Acute Promyelocytic Leukemia Cases Research Articles

Acute Respiratory Distress Syndrome in a Patient With Acute Promyelocytic Leukemia: Overlapping Between Differentiation Syndrome and COVID-19

Differentiation syndrome (DS) is a relatively common and severe complication in acute promyelocytic leukemia (APL) patients undergoing induction therapy with all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO). DS is a multisystem disorder with pulmonary involvement. The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is also a systemic disorder with similar pulmonary and other clinical manifestations as DS. Here, we report an APL case with overlapping between DS and COVID-19. After admission to the hospital, the patient was diagnosed with APL and underwent differentiation therapy with ATRA/ATO. In the meantime, COVID-19 was diagnosed with a positive polymerase chain reaction test of SARS-CoV-2 from an oropharyngeal swab. The patient developed acute respiratory distress syndrome, coagulopathy, and acute kidney injury, which fit the clinical pictures of both DS and COVID-19. The patient died at last and this complicate case imposed big challenges for clinicians due to the laboratory and imaging findings of DS disguised in the context of COVID-19. Therefore, comprehensive treatment strategy should be considered to balance the risk and benefit of differentiation therapy in the context of COVID-19.

Study of Bone Marrow Lymphocyte Subset in Acute Myeloid Leukemia.

Introduction Acute myeloid leukemia (AML) is a heterogenous disorder consisting of clonal expansion of myeloblasts. Tumor immunity plays an important part in the pathobiology of AML. Understanding the components of tumor immunity is important for understanding tumor pathogenesis and the principles of immunotherapy. Methods We studied 41 patients with AML, for total lymphocyte, CD4 positive helper T cells, CD8 positive cytotoxic T cells, and CD16/56 positive natural killer (NK) cells proportion. Quantification was done on bone marrow aspirate sample by flowcytometry. Whenever available, post induction bone marrow was also analyzed for the lymphocyte subset. Results No significant difference was noted in the percentage of blasts among the three risk categories: favorable, intermediate, and adverse. However, there was significant difference in the total lymphocyte among the risk stratification groups, being highest in the favorable group and lowest in the adverse group. CD8 positive cytotoxic T cells were significantly less in Acute Promyelocytic Leukemia (APML) cases ( p = 0.001). Total lymphocytes were, however, more numerous in APML ( p = 0.005). NK cell proportion was not significantly different between APML and non-APML patients. On completion of induction chemotherapy, bone marrow samples for 12 patients could be processed for lymphocyte subset. On comparing the baseline against the post induction bone marrow, it was observed that there was significant increment in the proportion of CD4 positive T lymphocytes ( p = 0.046). Conclusion There is a difference in lymphocyte subset amongst patients with AML. Larger studies including functional aspects are needed to better define the role of lymphocytes in disease pathogenesis.

Molecular characterisation of NPM1 and FLT3-ITD mutations in a central South African adult de novo acute myeloid leukaemia cohort.

BackgroundRecognition of molecular abnormalities in acute myeloid leukaemia (AML) has improved our understanding of its biology. NPM1 and FLT3-ITD mutations are recurrent in AML and clinically significant. NPM1 mutations are associated with a favourable prognosis, while FLT3-ITD mutations are an independent poor prognostic factor in AML.ObjectiveThis study described the prevalence and molecular characteristics of the NPM1 and FLT3-ITD mutations in a newly diagnosed AML patient cohort in central South Africa.MethodsThe study included 40 de novo AML patients. An NPM1 and FLT3-ITD multiplex polymerase chain reaction assay was optimised to screen patients for the respective mutations and were confirmed using Sanger sequencing. The prevalence of the NPM1 and FLT3-ITD mutations were determined, and mutation-specific characteristics were described in relation to patients’ demographic information and AML classifications.ResultsThe patients’ median age was 38.5 years, with 77.5% (n = 31) of patients being self-proclaimed Black Africans. AML with recurrent genetic abnormalities was most prevalent (57.5%; n = 23), of which acute promyelocytic leukaemia (APL) was most common (40.0%; n = 16). None of the patients had the NPM1 mutation. FLT3-ITD was present in 37.5% (6/16) of APL patients and in one (20.0%) of five AML patients with a t(8;21) translocation. Most patients had an FLT3-ITD allele ratio of ≥ 50% and ITD lengths of > 39 bp.ConclusionFLT3-ITD mutations were mainly found in APL cases at a similar prevalence as reported in the literature. High FLT3-ITD allele ratios and long ITD lengths predominated. No NPM1 mutations were detected.

PML-RARa of the Short but Not the Long/Variant Isoform Initiate from CD34+ TIM-3+ LSCs with Hierarchical Leukemic Organization

