Digital PCR in Myeloid Malignancies: Ready to Replace Quantitative PCR?

New techniques are on the horizon for the detection of small leukemic clones in both, acute leukemias and myeloproliferative disorders. A promising approach is based on digital polymerase chain reaction (PCR). Digital PCR (dPCR) is a breakthrough technology designed to provide absolute nucleic acid quantification. It is particularly useful to detect a low amount of target and therefore it represents an alternative method for detecting measurable residual disease (MRD). The main advantages are the high precision, the very reliable quantification, the absolute quantification without the need for a standard curve, and the excellent reproducibility. Nowadays the main disadvantages of this strategy are the costs that are still higher than standard qPCR, the lack of standardized methods, and the limited number of laboratories that are equipped with instruments for dPCR. Several studies describing the possibility and advantages of using digital PCR for the detection of specific leukemic transcripts or mutations have already been published. In this review we summarize the available data on the use of dPCR in acute myeloid leukemia and myeloproliferative disorders.

Ruxolitinib for the treatment of inadequately controlled polycythemia vera without splenomegaly: 80-week follow-up from the RESPONSE-2 trial.

RESPONSE-2 is a phase 3 study comparing the efficacy and safety of ruxolitinib with the best available therapy (BAT) in hydroxyurea-resistant/hydroxyurea-intolerant polycythemia vera (PV) patients without palpable splenomegaly. This analysis evaluated the durability of the efficacy and safety of ruxolitinib after patients completed the visit at week 80 or discontinued the study. Endpoints included proportion of patients achieving hematocrit control (< 45%), proportion of patients achieving complete hematologic remission (CHR) at week 28, and the durability of hematocrit control and CHR. At the time of analysis, 93% (69/74) of patients randomized to ruxolitinib were receiving ruxolitinib; while in the BAT arm, 77% (58/75) of patients crossed over to ruxolitinib after week 28. No patient remained on BAT by week 80. Among patients who achieved a hematocrit response at week 28, the probability of maintaining response up to week 80 was 78% in the ruxolitinib arm. At week 80, durable CHR was achieved in 18 patients (24%) in the ruxolitinib arm versus 2 patients (3%) in the BAT arm. The safety profile of ruxolitinib was consistent with previous reports. These data support that ruxolitinib treatment should be considered also as a standard of care for hydroxyurea-resistant/hydroxyurea-intolerant PV patients without palpable splenomegaly.

[Epidemiologic features of Philadelphia-negative chronic myeloproliferative disorders in Szabolcs-Szatmár-Bereg county, Hungary. Analysis of data of a 33-year period]. / A Philadelphia-negatív krónikus myeloproliferativ kórképek epidemiológiai jellemzoi Szabolcs-Szatmár-Bereg megyében. 33 év adatainak elemzése.

INTRODUCTION: In their previous work, the authors reported findings from 30 years on the incidence of hematological malignancies in Szabolcs-Szatmár-Bereg county, Hungary. Until now there are no other studies on this topic available in Hungary. AIM: Detailed analysis of epidemiologic features of patients with Philadelphia-negative chronic myeloproliferative disorders was carried out. METHOD: During a 33-year period (between January 1, 1983 and December 31, 2015) 4523 adult patients with hematologic malignancies were recorded in the leukaemia/lymphoma registry of Szabolcs-Szatmár-Bereg county. Among them, 255 patients with polycytaemia vera, 102 with primary myelofibrosis, and 331 with essential thrombocytaemia were registered. RESULTS: The incidence of polycythaemia vera and essential thrombocythaemia in Szabolcs-Szatmár-Bereg county showed an increasing tendency, with an overall incidence rate of 1.35 and 1.75/100 000 inhabitants/year, respectively; while the incidence of primary myelofibrosis decreased in the course of years (0.54/100 000 inhabitants/year). In cases of polycythaemia vera and primary myelofibrosis the male:female ratio was found to be equal, however essential thrombocythaemia showed a female dominance. The mean age of patients with polycythaemia vera was 65 (21-95) years, similar to essential thrombocythaemia with 65 (19-85) years, and to primary myelofibrosis with 65.5 (33-84) years. There were only two villages found in this county where the occurrence of patients with Philadelphia-negative chronic myeloproliferative disorders per one thousand inhabitants was significantly higher, than the average (1.22). In every familial cases of these, the manifestation of the disease in the second and the third generations became earlier than in the first genetration. The perceived average degree of the anteposition (anticipation) was found to be 22 years. CONCLUSION: The epidemiologic features of Philadelphia-negative chronic myeloproliferative disorders in Szabolcs-Szatmár-Bereg county are essentially similar to data published in the literature. Orv. Hetil., 2017, 158(15), 572-578.

