Moreover, 3’MLL was fused to sequences from chromosome 3q21.3 (FLJ40473). Subsequently, the MLL-NRIP3 fusion gene was identified with RT-PCR (Figure 2), using an MLL exon 8 specific forward primer (5'-CGTCGAGGAAAAGAGTGA-3') combined with an NRIP3 exon 3 specific reverse primer (5'-CAGGCCAAAGAGATGAGAT-3').

Since these results were not in concordance with the observed karyotype, additional FISH analysis was performed. A paint for chromosome 11 showed that there were chromosome 11 sequences present on the short arm of the der(3), and on a small region on the der(3)(q). The pericentric inversion of chromosome 11 was confirmed using probes on both sides of the centromeric region (Figure 2). The 11p telomeric probe was present on the top of the der(11)(p). The paracentric inversion of 11q was confirmed using probes on 11q13 and 11q21. The 11q telomeric probe was present on the top of the der(3)(p) confirming the t(3;11)(p21;q23). The break apart probes for NUP98 (11p15.4)(3) were unexpectedly detected on the top of the der(3)(p), near the 3’MLL localization. Subsequent hybridization using a more centromeric probe on 11p15.4 (RP11-21N2), which is 5 Mb telomeric to NRIP3, showed the signal on the der(11)(q) near the probe for 5’MLL based on inverted DAPI banding pattern. A probe covering part of the FLJ40473 region showed in addition to the normal location on 3q21, a weaker signal on the top of the der(3)(p) near the location of 3’MLL and NUP98 (Figure 2). We were not able to determine the origin of the small insertion of chromosome 11 sequences on the der(3)(q). However, FISH results clearly demonstrated that there were many more chromosome 11 and 3 rearrangements present than expected on the basis of the conventional karyotyping.

Figure 1.Karyogram of chromosomes 3 and 11. (A) Partial RBA-banded karyogram showing chromosomes 3 and 11. The der(3) and the der(11) are shown on the right. (B) Schematic representation of the normal chromosome 3 and 11 (middle) and the der(3) (left) and the der(11) (right). The band designations provided were all observed using specific FISH probes. The designation #11 is where the whole paint for chromosome 11 showed hybridization to the der(3). Used probes were (from pter to qter) for chromosome 3: RP11-438J1 (3p25), RP11-969E9 (3p21), RP11-451E6 (3p12.3), RP11-79F5 (3p12.1), RP11-456K4 (3q21), RP11-82C9 (3q26); for chromosome 11: RP11-120E20/348A20 (NUP98, 11p15.4), RP11-21N2 (11p15.4), RP11-102E22 (11p11.2), pLC11a (centromere 11), RP11-114D10 (11q12.2), 4179 (11q13), cos3.16 (11q21), MLL break apart (11q23.3), RP11-133I16 (11q23.3), 11qtel.

In this case with complex rearrangements of chromosome 3 and 11 a novel translocation partner of the MLL-gene was detected. We have shown that the translocation partner was found on chromosome 11 with LDI-PCR. This technique revealed the NRIP3 gene on 11p15 as a novel translocation partner of MLL in pediatric AML, while the 3’ part of MLL was translocated to chromosome 3. The latter is thought not to be of importance since the reciprocal MLL translocations are often not expressed. Furthermore, it has been suggested that the MLL translocation partners are not randomly selected but that they are part of a protein network serving common functional processes. For example, interactions have already been described between AF4 and AF9 and ENL and AF4/AF10, which play a functional role in leukemogenesis.4,5 So far, the function of NRIP3 is not known, although it is one of the genes to be frequently hypermethylated in non-small cell lung cancer, hence it may potentially play a role in the pathogenesis of other cancers.6 As this is the first case in which NRIP3 is involved as a translocation partner for MLL, no conclusions can be drawn with respect to the clinical relevance and prognostic value. However, our patient has been in continuous complete remission for more than seven years.

Acknowledgments:

J.F. van Galen and E. van Drunen for performing additional FISH analysis.

Footnotes

• Funding: projects of B.V.B are funded by the NWO 'Netherlands Organisation for Scientfic Research’. This work is also funded by grant 107819 from the Deutsche Krebshilfe to R.M.