Acute promyelocytic leukemia (APL) is successfully treated with PML-RARa-targeted therapies. APL is divided into two groups with distinct PML-RARa subtypes: Long/variant (the L/V type) and short (the S type) isoforms. The L/V type PML-RARa is observed ~70% of APL patients, whereas the S type isoform, which lacks nucleus localization sequence located in exon6 of PML gene, is involved in ~30% of APL cases. PML-RARa contributes to leukemogenesis presumably by repressing transcriptional activities via its DNA binding properties in nucleus. However, recent studies have shown that PML-RARa mainly localizes in nucleus in the L/V type APL, whereas it dominantly distributes in cytoplasms in the S type APL. This study was planned to clarify difference in the role of PML-RARa in leukemogenesis in APL patients with the L/V or the S type.We performed the transcriptome analysis of APL blast cells from 4 cases each of the L/V type and the S type. Surprisingly, APL cells of the L/V and the S type showed significantly different gene expression pattern by gene set enrichment analysis. For example, pathways for apoptosis involving BCL-2, prostaglandin and T lymphocyte maturation were enriched only in the S type APL, whereas cytokine pathways were mainly enriched in the L/V type APL. More interestingly, the gene sets associated with functional leukemic stem cells (LSCs) (K.Eppert, et al. Nat Med, 2011) were enriched in the S type APL (NES 1.72, FDR q-value 0.083).In most AML of FAB types except for M3 (APL), self-renewing LSCs are concentrated in the CD34+CD38- population, and transplantation of CD34+CD38- LSCs of AML patients resulted in reconstitution of human AML in xenograft models. Based on this data, it is widely accepted that AML is hierarchically organized and is maintained by LSCs, like normal hematopoiesis is initiated from normal HSCs. However, it has been reported that reconstitution of human APL is usually difficult in xenograft models (D.Thomas and R.Majeti. Blood, 2017), although human APL was reconstituted in rare cases by a recent report (S.Patel, et al. Leukemia, 2012). We have reported that self-renewing AML LSCs express TIM-3, a signaling molecule activating the β-catenin pathway (Y.Kikushige, et al. Cell Stem Cell, 2015), but TIM-3 expression was negative in leukemic cells in most cases of APL. However, because the S type but not the L/V type APL has LSC signatures by transcriptome analysis, we revisited to test the LSC activity of the S type APL.By FACS analysis, CD34+CD38- cells were rarely found in the L/V type APL patients, and they largely lacked TIM-3 expression and did not have PML-RARa transcript (n=10), suggesting that these CD34+CD38-TIM-3- cells may be residual normal hematopoietic stem cells (HSCs). In contrast, CD34+CD38- cells from the S type APL strongly expressed TIM-3 and PML-RARa transcript (n=12), suggesting that they may be functional LSCs. We then tested whether CD34+ cells of the S type APL can reconstitute human APL in a xenograft model. Purified 0.4-3.0 x106 CD34+ APL cells were transplanted into sublethally irradiated NSG mice. Strikingly in the S type APL, CD34+ but not CD34- cells reconstituted human PML-RARa+ APL in NSG mice in 3 out of 5 patient tested, whereas in the L/V type APL, CD34+ cells were incapable of reconstitution in all 3 cases tested. The hCD45+CD34+TIM-3+ cells harvested from the primary NSG recipients again reconstituted the S type APL in serial xenotransplantion experiments. These results collectively suggest that the S type APL have LSCs capable of self-renewal within the CD34+ leukemic population, and may use TIM-3 signaling for self-renewal as in other FAB types of AML.Thus, APL with the S type, but not the L/V type PML-RARa have expression signature related to LSCs, and have functional LSCs with TIM-3 expression. Therefore, the leukemic hematopoiesis of APL with the S type is hierarchically organized as in case of AML of other FAB types. The mechanisms by which these 2 types of PML-RARa exert different functions to the LSC property should be critical to understand pathogenesis of APL. DisclosuresNo relevant conflicts of interest to declare.

RARB Translocations in Acute Promyelocytic Leukemia without Rara translocation

Introduction: Acute promyelocytic leukemia (APL) is defined by unique morphological features, typically abnormal promyelocytes containing azurophilic granules. Genomic basis of APL is characterized by the translocation t(15;17)(q22;q21), inducing fusions between promyelocytic leukemia (PML) and retinoic acid receptor-a (RARA). APL cells with PML-RARA respond to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), and recent clinical trials have achieved excellent outcome for typical APL with PML-RARA . Although a certain fraction of APL cases lacks PMR-RARA, most of them had translocation including RARA and other genes, namely, PLZF, NPM, and TBL1XR1 . Eventually, all known APL-associated translocations involve RARA, accounting for 99% of APL. However, RARA rearrangement is unable to be detected in the rest of APL cases, and molecular mechanisms underlying this small subset is still unclear. We here analyzed APL cases without RARA translocation, and identified a novel RARB fusion in the majority of patients with RARA -negative APL.Materials and methods: We first performed whole-genome sequencing to identify potential genomic alterations in a case with RARA -negative APL, and successfully identified an in-frame fusion between TBL1XR1 and RARB . As RARB, encodes retinoic acid receptor-b, and RARA are both members of the nuclear receptor superfamily, we further searched for RARB alterations in additional 4 cases diagnosed APL morphologically lacking RARA translocation, using whole-transcriptome sequencing, RT-PCR, and FISH, according to sample availability. The five RARA -negative APL cases consist of one male and four females, and the median age of the cases were 3 years (range, 2-4 years of age). All cases were refractory to ATRA, and multiagent chemotherapy was given as acute myeloblastic leukemia but four cases suffered relapse or failed to achieve remission. To investigate functional effect of this novel fusion on leukemogenesis, we then transduced cell lines, bone marrow cells of mice, and human cord blood with TBL1XR1-RARB, RARB, and PML-RARA . Influence of all-trans retinoic acid (ATRA), arsenic trioxide (ATO) was also assessed.Results: Four of the five (80%) cases had RARB translocations (three had the TBL1XR1-RARB fusion (Figure), and one had RARB rearrangement detected by FISH although partner gene of the case could not be determined). The novel fusion protein, TBL1XR1-RARB, could homodimerize, and diminished transcriptional activity with a dominant negative effect on both RARA and RARB. Transduced U937 cell lines with TBL1XR1-RARB showed cell differentiation with increased expression of CD11b when treated with ATRA, and neutrophil differentiation was confirmed by morphology. These effect of TBL1XR1-RARB were cancelled by administration of ATRA, although the impact was partial compared to that of PML-RARA. ATO, leading PML-RARA degradation by biding PML, could not affect TBL1XR1-RARB transduced cell lines. Colony replating assay demonstrated that this TBL1XR1-RARB could enhance replating ability of murine bone marrow. Differentiation of human cord blood was blocked by TBL1XR1-RARB, as well as PML-RAR, which was also confirmed by morphological features.Conclusion: We found that the majority of RARA-negative APL had RARB translocation, defining a novel genetic subgroup in APL. Unlike wild type RARB, the TBL1XR1-RARB proteins homodimerize, suppress retinoic acid pathway, and are confirmed as having oncogenic potency, as well as PML-RARA. Our study confirmed that dysregulation in retinoic acid pathway was a hallmark of APL, however, response to ATRA was partial, which is concordant to clinical resistance to ATRA of RARA -negative APL. Thus, in the context of precision medicine, RARB alteration should be screened for RARA -negative APL cases. [Display omitted] DisclosuresKitamura:Daiichi Sankyo: Research Funding.