Cutaneous myeloid dendritic cell dyscrasia: A cutaneous clonal monocytosis associated with chronic myeloproliferative disorders and peripheral blood monocytosis.

Monocytes are critical components of the innate immune system and they can differentiate into dendritic cells (DCs). Cutaneous neoplasms of dendritic cell origin are uncommon and mostly represented by histiocytic lesions derived primarily from Langerhans cells. The myeloid DC (mDC) while recognized in the immunology literature does not have a well-defined neoplastic cutaneous counterpart. Eleven patients with a diagnosis of cutaneous mDC dyscrasia were evaluated. Routine hematoxylin and eosin stain were performed followed by selective phenotypic studies. The patients were older without a gender predilection and exhibited an asymptomatic papular skin rash with a waxing and waning course. The biopsies demonstrated a dermal based monomorphic small mononuclear cell infiltrate. The cells expressed CD14, CD11c, HLA-DR, as well as granzyme and lysozyme that defines terminally differentiated monocyte/dendritic cells. Expression of BDCA-3 (CD141) by the tumor cells indicated that they were myeloid dendritic cells (mDC2). Each patient had a prior or subsequent diagnosis of an abnormal bone marrow biopsy that included myelodysplastic syndrome, myelofibrosis, chronic myelomonocytic leukemia, and acute myelogenous leukemia. We propose the term cutaneous mDC cell dyscrasia for distinctive infiltrates of differentiated mDCs reflective of underlying myeloproliferative disease. The clinical course is variable and can be indolent although it is strongly correlated with myelodysplastic syndrome that included leukemia.

Hematocrit Self-Testing in Patients with Polycythemia Vera and Other Hematological Conditions: Assessing the Accuracy of the StatStrip Xpress® 2 LAC/Hb/Hct Device and User Opinion about the Device in Real-World Clinical Practice.

Maintaining hematocrit (Hct) levels below 45% can reduce morbidity and mortality in patients with polycythemia vera (PV). A device that patients can use to self-monitor Hct levels could enable timely interventions if Hct levels increase above 45%, and could improve quality of life (QoL). This study evaluated the accuracy of the StatStrip Xpress® 2 LAC/Hb/Hct meter (Hb/Hct meter) when used by healthcare professionals (HCPs) or patients in clinical practice. Blood samples from 68 visits for 60 patients with PV or other hematological conditions were collected and analyzed by HCPs using a laboratory hematological analyzer, and by patients (self-test) and HCPs (professional test) using the Hb/Hct meter at two Swiss centers. Accuracy was assessed as the mean difference in readings between two users/methods (mdiff, 90% confidence interval; Spearman correlation [r]). The Hct values were similar between the professional test and analyzer (n = 66 comparisons, mdiff = 0.1% [−0.5 to 0.8]; r = 0.95, p < 0.001), the self-test and professional test (n = 62 comparisons, mdiff = −0.2% [−1.1 to 0.7]; r = 0.93, p < 0.001), and the self-test and analyzer (n = 63 comparisons, mdiff = 0.0% [−0.8 to 0.7]; r = 0.94, p < 0.001). The hemoglobin values across users/methods were also similar. Reporting their opinion on the Hb/Hct meter at visit 1, 100% of the patients found it easy to use, and 97% were willing to use it at home. Of the patients with PV, approximately 71% and 56%, respectively, stated that they would feel safer using a self-testing device, and that it would improve their QoL. These findings demonstrate the potential of the Hb/Hct meter for HCP and patient use in real-world settings.

Sclerosing Extramedullary Hematopoietic Tumor: A Case Report.

