New Variant Translocation ( 8 ; 9 ; 21 ) ( q 22 ; p 24 ; q 22 ) in a Patient with Granulocytic Sarcoma Concurrent with Acute Myeloid Leukemia

Granulocytic sarcoma is a form of acute myeloid leukemia which may occur in any anatomical site. Isolated pancreatic granulocytic sarcoma is however, extremely rare. Translocation t(8;21) is the most common cytogenetic abnormality found in leukemia patients with granulocytic sarcoma and is associated with a relatively good prognosis when treated with chemotherapy. Variants of the t(8;21) are uncommon and account for approximately 3% to 4% of acute myeloid leukemia associated with t(8;21) and are rarely described in acute myeloid leukemia cases associated with granulocytic sarcoma. We report here a patient with acute myeloid leukemia and a novel variant t(8;9;21)(q22;p24;q22) with suspected granulocytic sarcoma in pancreas. A dual-color fluorescence in situ hybridization analysis with RUNX1T1 and RUNX1 probes, revealed the presence of an RUNX1/RUNX1T1 fusion signal in this translocation. To the best of our knowledge, a variant of t(8;21) in GS was rarely described and the involvement of the 9q22 region is the first time described here even in isolated AML-M2. We conclude that further accumulation of similar cases is needed and that genetic exploring of variants of t(8;21) may be helpful for a better understanding of molecular pathogenetic mechanism.


Introduction
Granulocytic sarcoma (GS) is an uncommon and localized extramedullary tumor composed of immature granulocytic cells.It is also known as extramedullary myeloid tumor or chloroma [1].It forms a solid malignant tumor consisting of myelocytes or granulocytes and is typically located in bone while occurrence is rare in other parts of the body such as in skin, soft tissue and lymph nodes [2].The tumor is a rare event of acute myeloid leukemia (AML).GS can occur at presentation, during treatment or in relapse of AML patients.Several chromosome rearrangements were associated with GS, especially t(8;21) and, less often inv (16)(p13;q22).In deed, it is reported in many series of patients with t(8;21) that GS occurs in approximately 18% of this population, which is four times the expected incidence in AML [3].
Molecular characteristics of the (8;21) translocation have been extensively studied.The genes involved in this translocation are the RUNX1 (AML1) gene on chromosome band 21q22 and the RUX1T1 (ETO) gene on chromosome band 8q22, leading to formation of a chimeric RUNX1/RUNX1T1 (AML1/ETO) fusion gene on the derived chromosome 8 [4].The fusion protein RUNX1/RUNX1T1 recruits N-CoR, sin3, and histone deacetylases which leads to transcription repression.These mechanisms probably play a contributing role in AML leukemogenesis [5].Variants of t(8;21)(q22;q22) involving a third or fourth chromosomes are seen in ~4% of AML cases [6].Patients with these variants show typical features of t(8;21) AML morphologically and, in the limited number of cases examined, the RUNX1/RUNX1T1 fusion transcripts detected were similar to those of t(8;21) [7].
Only few cases of GS of the pancreas had been reported in literatures [8].We hereby reported a case of suspected GS in the pancreas, in a male patient with AML.Cytogenetic studies showed a complex karyotype including a novel translocation (8;9;21)(q22; p24;q22) in all analyzed cells.

Patient
A 60-year-old Tunisian male, with no significant past medical history, was referred to the Aziza Othmana hospital, with a general malaise and leukocytosis.His complete blood count showed an anemic condition, with hemoglobin at 8.0 g/dL, total white blood cell count at 16,700/mm 3 , and platelet count at 28,000/mm 3 .

Cytogenetic Analysis of Bone Marrow Cells
BM samples were collected at presentation, referred to our laboratory.BM mononuclear cells were cultured for 48 hours and chromosome preparations were made by an R-banding method.Twenty metaphases from each specimen were analyzed and karyotypes as described previously [9] in accordance with the International System for Human Cytogenetic Nomenclature [10].