Revisiting Cytochemical Myeloperoxidase (MPO) Stain for Early Diagnosis of Acute Promyelocytic Leukemia (APML) in Areas with Limited Resources: An Experience from a Tertiary Care Center in India

Introduction- Acute Promyelocytic leukemia (APML) needs to be suspected early so that All-Trans Retinoic Acid (ATRA) therapy can be started as soon as possible. Flow cytometry shows absence of CD34 and HLADR in most cases and so can aid in diagnosis along with the usual morphological clues. However, it may have confusing patterns as in hypogranular APML and sometimes may be positive for HLA- DR and CD34. RT-PCR for detection of characteristic APML translocations (t(15;17),t(17;17) and others) is gold standard for APML diagnosis which is time consuming and often not possible especially in resource limited areas. Cytochemical MPO stain is a robust laboratory test that can be a valuable aid to morphology in early diagnosis of APML. In this study, the role of cytochemical MPO was evaluated for early diagnosis of APML.Method- This study included all the cases of APML that were diagnosed in a tertiary care center in India from May 2011 to May 2014. Cytochemical stains (MPO, Non- specific Esterase (NSE), Periodic- acid Schiff (PAS) stains were performed on peripheral smear and bone marrow aspirates. Flow cytometry was performed on peripheral blood/bone marrow aspirate in EDTA on Beckman Coulter Flow Cytometer (FC500) using a panel of markers. RT-PCR was not available. However, all the above cases responded immediately to trial of ATRA (diagnosis confirmed by response to treatment trial with ATRA). The study was approved by Institutional Ethics committee.Results & Discussion- 13 cases of APML (6 males, 7 females) were identified with age ranging from 2-36 years. On flow cytometry, side scatter was moderate in 4 cases and high in 9 cases. The former were AML M3v (hypogranular variant) whereas the latter were classic APML. CD13 was heterogeneously dim to moderate positive in 12/13 cases .CD 33 was homogenously positive in all 13 cases (dim positive in 3; moderate positive in 10). CD34 was positive in 1/13 case and HLA-DR was positive in 2/13cases. All cases showed 3+ cytochemical MPO positivity, diffuse flush PAS positivity and NSE positivity. All cases went into immediate remission on treatment with ATRA.Thus even though flow cytometry was helpful in diagnosing APML, there were still some variations in the patterns. It is concluded that, though flow cytometric pattern of APML is often characteristic, occasionally it may have moderate side scatter and positive HLA-DR and CD34. In such situations and also in places with limited availability of flow cytometry/ RT PCR , strong (3+) cytochemical MPO staining in addition to characteristic morphologic features helps in the quick suspicion of APML and early treatment trial with ATRA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proteomic Profiling Reveals Protein Expression Patterns Correlated with Outcome in Acute Promyelocytic Leukemia (APL)

Background: Acute promyelocytic leukemia (APL) is the most favorable prognostic form of acute myeloid leukemia (AML). However, 5-20% of the patients relapse, which indicates that the clinical need to identify those who are likely to relapse remains. In this study we aimed to define protein expression patterns of APL with the goal to find certain protein patterns that would be prognostic for outcome.Methods: Custom reverse phase protein arrays (RPPA) were performed on 20 newly diagnosed APL patients, 205 newly diagnosed AML patients, and 10 normal CD34+ bone marrow samples, probed with 230 strictly validated antibodies. First, proteins were divided into 31 functionally related groups to analyze them in the context of other proteins. Progeny clustering analysis was applied to each of the functional groups to identify an optimal number of protein clusters; cases with similar expression of proteins. Then, block clustering was done on a binary matrix that indicated the protein cluster membership for all protein groups, for selected APL patients only, to assess whether we could identify subsets of APL cases (signatures) that expressed similar patterns of correlated protein clusters from various protein groups. Survival curves were generated for the identified signatures and individual proteins were compared between signatures using the student t-test with the Bonferroni correction (P<0.05).Results: Protein expression levels were measured for APL and AML patients relative to the normal CD34+ samples. Progeny clustering analysis revealed protein clusters for each functionally related protein group. All protein clusters that included APL patients were also found in AML patients. Only for the functional protein groups “Apoptosis regulating”, “HIPPO” and “TP53”, we observed protein patterns specific to APL. Block-clustering on APL patients showed strong correlations between various protein clusters and identified two subsets of patients, which we called protein signatures. Interestingly, survival curves showed that all of the relapse patients (N=4/20, 80.0%) were present in signature 2 (N=4/13, 30.8% vs. signature 1 N=0/7, 0%). Due to the small number of patients in our cohort, no level of significance was reached (P=0.084). Moreover, the number of promyelocytes in the bone marrow was lower in signature 1 (median 47 vs. 71 overall, P=0.070) and fibrinogen levels were higher in signature 1 (median 235 vs. 159, P=0.052) compared to signature 2. Direct comparison of individual proteins between signature 1 and 2 found 38 differentially expressed proteins. Notably, most protein had opposite protein levels relative to the normal cells in both signatures. For instance, ERG, STMN1, SSBP2, PRKAA1_2.pT172 and hnRNPK were higher in signature 2 and lower in signature 1, whereas CTSG, ITGB3 and ITGA2 were higher in signature 1.Conclusion: We demonstrated that although APL is genetically and therapeutically unique form of AML, most expression patterns were comparable between APL and AML. Most interestingly, we found that a subset of APL patients could be distinguished as high-risk solely based on their protein expression patterns. By knowing the signature membership at an early stage could, we could then decide whether additional therapy is required or not. Recognition of proteins that are higher or lower expressed in both signatures could be used to identify novel therapeutic targets. However, validation of our findings in a larger cohort is required. DisclosuresNo relevant conflicts of interest to declare.

Hepatotoxicity Profile of Single Agent As2O3 in the Management of Newly Diagnosed Cases of Acute Promyelocytic Leukemia: Impact on Outcome and Correlation with Genetic Polymorphisms.

