the natural history of acute myeloid leukamia ( aml ) is typically unfavorable with rapid progression leading to death in the absence of specific therapy . spontaneous remission ( sr ) is a rare but well - documented event with hundreds of cases published to date . despite reported cases and progress of conventional cytogenetic and molecular biology , ever since the generalization and rapid start of induction chemotherapy after acute myeloid leukemia diagnosis , sr has been very rarely observed . in particular , aml with monocytic differentiation ( aml m5 ) represents the largest subgroup of aml with spontaneous remission reported in the literature , with seven cases published since 1980 17 but the existence of a link between aml m5 and sr remains elusive . for the first time , we report the cases of three patients with similar genetic characteristics diagnosed with aml m5 according to the fab classification , who underwent rapid and transient sr . aml diagnoses were established by conventional morphologic , histologic , cytochemical , immunophenotypic , and genetic criteria . metaphases were obtained and analyzed using standard techniques of colchicine arrest , hypotonic treatment , and 3:1 v / v methanol / acetic acid fixation . karyotypes were reported using the international system for human cytogenetic nomenclature , based on the analysis of at least 20 metaphases when possible . dna was extracted from bone marrow using proteinase k , followed by salt and ethanol precipitation and stored at 20c in 10 mmol / l tris  cl 1 mmol / l edta ( ph 8) buffer . pcr reactions were performed on 100 ng of dna using npm1-f ( 5- tgg ttc ctt aac cac att tct tt-3 ) and npm1-r ( 5- ttc cat aca tac tta aaa cca agc a-3 ) to exon 12 of npm1 . , two fragments were amplified using pp1-f : 5-tcgccatgccgggagaactctaac-3 ; pp1-r : 5-ctggtaagggaagaggccggccag-3 and pp2-f : 5-ccgctggtgatcaagcagga-3 ; pp2-r : 5-gggcaagcctcgagatcc-3 primers . only the hotspot region was sequenced for flt3 ( flt3-f : 5-ccaggaacgtgcttgtcacccac-3 and flt3-r : 5-tcaaaaatgcaccacagtgag-3 ) . all the pcr products were visualized on 1.5% agarose gel electrophoresis after gel red staining . all pcr - amplified samples were purified by standard methods and sequenced on abi prism 3130 genetic analyzer ( life technologies , carlsbad , ca ) . in january 1998 a 24-year - old female patient with no medical history presented with acute febrile polyarthralgia and fatigue . physical examination showed reduced performance status ( ecog = ; 2 ) and high grade fever ( 40c ) but no sign of organ infiltration and no lymphadenopathy . a full - body ct scan revealed no abnormalities . upon admission , blood cell count showed moderate pancytopenia ( hb 10 g / dl , platelets 45 g / l and wbc 2 bone marrow aspirate was very poor and showed 58% of blast cells with monocytic appearance and lobulated nuclei . the bone marrow biopsy found a blast infiltration by cells of monocytic morphology expressing myeloperoxidase ( mpo ) . an empirical broad spectrum antibiotic therapy was started and led to prompt clinical improvement with fever defervescence , normalization of hemogram values and a complete sr documented by bone marrow examination ( blasts < 5% ) . no colony - stimulating factors were administered and the patient was able to return home . second leukemia relapse occurred 1 year later with hypercellular bone marrow aspirate showing 80% , mpo negative , esterase - positive monoblastic cells . the patient then underwent stem cell transplant with a fully hla - matched unrelated donor and standard myeloablative regimen . npm1 mutation analysis was performed on dna extracted from a fixed cytogenetic pellet and revealed a favorable npm1 mutation in this patient ( c.861_862instgtc ; identified in cosmic database as cosm20861 ) . no mutations were found in either cebpa or flt3 ( neither internal duplication ( it d ) nor mutations in tyrosine kinase domain ( tkd ) ) . a 33-year - old male patient with no medical history was admitted in january 2013 . he had presented fever , fatigue , moderate weight loss , and anorexia for 1 month before hand . g / l with 5% of blast cells ) , and normal platelet count ( 175 bone marrow aspirate showed reduced cellularity with 10.5% of mpo positive blast cells and 14.5% of monocytes with dysgranulopoiesis . a bone marrow biopsy confirmed aml m5a diagnosis according to the fab classification with flow cytometry revealing cd68 + , cd117 + , cd15 + , cd34 , cd163 , and mpo blast cells . the karyotype was normal , fish analysis showed no mll rearrangements , and molecular biology identified a type j npm1 mutation ( an insertion of tatg , fig . 1 ) without flt3-itd , flt3-tkd , or cebpa mutation . due to the patient 's septic condition , antibiotic therapy with piperacillin / tazobactam was introduced and cytostatic drugs were not immediately started . sr was observed in the following days with normalization of complete blood count ( cbc ) and clearance of blasts cells in the blood and bone marrow . blast cell morphology , cytogenetics , and molecular biology were identical to that of first diagnosis . the pulmonary aspergillosis has resolved and the patient is still alive in complete hematological remission 17 months after three courses of high - dose cytarabine . a 74-year - old woman was hospitalized in our leukemia unit in january 2014 following the discovery of anemia and leukocytosis with monocytosis in the context of febrile pneumonia . this patient had a history of high blood pressure , diabetes , and high cholesterol . cbc revealed a hemoglobin level of 8.8 g / dl , a white blood cell count of 21 g / l with 8% of neutrophils and 15 bone marrow aspirate displayed 14% of blasts comprising granular blasts and monoblasts , and 41% of promonocytes . the karyotype was normal , fish analysis identified no mll rearrangements , and molecular biology revealed a type a npm1 mutation ( duplication of tctg , fig . 