Sclerosing extramedullary hematopoietic tumor (SEMHT) is a rare disease that was originally named fibrous hematopoietic tumor or myelosclerosis. The process typically occurs in patients with a history of chronic myeloproliferative disorders and may afflict the skin, lung, breast, gastrointestinal tract, breast, kidney, lymph node, and thyroid gland. In this article, we report the case of a 37-year-old female with more than 5 years history of acute B-cell lymphoblastic leukemia who presented with right upper quadrant pain and tenderness. Computed tomography scan showed multiple new ring-enhancing low-density lesions within the right and left hepatic lobes. A computed tomography-guided liver core biopsy was performed on one of the new liver lesions and showed extramedullary hematopoiesis with atypical megakaryocytes and sinusoidal capillarization with associated fibroblastic proliferation. Numerous atypical megakaryocytes were scattered in the background of haphazard collagen deposition. No significant blasts or a leukemic process were identified. Bone marrow aspiration and biopsy showed extensive reticulin fibrosis (MF-3), trilineage dysplasia, increased blasts (10% to 19%), and hypercellularity (close to 100%), which was consistent with MDS-EB-2. Cytogenetics was reported as follows: 44~46,XX,-3,add(3)(p13),-5,-6,-7,17,del(17)(p12),+1~5mar[cp9]/46,XX. Molecular analysis was negative for both JAK2 V617F and CALR exon 9 activating mutations. In summary, we contributed a new case of SEMHT diagnosis in a synchronous presentation with poor clinical evolution associated chromosome 7 deletion and intact JAK2 and CALR exon 9. Care should be taken when diagnosing intraabdominal and retroperitoneal soft tissue masses with the history of hematological disorders.

FAS/FASL gene polymorphisms in Turkish patients with chronic myeloproliferative disorders.

INTRODUCTION: Chronic myeloproliferative disorders (CMPD) are chronic myeloid hematological disorders, characterized by increased myeloid cell proliferation and fibrosis. Impaired apoptotic mechanisms, increased cell proliferation, uncontrolled hematopoietic cell proliferation and myeloaccumulation may contribute to the pathogenesis of CMPD. The aim of our study was to show the possible role of FAS/FASL gene polymorphisms in CMPD pathogenesis and investigate the association with clinical parameters and susceptibility to disease. MATERIAL AND METHODS: We included 101 (34 polycythemia vera (PV), 23 primary myelofibrosis (PMF), 44 essential thrombocythemia (ET)) CMPD patients diagnosed according to the WHO classification criteria and 95 healthy controls in this study. All the patients and the controls were investigated for FAS/FASL gene expression, allele frequencies and phenotype features, and also FAS mRNA levels were analyzed. RESULTS: Chronic myeloproliferative disorders patients showed increased FAS-670AG + GG genotype distribution compared with the control group (p < 0.05). While the A allele was more frequent in both groups, AG genotype was more frequent in CMPD patients. There was no association between FAS-670A>G gene polymorphism and some clinical parameters such as splenomegaly and thrombosis (p > 0.05). No statistically significant difference in FASL+843C>T genotype or allele frequency was found between groups (p > 0.05). Moreover, no statistically significant difference was detected in FASL and JAK2V617F mutations (p > 0.05). FAS mRNA expression was 1.5-fold reduced in patients compared to healthy subjects. CONCLUSIONS: According to our findings, FAS/FASL gene expression may contribute to the molecular and immunological pathogenesis of CMPD. More investigations are needed to support these data.

Janus kinase 2 mutations in cases with BCR-ABL-negative chronic myeloproliferative disorders from Turkey.