As2O3 is an effective therapeutic agent in the management of acute promyelocytic leukemia (APL). Concerns have been raised about its potential cardiac and hepatotoxicity. We retrospectively analyzed the hepatotoxicity profile in newly diagnosed patients treated at our center, its impact on survival and also studied the effect of genetic polymorphisms on its incidence. Polymorphisms that were studied included some members of the GST family of genes (GSTM1, GSTT1and GSTA1) and that of the MTHFR gene (MTHFR C677T and MTHFR A1289C), these could potentially have a role in the bio-transformation of As2O3 and in handling reactive oxygen species generated by it.Between January 1998 and April 2005, 83 newly diagnosed patients with APL were treated with a regimen of single agent As2O3 in induction, consolidation and maintenance at doses of 10 mg/day for adults and 0.15mg/kg/day for the pediatric population. [Display omitted] There were 7 early deaths (6 IC bleeds, 1 sepsis), these patients were not evaluable for toxicity. Liver function tests (LFT) were done weekly in induction, once in 2 weeks in consolidation, prior to every course of maintenance and at three monthly intervals on follow up.Of the 76 evaluable patients in 46 (60.5%) there was no evidence of hepatotoxicity, grade 1, 2, 3 and 4 toxicity (NCI-CTC v2.0) was seen in 14(18.4%), 8(10.5%), 4(5.3%) and 4(5.3%) patients respectively. Of the 4 patients with grade 4 toxicity; in 3 there was documented acute hepatitis B infection. Onset of hepatotoxicity occurred in induction in 19 (63.3%), during consolidation in 3 (9.9%), during maintenance in 6 (20%) and following completion of therapy in 2 (6.6%). Of the 30 (39.5%) patients who developed hepatotoxicity in the majority the prominent abnormality was transaminitis (29 – 96.6%), it was transient (<4 weeks duration) in 20 (66.7%) and resolved on continuing As2O3 in 20 (66.7%). For all patients with grade 3 – 4 toxicity As2O3 was withheld till the LFT had normalized. One patient with grade 2 toxicity died on day 42 secondary to uncontrolled sepsis. Two patients have persisting LFT abnormality post completion of therapy (one each grade 1 and grade 2). There were no cases of acute hepatic failure or mortality attributable to hepatic toxicity.There was a significant association of hepatotoxicity with the MTHFR A1298C polymorphism (p=0.033). There was no statistical correlation of hepatotoxicity and null phenotype of GSTT1 or GSTM1 or with polymorphisms of GSTA1and MTHFR C677T. Occurrence of hepatotoxicity had no impact on OS in this group of patients (Log rank test p=0.666). In patients who developed hepatotoxicity EFS was superior to those that did not (97% vs 73%) though this did not reach statistical significance (p=0.116).Conclusion: The hepatotoxicity profile of single agent As2O3 as used in this regimen in the treatment of newly diagnosed cases of APL is mild and transient in the majority of patients. Its occurrence correlates with presence of the MTHFR A1298C polymorphism and it does not have an adverse impact on EFS or OS.

Disparity in utilization of multiagent therapy for acute promyelocytic leukemia (APL): A large National Cancer Database (NCDB) analysis.

6587 Background: Clinical trials have demonstrated a high rate of cure in APL with the use of multiagent therapy; however, overall survival in real world practice is significantly lower than that in the trials (Blood 2020; 136 (s 1): 13-14). We performed a large NCDB analysis to determine the appropriateness of treatment as a possible explanation for worse survival outside of the clinical trials. Methods: We included a total of 7190 APL cases reported to NCDB between 2004-2015. Multiple logistic regression analysis was used to evaluate the effect of covariates on probability of multiagent therapy use. Results: Only 64% of total patients received multiagent therapy; 29% received singe agent therapy and 4% received unknown therapy. 3% (n = 207) did not receive any treatment for reasons including early death (n = 8), patient refusal (n = 15), perceived contraindication (n = 12) and unknown reasons (n = 182). Compared to patients &gt; 60 years, younger patients aged 0-18 years (hazard ratio [HR] 3.2, 95% confidence interval [CI] 1.8-5.5, p &lt; 0.001), 19-40 years (HR 1.6, 95% CI 1.03-2.54, p = 0.03) and 41-60 years (HR 1.6, 95% CI 1.3-1.9, p &lt; 0.001) were more likely to receive multiagent chemotherapy. Patients with Medicaid were more likely to receive multiagent therapy compared to those with private insurance (HR 1.2, 95% CI 1.01-1.42, p = 0.04), possibly because patients with Medicaid are younger. The likelihood of receiving multiagent therapy decreased in uninsured patients (HR 0.6, 95% CI 0.5-0.8, p &lt; 0.001). Compared to academic cancer centers, patients treated at community cancer center (HR 0.5, 95% CI 0.3-0.7, p = 0.001), comprehensive community cancer center (HR 0.7, 95% CI 0.6-0.8, p &lt; 0.001)) and integrated network cancer center (HR 0.8, 95% CI 0.6-0-0.9, p = 0.01) were less likely to be treated with multiagent therapy. Lower comorbidity index increased the likelihood of receiving multiagent therapy. The likelihood of receiving multiagent therapy was not influenced by sex, race, annual income, distance traveled to treatment facility and high school education. Conclusions: To our knowledge, this is the first large scale analysis of utilization of multiagent therapy in APL in real world practice. In our study, 3% of patients did not receive treatment, a much smaller proportion of patients compared to acute myeloid leukemia, where a quarter to a third of patients do not receive any chemotherapy (Blood Adv; 2018 (2): 1277–1282). However, 29% of APL patients received suboptimal treatment with single agent therapy. The use of single agent therapy was higher in older adults and those with greater comorbidity. About half of the patients were treated outside of academic centers, which was associated with a higher probability of receiving single agent therapy. Uninsured patients were more likely to receive single agent therapy. Our findings highlight disparity based on insurance and health system factors.

