KMT 2 A-ARHGEF 12 , a Therapy Related Fusion with Dismal Prognosis

Nada Assaf (  assaf.nada@outlook.com ) CH Versailles: Centre Hospitalier de Versailles https://orcid.org/0000-0002-3352-2660 Raphael Liévin CH Versailles: Centre Hospitalier de Versailles Fatiha Merabet CH Versailles: Centre Hospitalier de Versailles Victoria Raggueneau CH Versailles: Centre Hospitalier de Versailles Jenifer Osman CH Versailles: Centre Hospitalier de Versailles Marc Spentchian CH Versailles: Centre Hospitalier de Versailles Rathana Kim Hôpital Saint-Louis: Hopital Saint Louis Florian Renosi Université de Bourgogne: Universite de Bourgogne Francine Garnache Université de Bourgogne: Universite de Bourgogne Virginie Eclache Hôpital Avicenne: Hopital Avicenne Isabelle Luquet IUCT Oncopole: Institut Universitaire du Cancer Toulouse Oncopole Lilia Corral Abascal Centro ricerca tettamenti monza Mariella D’Angiò University of Milan–Bicocca: Universita degli Studi di Milano-Bicocca Patrizia Larghero University of Frankfurt: Goethe-Universitat Frankfurt am Main Claus Meyer University of Frankfurt: Goethe-Universitat Frankfurt am Main Rolf Marschalek University of Frankfurt: Goethe-Universitat Frankfurt am Main Philippe Rousselot CH Versailles: Centre Hospitalier de Versailles Christine Terré CH Versailles: Centre Hospitalier de Versailles


Introduction
The KMT2A gene, previously known as MLL, is located at chromosome 11q23.3. KMT2A acts as a catalytic, binding and recognizing part of the COMPASS complex, a promoter of embryonic development and early hematopoiesis. [1] KMT2A rearrangements can occur in a wide variety of gene transformations including single nucleotide insertions or deletions, translocation of the KMT2A region into partner chromosomes or vice versa. Additional rearrangements, including gene duplication or reciprocal translocations, have also been shown to be involved in the modi cation of the KMT2A protein product. [2] With the exception of internal partial tandem duplications, rearrangements at the KMT2A locus lead to the formation of hybrid genes composed of portions of KMT2A and portions of many partner genes. [3] To date, more than one hundred different KMT2A fusion alleles have been described. (2) The KMT2A gene exhibits two different breakpoint cluster regions (BCR): major and minor. (4) Breakpoints in the major BCR localize mainly at introns 9-11, while breakpoints in the minor BCR occur within KMT2A introns 21-23. In many cases, chromosomal rearrangements of KMT2A result in the overexpression of HOX genes leading to increased hematopoietic stem cell proliferation and decreased regulated apoptosis. [3] The clinical consequences of the frequent KMT2A fusion are well known [4] , however little is known about the effect of rare translocation partner genes (TPG). We hereby present the diagnostically challenging case of an adult therapy related B lymphoblastic leukemia (B-ALL) harboring the rare KMT2A-ARHGEF12 fusion at an unusual breakpoint region.
In the presence of speci c bone lesions, a diagnosis of Multiple Myeloma (MM), International Staging System 3 was made. He was treated according to the CASSIOPEIA study [5] , standard arm, with 4 cycles of bortezomib, thalidomide and dexamethasone. After complete remission, autologous hematopoietic stem cell transplant (HSCT) was performed following a conditioning regimen with melphalan 200 mg/m². Thalidomide was omitted during consolidation due to the development of stage 2 neuropathy. Until September 2019, our patient was placed on maintenance daratumumab with serial follow up by ow cytometry.
Upon presentation for the last cycle of daratumumab, the patient was found to have petechia. He was asymptomatic and denied fever, night sweats or recent weight loss. His vital signs were normal. There was no lymphadenopathy, splenomegaly, spontaneous/provoked bone pain nor evidence of external plasmacytoma. The neurologic exam was negative, except for residual peripheral stage 1 neuropathy. Complete blood count showed macrocytic anemia (hemoglobin = 7.4 g/dL; mean corpuscular volume = 105fL), thrombocytopenia (platelets = 23 x 10 9 /L) and leukocytosis (white blood cells = 21.8 x 10 9 /L). The peripheral blood was also marked with recent (< 2 months) monocytosis (absolute number = 2.02 x 10 9 /L; percentage = 16%) and eosinophilia (absolute number = 0.63 x 10 9 /L; percentage = 5%). Circulating blasts were found, accounting for 1% of the total white blood cell population. Serum protein electrophoresis/immuno xation was negative. Proteinuria and MRD for MM were undetectable.
In addition, plasmacytoid dendritic cells (pDCs) were observed accounting for 5% of the total marrow cell population by morphology. This population consisted of mature large cells characterized by an abundant, clear cytoplasm with extending projections and a small nucleus with coarse chromatin and absent nucleoli (Fig. 1, panels A-D). The pDCs, accounting for 8% of cells by ow cytometry, were positive for CD123 (strong), CD4 and the speci c markers CD303, CD304, FcER1 and Lamp-5 (BAD-LAMP). CD56 and cTCL1 were negative. These observations were con rmed by a bone marrow biopsy and referral to a specialized center (Etablissement Français du Sang Bourgogne-Franche-Comté, Besançon, France).
Next Generation Sequencing (NGS) performed on the bone marrow sample revealed the presence of NRAS G13D, RUNX1 F136C, DNMT3 N757D and R792 mutations.
B-ALL molecular analysis revealed the absence of fusion transcripts using a targeted approach by reverse transcriptase multiplex ligation-dependent probe ampli cation (RT-MLPA) [6] . No clonal Ig-TCR rearrangements were identi ed using EuroMRD guidelines. [7] No abnormalities were observed on conventional karyotyping (46,XY[20]). As recommended by the GFCH (Groupe Francophone de Cytogénétique Hématologique) for normal karyotype B-ALL, FISH for the detection of KMT2A rearrangements (break apart double color 11q23, Metasystems Probes, Heidelberg, Germany) was performed. In contrast with RT-MLPA results, a rearrangement was detected in 96/100 examined cells with loss of the 3'signal ( Fig. 1, panel E). Referral to the German Diagnostic Center of Acute Leukemia (DCAL) Goethe-University Frankfurt allowed the characterization of the fusion gene partner using capture probes on the entire KMT2A locus. [8] A novel KMT2A (intron 35) fusion with ARHGEF12 (intron 1) was identi ed. Speci c PCR probes matching our patient's KMT2A rearrangement were then designed for serial follow up (Table 1). Table 1 Follow up PCR probes for minimal residual disease detection.