OBJECTIVE: We aimed to investigate the frequency of Janus kinase 2 (JAK2) mutations in cases with chronic myeloproliferative disorders (CMDs), and the relationship between the presence of JAK2 mutation and leukocytosis and splenomegaly, retrospectively. MATERIALS AND METHODS: Patients, who were diagnosed with BCR-ABL-negative CMDs according to diagnosis criteria of the World Health Organization and followed up at the hematology clinic between 2013 and 2015, were investigated in terms of the frequency of JAK2 mutation in cases with CMDs, and the relationship between the presence of JAK2 mutation and leukocytosis and splenomegaly, retrospectively. RESULTS: In total, 100 patients, who were diagnosed with BCR-ABL-negative CMDs, were evaluated retrospectively. The mean age of the patients with JAK2 positivity was significantly higher compared to patients with negative. JAK2-positivity rates in the age groups were significantly different. Gender, diagnosis, splenomegaly, and leukocytosis were not statistically different for JAK2 positivity between the groups. CONCLUSION: JAK2 V617F mutation is more commonly seen in older age as a risk for complications related to CDMS. Splenomegaly and leukocytosis are not associated with JAK2 V617F mutation.

Bone Health in Patients With Hematopoietic Disorders of Bone Marrow Origin: Systematic Review and Meta- Analysis.

Blood cell production and bone homeostasis are physically interlinked systems that exhibit active cross-talk. We examined how bone health is affected in patients with hematopoietic disorders due to abnormal proliferation of bone marrow cells. The electronic databases Medline, Embase, PubMed, BIOSIS Previews, Web of Science, and Cochrane were searched for studies presenting numerical values for trabecular bone volume or bone mineral density in control and patients with hematopoietic disorders. We identified 5 studies for beta-thalassemia, 6 for sickle cell anemia, 2 for polycythemia vera and essential thrombocythemia, 3 for chronic myelogenous leukemia, 6 for myelofibrosis, 5 for multiple myeloma, and 4 studies each for systemic mastocytosis, lymphocytic leukemia, and hemochromatosis. The effect of the disease state on bone density was significant and negative for beta-thalassemia (r = -2.00; 95% confidence interval [CI] -3.41, -0.58; p < 0.005), sickle cell anemia (-0.91; -1.36, -0.47; p < 0.00005), chronic myelogenous leukemia (-0.55; -0.88, -0.22; p < 0005), mastocytosis (-0.99; -1.16, -0.82; p < 0.00001), lymphoblastic leukemia (-0.69; -0.98, -0.40; p < 0.00001), multiple myeloma (-0.67; -0.99, -0.35; p < 0.00005), and hemochromatosis (-1.15; -1.64, -0.66; p < 0.00001). The changes were negative but not significant for polycythemia vera (-0.16; -0.38, 0.05; p = 0.069) and essential thrombocythemia (-0.33; -0.92, 0.26; p = 0.14). In myelofibrosis, disease state was associated with increased bone density (0.74; 0.12, 1.36; p < 0.05). Bone density change significantly and negatively correlated with the level of ferritin and bone marrow cellularity but not with hemoglobin or erythropoietin. Thus, independent of hematopoietic lineage, abnormal proliferation of bone marrow cells appears to be associated with bone loss. Iron metabolism may independently contribute to bone homeostasis. © 2016 American Society for Bone and Mineral Research.

Splanchnic vein thrombosis, the onset manifestation in JAK positive Chronic Myeloproliferative Disorders Neoplasms.

BACKGROUND: Patients with Myeloproliferative Neoplasms-(MPN) have a high risk of thrombotic complications. Portal vein thrombosis is a severe complication, which in many cases, appears at the onset of the disease; the risk factors are related to the presence of qualitatively altered thrombocytes and leucocytes, leading to their activation and appearance of leukocytes-platelet-aggregates; anomalies of portal vein endothelial cells are also implicated. The presence of JAK2V617F mutation increases the risk for splahnic thrombosis. METHODS AND RESULTS: We present three patients with portal vein thrombosis and Budd Chiari syndrome, who were further diagnosed with MPN-the thrombosis was the onset event of the disease. CONCLUSION: Patients were diagnosed with thrombosis of the portal vein before being diagnosed with MPN. Splenectomy was not associated with risk of thrombosis for the two cases in which it was performed; for one case, splenectomy was a therapeutic method to resolve portal hypertension. All patients had homozygous JAK2 mutation, which is associated in recent studies with increased risk of portal, mesenteric thrombosis. The high number of platelet was difficult to control for all patients. Bone marrow biopsy and determination of JAK status are valuable investigations for patients who have splenoportal thrombosis, with no apparent identifiable cause.