Diagnosing Diamond-Blackfan anaemia: 'Vorsprung durch Technik'.

Especially since the turn of the millennium, the growth of precision medicine1 has promised a change in our approach to patient care. The prospect now exists of an objective, accurate and detailed diagnosis, leading to the selection of the appropriate therapy at the appropriate dose for the needs of the patient. This marks a sea-change in the approach to diagnosis and treatment over the period of publication of this Journal, as can be seen from the very first paper published – even the title points to acute leukaemia in general in a way that soon became obsolete.2 However, some conditions persist even today in being diagnosed by exclusion, or in Justice Potter Stewart’s famous definition of pornography, using the principle of ‘I know it when I see it’.3 With, as is made clear by van Dooijeweert et al.4 in this issue of the Journal, with no validated functional screening test, Diamond–Blackfan anaemia (DBA) falls into this category. The results of the study presented here showing a metabolic signature for DBA therefore have great potential interest. The promise of being able to characterise a condition using genomics or metabolomics is seductive. Gone is all subjectivity, replaced by a single test that gives an accurate answer. Even better, the signature offers the lure of the predictive marker: identifying therapeutic targets for tailored therapy and tracking response to that therapy. The success or failure of a treatment can be detected early, while simultaneously the chances of a treatment being ineffective in a particular patient are reduced. However, the derivation of such signatures, especially in rarer conditions is not without challenges. The number of different biomarkers that can be analysed in the laboratory means that one is presented with more potentially explanatory variables than patients on which to test them. If sufficiently many markers are available (with data on many genes or many proteins), good discrimination can be seen even if these markers are chosen entirely at random from the set available. For example, on a set of 59 patients with chronic myeloid leukaemia it was shown empirically that it is possible for 30 randomly selected gene expression probes to predict response correctly, in that dataset, on average 90% of the time.5 This is a manifestation of a familiar high school mathematics problem: with n unknowns and n independent simultaneous equations there exists a unique solution. The high degree of accuracy in discriminating responders from non-responders is because the variables chosen fit the data, as opposed to the disease. The other challenge comes in the design of the experiment: in terms of diagnosis one is typically aiming to distinguish cases from controls. Often these controls will be healthy, or use a convenience sample of material from unrelated procedures. Laying aside the issue that one may in fact be identifying a factor associated with the selection of the controls,6 do the controls chosen actually represent the population in which one is looking for the correct diagnosis? For example: if one were looking to identify patients with acute promyelocytic leukaemia (APL) in one’s clinic, then it would be important to distinguish between APL and acute myeloid leukaemia (AML). A test that was derived based only on APL cases and healthy controls might identify factors common to AML and APL that are not present in healthy controls – rendering it all but useless in the clinic. In other words the test needs to distinguish between those with the condition and those who might reasonably be suspected to have the condition but do not.6 So how does the work presented by van Dooijeweert et al.4 fare when considered on these two points? There are two particular features here that provide grounds for optimism. First, although small, there is a validation cohort that shows similar detection rates to the original training set. And second, and indeed rarer, is a comparison between DBA and congenital dyserythropoietic anaemia (CDA) demonstrating a different signature for DBA than for CDA. Both of these features are heartening: that this signature fits the condition, as opposed to being a chance ‘best fit’ to the dataset provided; and second, that the signature can distinguish DBA from other similar conditions – the test that a diagnostic would need to pass in a clinical setting. As the authors themselves admit, there is more work to be done. Larger independent validation in a real-life clinical setting will reinforce the findings. And at present there is no indication of how the signature can work to identify therapeutic options, or as a serial marker of disease activity. But the findings here provide proof of principle for an approach that is less invasive than current techniques, and may lead to an objective set of diagnostic criteria, and a gateway to choosing therapy and monitoring its effect.

Successful treatment of acute promyelocytic leukemia in a patient with prosthetic heart valves: a case report and review of the literature

BackgroundAlthough life-threatening hemorrhage is a usual manifestation of acute promyelocytic leukemia (APL), thrombotic events seem to be more common in APL compared to other subtypes of acute leukemia. The treatment and prophylaxis of thrombosis are controversial due to the high risk of bleeding caused by disseminated intravascular coagulation (DIC) and thrombocytopenia. To the best of our knowledge we report the first case of APL in a patient with prosthetic heart valves successfully treated with a combination of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). We hope this case report helps clinicians to manage different spectra of coagulopathy in APL successfully.Case presentationA 38-year-old Asian man presented with diagnosis of APL confirmed by bone marrow biopsy. He was on warfarin due to prosthetic mitral and aortic valves. He was at risk of both hemorrhagic events due to DIC and life-threatening valve thrombosis. Our management regimen included unfractionated heparin adjusted according to the platelet count to prevent both valve thrombosis and bleeding events. The patient tolerated treatment well without any hemorrhagic or thrombotic events, and complete molecular remission was achieved by ATRA and ATO without the need for chemotherapeutic agents.ConclusionAlthough this case is exceptional, a precise evaluation may be needed to select the appropriate dose and type of anticoagulant to treat a patient with APL.