Primer Sequence
MLL_intron35-Fprimer ATCCTGCACCTCCTTGTGTTG ARHGEF12_intron1-Rprimer AAGCTTGCTAGGTGTTAAGTTTGG MLL-ARHGEF12_junction-Probe ACTGCTTCCAGCGGGTCACAGATACC Treatment with Tagraxofusp, an anti-CD123 cytotoxin -targeting B lymphoblasts and pDCs-was initiated in preparation for allogenic HSCT which could not be performed due to disease complication by bacterial septicemia resulting in patient death.

Discussion
Diagnostic challenge: Myelomonocytic hyperplasia, B lymphoblasts and mature pDCs along with preservation of some normal hematopoiesis were present in our patient's bone marrow. Dysplasia and monocytosis along with the absence of a BCR-ABL1 fusion transcript, PDGFRA, PDGFRB and JAK2 rearrangements suggest a diagnosis of Chronic Myelomonocytic Leukemia (CMML) [4] , con rmed by ndings persistence for > 3 months. The presence of B lymphoblasts suggest disease transformation.

Expansion of plasmacytoid dendritic cells presents in 2 distinct pathologic forms: Blastic pDC Neoplasm (BPDCN) and Mature pDC Proliferation (MPDCP).
These entities are distinguished by the mature morphology of expended cells in MPDCP and the absence of CD56 expression. [4,9] MPDCP is associated with the presence of a myeloid neoplasm (frequently CMML), but also with MDS or acute myeloid leukemia (AML) with monocytic differentiation. In CMML, nodules of mature pDCs can be seen in around 20% of bone marrow biopsies. [4] MPDCP constitutes a rare entity, with around 80 cases described in the medical literature. [9] Patients are predominantly males above 60 years of age. [9] Treatment and prognosis depend on the underlying myeloid condition rather than the associated MPDCP. [9] KMT2A rearrangement: A well-recognized characteristic of KMT2A rearrangements is their ability to associate with multiple TPGs. Using long-distance inverse PCR analysis, Meyer et. al report that 9 different fusion genes account for 90% of KMT2A rearrangements while the remaining 10% are spread among not less than 100 TPGs. [2] Thus, diagnosis using predesigned PCR probes complementary to the most common variants results in around 10% false negative rate. In addition, around 15% of KMT2A transformations are cryptic resulting in a visually normal chromosome 11 [10] , re ected in in our case. As such, re ex testing using break apart dual color FISH is recommended in all suspected B-ALL and mixed lineage acute leukemia cases to rule out a KMT2A rearrangement. [11] Once the partner gene is identi ed, speci c PCR probes can be designed and used for MRD monitoring. This approach was veri ed in several prospective studies. [12][13][14] The KMT2A-ARHGEF12 fusion was rst described by Kourlas et al. in a case of complex karyotype AML [15] (Table 2; case 1). To date, only three additional cases were reported in the literature [16][17][18] (Table 2; cases 2-4). Two other patients with this rare fusion were diagnosed at the DCAL (Table 2; cases 5-6). The majority are treatment related malignancies or occurring after long term herbicides exposure. This suggests a molecular trigger for this unusual fusion. ARHGEF12 is located on chromosome 11, telomeric to KMT2A. The rearrangement mechanism thus involves a 2MB interstitial deletion resulting in KMT2A-ARHGEF12 fusion, usually occurring within the major BCR of KMT2A. In our case (Table 2; case 7), the rearrangement resulted from the in-frame fusion of intron 35 of KMT2A and intron 1 of ARGHEF12, where nearly the entire KMT2A protein is fused to the entire ARGHEF12 protein. This KMT2A breakage, occurring outside the major and minor BCR (8) , has never yet been described, to the best of our knowledge. In addition to the identity of the TPG, the location of the KMT2A breakpoint was found to affect the clinical behavior and prognosis of the hematological malignancy. [2,19] Failure of complete remission and short overall survival was observed for all reported adult patients with KMT2A-ARHGEF12 fusion ( Table 2). The clinical effect of the rare breakpoint found in our patient remains to be elucidated.  [15] Case 2 [16] Case 3 [17]

Conclusion
In conclusion, reporting unusual genetic aberrations is essential to improve our knowledge of the biology and clinical features of hematologic disorders. Additional information is needed for the determination of the prognosis and adequate treatment modalities of rare KMT2A rearrangements.