FISH Analysis
A dual-color FISH assay using RUNX1T1 and RUNX1 specific probes (Vysis, Downers Grove, IL) was performed on BM cells, as previously described [11].The RUNX1T1 probe was directly labeled with Spectrum Orange, and the RUNX1 probe was directly labeled with Spectrum Green.The hybridizations were performed on fixed cell pellets.
After hybridization and washing, cells were counterstained with DAPI (4',6-diamidino-2-phenylindole), then were examined with a fluorescent microscope equipped with appropriate filters and Cyto-Vision FISH system image capture software (ZeissAxioskop 2 plus).
In a normal case, the hybridization with the RUNX1T1 (orange) and the RUNX1 (green) probes showed two orange and two green signal patterns.However, in a case with the t(8;21), the hybridization with these probes showed one or two fusion signals (orange/green or yellow) corresponding to one or two fusion genes RUNX1/ RUNX1T1, one orange and one green signal patterns corresponding to the normal copies of the RUNX1T1 and the RUNX1 genes, respectively.
Slides contain no metaphases so the interphase FISH analysis for the RUNX1/RUNX1T1 probe showed one normal orange (RUNX1T1) and one normal green (RUNX1) signals on normal chromosomes 8 and 21 and one orange-green fusion signal corresponding to the co-localization of RUNX1T1 and RUNX1 signals on probably the rearranged chromosome 8 as, thus confirming the presence of the RUNX1/RUNX1T1 fusion gene in this translocation.FISH results revealed also the presence of a green signal on the derivative chromosome 21 and of one small orange signal (RUNX1T1) which would be probably located der(9) (Figure 1(B)).

Discussion
GS is an uncommon manifestation of AML, and the incidence of extramedullary disease in AML patients is about 3% -8%.Isolated granulocytic sarcomas located in the pancreas are exceptional, only few cases were previously described [2] [12], and have often led to initial erroneous diagnosis.Immunohistochemical methods are essential in order to obtain correct diagnosis [13].
Although, karyotype analyses were not reported in many cases of GS in the literature, the vast majority of abnormal karyotypes in patients with AML involved t(8;21).However, the prognostic significance of the presence of GS in these patients is not clearly defined.It has been reported that AML-M2 patients with positive RUNX1/RUNX1T1 showed good response to the treatment, with relatively high complete remission rate and long duration of remission [14].
In this study, we report a male patient with a suspicious pancreatic GS concurrent with AML type FAB-M2.Because the general state of the patient was quickly altered, confirmation of the diagnosis by pathology and/or immunohistochemistry evaluation of the tumor was, infortunately, not possible.The karyotype revealed a novel three-way translocation t(8;9;21)(q22;p24;q22).Fluorescence in situ hybridization studies revealed the presence of the RUNX1/RUNX1T1 chimeric gene which clearly indicates that the RUNX1/RUNX1T1 fusion from t(8;21) is one of the main causes of leukemogenesis in the variant translocations associated with t(8;21).As far as we know, this is the first time in which we describe a variant t(8;21) in GS with AML and we report the involvement of the 9p24 in both AML and GS.The participation of the chromosome 9 in a variant t(8;21) has never been described before in GS with AML however, Kawakami et al. [15] recently, reported the involvement of 9q34 breakpoint in a novel variant of t(8;21) in a case with AML-M2.However, many other chromosomes were found implicated in a three-way translocation with t(8;21) as described in Table 1 including the case we reported here.
The phenomenon is already difficult to explain.The mechanism of the occurrence of GS with t(8;21) may be related to the deregulation of Core Banding transcription factors involved in cell recognition and adhesion.The involvement of a third chromosome complicated much more the molecular pathogenetic mechanism.
Generally, chromosome 9p24 abnormalities are rare and sometimes involve JAK2 tyrosine kinase which is well known.The subset with JAK2 translocations are usually associated with myeloproliferative neoplasms and harbor a missense somatic mutation (JAK2V617F), suggesting a cause-effect relationship [31].Functionally, the V617F mutation enhanced the JAK2 kinase activity, and conferred erythropoietin hypersensitivity in the affected cells [31] [32].Many of the effects of JAK2 activation in the cells are mediated by the signal transducers and activators of transcription (STAT), and JAK2-STAT signaling is frequently activated both in hematologic malignancies and solid tumors [33] [34].
The prevalence of JAK2-V617F in de novo AML is low, around 3%.Recently, Lee et al. [32] and Vicenti et al. [35]  Although, the consequence of this mutation in leukemia pathogenesis remains elusive at this stage, these data could suggest, that this mutation is an additional genetic event in some patients with AML that contributes to the myeloid proliferation by diverse mechanisms [36], but has no impact on the evolution of the disease as demonstrated by Vicente et al. [35].
To the best of our knowledge, we report here a novel variant translocation (8;9;21)(q22; p24;q22) never described before in AML with GS and even in the isolated AML-M2.

Conclusion
We conclude that, the characterization of genetic aberrations in AML concurrent or not with GS has substantially improved our understanding of the pathogenesis of this disease and is of growing importance for more efficient risk assessment for individual patients.Thus, further accumulation of similar cases with variants of t(8;21) is needed.