Acute Promyelocytic Leukemia: A Long-Term Retrospective Study in Mexico

BackgroundThe present retrospective study reviewed acute promyelocytic leukemia (APL) cases recorded in Mexico between January 2007 and January 2017. The primary objective of the study was to evaluate overall survival (OS) in Mexican patients with APL. Secondary objective was to evaluate the impact of induction treatment with different anthracyclines on OS, event-free survival (EFS) and complications in this patient population.MethodsThe medical charts of patients referred to medical institutions in Mexico from January 2007 through January 2017 for the treatment of suspected APL were reviewed retrospectively. Patients aged 15 - 75 years, in whom the diagnosis of APL was confirmed, who had an Eastern Cooperative Group performance status of 0 - 2, and who were eligible for combined treatment with intensive chemotherapy and all-trans retinoic acid (ATRA), were included in the study. Study participants received induction and consolidation treatment with ATRA plus either daunorubicin or idarubicin, followed by 2 years of single-agent ATRA as maintenance therapy. Patients who were unable to pay for ATRA treatment received anthracycline-based induction and consolidation, with methotrexate plus mercaptopurine as maintenance therapy.ResultsA total of 360 patients from 21 public and private hospitals were included in the study. The median age of the population was 37 years, and 51% were male. Of the 360 patients, 205 (57%) vs. 155 (43%) received daunorubicin vs. idarubicin as induction treatment for APL. ATRA was administered to 201 (98%) patients in the daunorubicin group vs. 138 (89%) in the idarubicin group (P = 0.001), and was initiated at diagnosis in 92% vs. 73% of recipients, respectively (P = 0.0001). At 150 months, OS and EFS for the entire population were 84% and 79%, respectively. Both OS (90% vs. 76%, P = 0.003) and EFS (85% vs. 72%, P = 0.001) were significantly prolonged in daunorubicin vs. idarubicin recipients. Rates of complications were similar in the two groups.ConclusionsAs arsenic trioxide (ATO) is not currently available in Mexico, anthracycline plus ATRA is the mainstay of treatment for APL here. Our results confirm the efficacy of this strategy, with high OS and EFS rates being observed 12.5 years after diagnosis.

A Rare Case of Pancytopenia Post Heart Transplant

Introduction Hematologic malignancy following solid organ transplant is uncommon and no cases have been reported in heart transplant recipients. We present a case of acute promyelocytic leukemia (APL) after orthotopic heart transplant. Case Report 57 year-old, with dilated cardiomyopathy secondary to sarcoidosis and no history of malignancy underwent successful orthotopic heart transplant. He had one episode of cell mediated rejection which was treated with pulse dose steroids. He was on tacrolimus, cellcept and prednisone for maintenance immunosuppression. Ten months post-transplant, routine laboratory test revealed pancytopenia which was attributed to medication side effects. His cellcept and valcyte were discontinued. He received Filgrastim. Cytomegalovirus, Epstein-Barr virus, and Parvovirus B-19 infections were ruled out. He had recurrent episodes of decline in his absolute neutrophil counts and was referred to hematology. A bone marrow aspirate and smear showed left shift granulocytic maturation arrest in a promyelocytic/myelocytic stage and cells suggestive of Auer rod. FISH cytogenetics, with 23% positive for PML-RARA fusion gene, confirmed the diagnosis of APL. He was initiated on therapy with All-trans retinoic acid (ATRA). Treatment with arsenic trioxide was deferred due to concerns for cardiotoxicity. His treatment course was also remarkable for differentiation syndrome which responded to hydroxyurea and dexamethasone. Overall, he had good response to ATRA and repeat BMB showed normocellular marrow with trilineage hematopoiesis with no evidence of APL. Summary Clinicians should maintain a high degree of suspicion when patients with a history of OHT develop unexplained pancytopenia. A small case series revealed the population most commonly affected with post-transplant APL were males, with a median age of 50 years at diagnosis and within a median interval of two years post solid organ transplant. Our patient was able to achieve remission without the standard chemotherapeutic agents used to treat APL associated with cardiotoxicity such as arsenic trioxide and anthracycline. To our knowledge, this case is the first reported of post orthotopic heart transplant acute promyelocytic leukemia.

Pediatric Acute Promyelocytic Leukemia: Epidemiology, Molecular Features, and Importance of GST-Theta 1 in Chemotherapy Response and Outcome.

Previous studies have suggested a variation in the incidence of acute promyelocytic leukemia (APL) among the geographic regions with relatively higher percentages in the Latin American population. We aimed to explore the population burden of pediatric APL, gathering information from the population-based cancer registry (PBCR) and the diagnosis of APL obtained through incident cases from a hospital-based cohort. The homozygous deletion in glutathione S-transferases (GSTs) leads to a loss of enzyme detoxification activity, possibly affecting the treatment response. Mutations in the RAS pathway genes are also considered to be a key component of the disease both in the pathogenesis and in the outcomes. We have assessed mutations in a RAS–MAP kinase pathway (FLT3, PTPN11, and K-/NRAS) and GST variant predisposition risk in the outcome. Out of the 805 children and adolescents with acute myeloid leukemia (AML) who are registered in the PBCR, 35 (4.3%) were APL cases. The age-adjusted incidence rate (AAIR) was 0.03 per 100,000 person-years. One-hundred and sixty-three patients with APL were studied out of 931 AML cases (17.5%) from a hospital-based cohort. Mutations in FLT3, KRAS, and NRAS accounted for 52.1% of the cases. Patients with APL presented a 5-year probability of the overall survival (OS) of 67.3 ± 5.8%. A GST-theta 1 (GSTT1) null genotype conferred adverse prognosis, with an estimated hazard ratio of 2.8, 95% confidence interval (CI) 1.2–6.9. We speculate that the GSTT1 polymorphism is associated with therapeutics and would allow better OS of patients with APL with a GSTT1 null genotype.

Tetraploid acute promyelocytic leukemia with double translocation t (15,17) PML/RARA: the first case report in Croatia and Europe

Acute promyelocytic leukemia (APL) is characterized by the translocation t (15;17)(q22;q21) cytogenetic abnormality in the majority of cases. In most of the cases the cells of APL have normal, diploid karyotype. There are very few cases presented with very rare tetraploid karyotype with double translocation t(15;17)(q22;q12). We report the first case of tetraploid APL with double translocation t(15, 17) in Europe. A 66-year old male patient presented with dyspnea and unexplained dental bleeding. Blood work showed a white blood cell count of 1x109/L, hemoglobin was 124 g/ L, platelet count was 61x109/L and fibrinogen level was low (1.4 g/L). Cytogenetics showed a tetraploid karyotype. Fluorescence in situ hybridization analysis proved existence of clonal cells with translocation t (15,17) in 15% of metaphase nuclei and tetraploid subclonal cells with the same translocation in 70% of metaphase nuclei. Findings were consistent with APL, tetraploid variant and the patient started all-trans retinoic acid (ATRA) treatment. The patient achieved complete remission in 2 months and completed three consolidation therapy cycles with ATRA, idarubicin or mitraxontrate. Currently, the patient is undergoing maintenance therapy with ATRA, 6-mercaptopurine and weekly methotrexate.