2 ) with neither flt3-itd nor flt3-tkd , nor cebpa mutation . antibiotic therapy with amoxicillin / clavulanic acid was started for the pneumonia and we observed a leukemia sr with normalization of cbc and disappearance of bone marrow blasts ( despite persistence of approximately 15% of dystrophic monocytes ) . given the sr , the patient was allowed to return home . flow cytometry performed on the bone marrow identified a cd13 + , cd33 + , cd117 + , cd64 + , and cd14 + blast population , in favor of a monocytic differentiation . cytogenetics and molecular biology were identical to that of the initial diagnosis . following aml type chemotherapy , electropherogram as generated by sanger sequencing showing type a npm1 exon 12 mutation in dna extracted from bone marrow . in january 1998 a 24-year - old female patient with no medical history presented with acute febrile polyarthralgia and fatigue . physical examination showed reduced performance status ( ecog = ; 2 ) and high grade fever ( 40c ) but no sign of organ infiltration and no lymphadenopathy . a full - body ct scan revealed no abnormalities . upon admission , blood cell count showed moderate pancytopenia ( hb 10 g / dl , platelets 45 g / l and wbc 2 bone marrow aspirate was very poor and showed 58% of blast cells with monocytic appearance and lobulated nuclei . the bone marrow biopsy found a blast infiltration by cells of monocytic morphology expressing myeloperoxidase ( mpo ) . an empirical broad spectrum antibiotic therapy was started and led to prompt clinical improvement with fever defervescence , normalization of hemogram values and a complete sr documented by bone marrow examination ( blasts < 5% ) . no colony - stimulating factors were administered and the patient was able to return home . second leukemia relapse occurred 1 year later with hypercellular bone marrow aspirate showing 80% , mpo negative , esterase - positive monoblastic cells . the patient then underwent stem cell transplant with a fully hla - matched unrelated donor and standard myeloablative regimen . npm1 mutation analysis was performed on dna extracted from a fixed cytogenetic pellet and revealed a favorable npm1 mutation in this patient ( c.861_862instgtc ; identified in cosmic database as cosm20861 ) . no mutations were found in either cebpa or flt3 ( neither internal duplication ( it d ) nor mutations in tyrosine kinase domain ( tkd ) ) . a 33-year - old male patient with no medical history was admitted in january 2013 . he had presented fever , fatigue , moderate weight loss , and anorexia for 1 month before hand . g / l with 5% of blast cells ) , and normal platelet count ( 175 bone marrow aspirate showed reduced cellularity with 10.5% of mpo positive blast cells and 14.5% of monocytes with dysgranulopoiesis . a bone marrow biopsy confirmed aml m5a diagnosis according to the fab classification with flow cytometry revealing cd68 + , cd117 + , cd15 + , cd34 , cd163 , and mpo blast cells . the karyotype was normal , fish analysis showed no mll rearrangements , and molecular biology identified a type j npm1 mutation ( an insertion of tatg , fig . 1 ) without flt3-itd , flt3-tkd , or cebpa mutation . due to the patient 's septic condition , antibiotic therapy with piperacillin / tazobactam was introduced and cytostatic drugs were not immediately started . sr was observed in the following days with normalization of complete blood count ( cbc ) and clearance of blasts cells in the blood and bone marrow . blast cell morphology , cytogenetics , and molecular biology were identical to that of first diagnosis . the pulmonary aspergillosis has resolved and the patient is still alive in complete hematological remission 17 months after three courses of high - dose cytarabine . a 74-year - old woman was hospitalized in our leukemia unit in january 2014 following the discovery of anemia and leukocytosis with monocytosis in the context of febrile pneumonia . this patient had a history of high blood pressure , diabetes , and high cholesterol . cbc revealed a hemoglobin level of 8.8 g / dl , a white blood cell count of 21 g / l with 8% of neutrophils and 15 bone marrow aspirate displayed 14% of blasts comprising granular blasts and monoblasts , and 41% of promonocytes . the karyotype was normal , fish analysis identified no mll rearrangements , and molecular biology revealed a type a npm1 mutation ( duplication of tctg , fig . antibiotic therapy with amoxicillin / clavulanic acid was started for the pneumonia and we observed a leukemia sr with normalization of cbc and disappearance of bone marrow blasts ( despite persistence of approximately 15% of dystrophic monocytes ) . given the sr , the patient was allowed to return