Association of PML-RARα Fusion mRNA Type With Pretreatment Hematologic Characteristics But Not Treatment Outcome in Acute Promyelocytic Leukemia: An Intergroup Molecular Study

AbstractIn each case of acute promyelocytic leukemia (APL) one of three PML-RARα mRNA types is produced, depending on the break/fusion site in the PML gene that is linked to a common RARα gene segment: a short (S)-form type, PML exon 3 RARα exon 3; a long (L)-form type, PML exon 6 RARα exon 3; or a variable (V)-form type, variably deleted PML exon 6 RARα exon 3. We evaluated whether PML-RARα mRNA type is associated with distinct pretreatment clinical characteristics and therapeutic outcome in previously untreated adult APL patients registered to protocol INT 0129 by the Eastern Cooperative Oncology Group, the Southwest Oncology Group, and the Cancer and Leukemia Group B. Of 279 clinically eligible cases, 230 were molecularly evaluable, and of these, 111 were randomized to receive remission induction therapy with all-trans retinoic acid (ATRA) and 119 with conventional chemotherapy. Nine cases not excluded by central pathology review were PML-RARα negative, and notably, none of five of these cases treated with ATRA achieved complete remission (CR). Among 221 PML-RARα–positive cases, there were 82 S-form cases (37%), 121 L-form cases (55%), and 18 V-form cases (8%). Before any antileukemic therapy, the S-form type, compared with the L-form type, was associated with higher values for the white blood cell (WBC) count (median 2,500/μL v 1,600/μL; P = .009), the percentage of blood blasts plus promyelocytes (median 29% v 8.5%; P = .03), and the absolute blood blasts plus promyelocytes (884/μL v 126/μL; P = .019). Also, an increased percentage of S-form versus L-form cases had the M3 variant phenotype, 24% v 12% (P = .036). There were no differences between S-form and L-form cases in either CR rate (79% v 69%; P = .14) or disease free survival distribution (multivariate analysis adjusting for the association of S-form type and higher WBC count; P = .40). We conclude that the S-form type is associated with previously-identified adverse risk WBC parameters but that the identification of the S-form or L-form type of PML-RARα mRNA, per se, does not predict clinical outcome or add to the value of an increased WBC count as a negative prognostic indicator in APL patients.

Nanopore sequencing sheds a light on the FLT3 gene mutations complexity in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) patients carry in 27% of cases an activating mutation of the fms-like tyrosine kinase-3 (FLT3) gene: internal tandem duplication (ITD) or tyrosine kinase domain (TKD) point mutation. The simultaneous presence of both types of mutations, so-called FLT3 dual mutations, has been reported in 2% of APL, but this circumstance has never been studied. We studied a cohort of 74 APL cases, performing an in-depth analysis of three FLT3 dual mutant cases. Nanopore sequencing (NS) allowed us to characterize their complex mutational profile, showing the occurrence of multiple activating FLT3 mutations on different alleles in the leukemic promyelocytes and suggesting a cumulative impact of these events on the constitutive activation of the FLT3 pathway in APL cells. NS approach not only sheds light on the FLT3 mutational complexity in APL, but may also be useful to better clarify the FLT3 mutations landscape in acute myeloid leukemia.

Microfluidic-Based Detection of AML-Specific Biomarkers Using the Example of Promyelocyte Leukemia.

A microfluidic assay for the detection of promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) fusion protein was developed. This microfluidic-based system can be used for rapid personalized differential diagnosis of acute promyelocyte leukemia (APL) with the aim of early initiation of individualized therapy. The fusion protein PML-RARα occurs in 95% of acute promyelocytic leukemia cases and is considered as diagnostically relevant. The fusion protein is formed as a result of translocation t(15,17) and is detected in the laboratory by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic methods require many laboratory steps with specialized staff. The developed microfluidic assay includes a sandwich enzyme-linked immunosorbent assay (ELISA) system for PML-RARα on surface of magnetic microparticles in a microfluidic chip. A rapid detection of PML-RARα in cell lysates is achieved in less than one hour. A biotinylated PML-antibody on the surface of magnetic streptavidin coated microparticles is used as capture antibody. The bound translocation product is detected by a RARα antibody conjugated with horseradish peroxidase and the substrate QuantaRed. The analysis is performed in microfluidic channels which involves automated liquid processing with stringent washing and short incubation times. The results of the developed assay show that cell lysates of PML-RARα-positive cells (NB-4) can be clearly distinguished from PML-RARα-negative cells (HL-60, MV4-11).

The Utility of Leucocyte Cell Population Data and Scattergram in Rapid Identification of Acute Promyelocytic Leukemia

Introduction: Acute promyelocytic leukemia (APL) is one of the hematological emergencies requiring early detection and treatment. The hematology laboratory plays a pivotal role in management. The correct morphological identification of APL is made even before the confirmation by the cytogenetic or molecular analysis. Automated hematology analyzers are the backbone of the hematology laboratories. Other than providing routine counts and differentials, they are also capable of producing sufficient information in the form of leucocyte cell population data (CPD) scatter plots depicting the leucocyte subpopulation. These data are primarily underutilized, particularly in assessing the lineage of various hematopoietic neoplasm. Here we analyzed the utility of leucocyte CPD and the pattern of scatter plot in Sysmex XN 1000 analyzer for the detection of acute promyelocytic leukemia. Materials and Results: We included 100 controls and 100 AML cases. Leucocyte CPD parameters were compared along with scatter plots from the WDF channel from Sysmex XN1000 analyzer were recorded and analyzed. Immunophenotyping and molecular analysis confirmed the diagnosis in all but two cases, where the patients expired within a few hours of admission in the emergency. Out of a total of 100 cases, 22 were labeled as APL, 63 as AML, and 15 as AML M4/M5. Among the APL cases, in 19 cases, immune-phenotyping was done on peripheral blood, and the remaining three bone marrow aspirate samples were used. The scatter-plot analysis of APL cases showed a characteristic pattern which differentiated this entity from other acute myeloid leukemias. In the case of APL, on Side Fluorescence Light Scatter (SFL) Vs. Side scatter (SSC), the abnormal promyelocytes occupy the space above neutrophils and to monocytes' right, where the immature precursors of myeloid are usually found. In cases where the total leukocyte count was within the normal range, a hiatus between these abnormal promyelocytes and neutrophils are seen. This finding was consistent in all cases irrespective of the morphological variation. On the other hand, other variants of acute myeloid leukemias along with monoblastic leukemias, the scatter plots show the blasts in the monocytic region with extension to the ceiling. On comparison, the myeloblast and monoblasts lie more towards the y-axis than abnormal promyelocytes. All the CPD parameters were compared against the control group and the AML group. The cell population data showed a significant difference in all the parameters representing the side fluorescence, size and granulations except LY-WX (width of dispersion of side scatter) when compared to normal healthy controls. Amidst all the AML cases, APL significantly differed from other cases in NE-SFL, LY-WY, and MO-WZ. AML M4/M5 and APL also varied in LY-Y, NE-WY, NE-WZ, and MO-WY. AMLs excluding AML M4/M5 showed additional variation in MO-Y and MO-Z. Discussion: APL cases have a characteristic scatter plot patter on SFL Vs. SSC. The scatter-plot shows a tear-drop like a collection of cells. These cells have high side scatter and high lateral fluorescence owing to the large size of the cells, apple-core like the nucleus, and dense granulations. The differences in the CPD parameters among APL cases and controls and other acute myeloid leukemia can be attributed to these morphological features of the abnormal promyelocytes. Haider et al. found a significant difference between NE-SFL and other myeloid leukemia. [1] This was in corroboration with our findings. Another research done by Park et al. showed that NE-SFL and MO-WX had the highest sensitivity and specificity in differentiating APL from other AML. [2] Conclusion: We conclude that the new age automated cell counters provide numerous data that remains unexplored and can be utilized further along with the information from the scatter plots to make a rapid diagnosis of Acute Promyelocytic Leukemias.

Epidemiology of Acute Myeloid Leukaemia in New Zealand: A National Cancer Registry Analysis

Background: Acute myeloid leukaemia (AML) is a blood cancer characterised by the expansion of a malignant myeloid progenitor. The estimated age-standardised incidence rate in many western countries has remained static over the last 2 decades at 3-4 per 100,000, whilst long-term survival has improved, especially for the younger individuals. However, disparities remain for the older individuals, people of ethnic minority background, and those who are more socio-economically deprived. Previous evaluation of population data from New Zealand has shown a similar pattern, but a more recent analysis has not been done. Here we present the incidence and long-term survival of patients with AML in New Zealand (NZ), using the New Zealand Cancer Registry (NZCR). Method: The NZCR was established in 1948 and it became mandatory by law to report all new cases of malignancy by 1994. We extracted all AML cases from the registry between 1 January 1997 and 31 December 2016. Cases with an ICD-10-CM code for acute myeloid leukaemia and its subtypes including acute promyelocytic leukaemia (e.g. C92.0) were included. Individuals residing overseas or without an address were excluded, and individuals with a diagnosis of acute promyelocytic leukaemia (APML) were analyzed separately. The socio-economic status of the individual was estimated based on their domicile area using the New Zealand 2013 Index of Deprivation (NZDep2013) which is a geographically based composite measure of deprivation. Overall survival was calculated from the date of diagnosis to the date of death or last follow-up (31 December 2016). Multivariable Cox-proportional hazard models were used to evaluate potential associations with survival time in NZ AML cases. Results: During this 20-year period, 154 cases of APML and 2876 cases of AML (excluding APML) were reported to the registry on individuals residing in New Zealand. Of the AML cases, 53% were male and the median age at the time of diagnosis was 67 (IQR 52-77), with a small positive correlation between year of diagnosis and age at diagnosis (Spearman's rho=0.05, p=0.009). The majority of cases (77%) were of European descent, 12% were New Zealand Maori, and 6% were Pacific Islanders. Individuals of European descent were significantly older at diagnosis compared to other ethnicities (median of 70 vs 51 for Maori, 56 for Pacific Islanders, and 58 for all other ethnicities, p&amp;lt;0.001). AML appeared to disproportionally affect those more socio-economically deprived, with 23% of cases reported in the most deprived 20% of the population, compared with only 16% of the cases in the least deprived 20%. The annual crude incidence remained stable during this period at an average of 3.42 per 100,000 (ranging from 2.57 to 4.29, figure 1), and was significantly higher in the older adults (figure 2). Age-standardised rates were lower (figure 1), with an average of 2.6 (range 1.9 to 3.4) cases per 100,000, and a small but significant average annual decrease over the study period. The estimated 1, 2, and 5-year survival for the entire cohort was 38%, 27%, and 22%, respectively. Age at diagnosis was a significant predictor of inferior survival, with a hazard ratio (HR) for all-cause mortality of 2.06, 3.95, 6.39 and 10.84 for the 50-59, 60-69, 70-79 and &amp;gt;80 age groups, respectively, compared to those aged &amp;lt;50. Shorter overall survival was also noted in individuals in the more socio-economically deprived 50% of the population (HR 1.13, 95% CI 1.03-1.23). Conclusion The incidence of AML in New Zealand has remained static in the last 2 decades, consistent with data from other western countries. Lower age-standardised rates and the small decrease in these observed over the study period are likely to reflect the increasingly and comparatively older population in NZ. Maori and Pacific Islanders appeared to present at a younger age than individuals of European descent. Age at diagnosis and socio-economic deprivation were shown to be an adverse prognostic factor for overall survival. Further in-depth analysis is required to determine the cause of these observations at a population level. Disclosures Chan: AbbVie:Membership on an entity's Board of Directors or advisory committees;Janssen:Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding, Speakers Bureau;Celgene:Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company);Amgen:Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company);Roche:Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company).