home . flow cytometry performed on the bone marrow identified a cd13 + , cd33 + , cd117 + , cd64 + , and cd14 + blast population , in favor of a monocytic differentiation . cytogenetics and molecular biology were identical to that of the initial diagnosis . following aml type chemotherapy , the patient is alive , in good health and in cr . electropherogram as generated by sanger sequencing showing type a npm1 exon 12 mutation in dna extracted from bone marrow . spontaneous remission of acute myeloid leukemia is an extremely rare and almost always transient event , with a mean duration in the literature of 7.7 months ( range 136 ) 8 . since 1979 , approximately thirty cases of aml sr have been reported 8,9 , although the underlying mechanisms involved remain unclear . a potential role of bacterial and/or fungal infections and blood transfusions was suggested in sr occurrence 10 by triggering an immune and antileukemic response . activation of the immune system and cytokines such as tumor necrosis factor ( tnf ) , interferon gamma ( ifn- ) , and interleukin-2 ( il-2 ) released during infections , in conjunction with an increase in the antibody - mediated cytotoxicity of natural killer ( nk ) cells and cytotoxic activity of t lymphocytes and macrophages , may play a role in the occurrence of sr 1,2,11,12 . indeed , immune response might be a potential mechanism of prolonged disease control : in 2012 , the case of a german patient who presented aml with a 10-year - long sr was reported . in this patient , it was demonstrated in vitro that nk cells displayed cytotoxic activity against the k562 myeloid leukemia cell line with upregulation of cd107a in flow cytometry 13 . in addition , infection is often accompanied by a hypergammaglobulinemia , which has been associated with aml sr in some cases 13 . the potentially beneficial effect of systemic infections in aml led to preliminary trials using bacterial extract inoculations or vaccination programs , which did not show a significant effect on survival or occurrence of remission 14,15 . our patients did not receive any blood transfusions but all presented with infections that responded to antibiotic treatment with normalization of cbc and occurrence of sr . unfortunately , no blood cytokine monitoring was performed in our three patients . to date , most cases of aml sr have been described in patients with normal karyotypes 3,11 . to our knowledge , this is the first report of three cases of sr in aml m5 according to the fab classification with normal karyotype and npm1 mutations . npm1-mutated aml represents a separate entity included in the aml who classification since 2008 , with a favorable prognosis in patients with normal karyotype aml ( aml - nk ) in the absence of the flt3-itd mutation 16 . the npm1 mutation is the most common genetic alteration found in aml - nk and is present in approximately one - third of adult aml 17 . npm1 mutations are located in the last exon ( exon 12 ) and consist in duplication or insertion of small nucleotide sequences . more than 50 mutations have been reported to date , leading to a npm1 protein variant with modification of the last seven residues , as well as four additional residues 13,17,18 . physiologically , npm1 encodes a nucleocytoplasmic shuttling protein ( npm1wt ) involved in the regulation of ribosome genesis and centrosome duplication 19 . npm1 mutation is associated with the aberrant cytoplasmic localization of npm1 , thereby disrupting its function and perturbing multiple cellular pathways 18,20 . the potential role of npm1 mutations in the occurrence of sr is not known but it has been previously reported that patients with aml - nk and mutated npm1 had a better response rate to induction chemotherapy 17,18 which could advocate for increased chemosensitivity of blast cells with npm1 mutation . recent findings suggest that npm1 mutations are associated with increased sensitivity to oxidative stress and that mutant npm1 is a target of arsenic trioxide - mediated oxidative stress 21,22 . systemic infections , usually described in cases of sr , are associated with exuberant activation of the immune system and high production of reactive oxygen species ( ros ) . thus , we hypothesize that the three cases of sr presented herein might be linked by the presence of mutant npm1 , and therefore be more susceptible to oxidative stress than cases with wild - type npm1 . moreover , we can speculate that there is a link between sr and ros that still need to be demonstrated . sr is an exceptional phenomenon , often transient and relatively well documented but the underlying molecular mechanisms are still unknown . npm1-mutated aml m5 patients who developed spontaneous remission after antibiotic treatment of febrile neutropenia present at diagnosis seem to form a subtype with favorable prognosis and chemo sensitive disease . a better understanding of this rare process could uncover new potential therapeutic targets for acute leukemia .
key clinical messagepatients with npm1-mutated aml m5 who develop spontaneous remission ( sr ) after antibiotic therapy at diagnosis seem to form a favorable prognosis and chemo sensitive subtype . 
 we report three cases of aml m5 patients with the same genotype that experienced transient sr and are now leukemia free after standard treatment .