Minimal Residual Disease Detection at RNA and Leukemic Stem Cell (LSC) Levels: Comparison of RT-qPCR, d-PCR and CD26+ Stem Cell Measurements in Chronic Myeloid Leukemia (CML) Patients in Deep Molecular Response (DMR).

A Deep Molecular Response (DMR), defined as a BCR::ABL1 transcript at levels ≤ 0.01% by RT-qPCR, is the prerequisite for the successful interruption of treatment among patients with Chronic Myeloid Leukemia (CML). However, approximately 50% of patients in Treatment-Free Remission (TFR) studies had to resume therapy after their BCR::ABL1 transcript levels rose above the 0.1% threshold. To improve transcript detection sensitivity and accuracy, transcript levels can be analyzed using digital PCR (dPCR). dPCR increases BCR::ABL1 transcript detection sensitivity 10-100 fold; however, its ability to better select successful TFR patients remains unclear. Beyond the role of the immune system, relapses may be due to the presence of residual leukemic stem cells (LSCs) that are transcriptionally silent. Flow cytometry can be used to identify and quantify circulating bone marrow Ph+ LSCs CD34+/CD38- co-expressing CD26 (dipeptidylpeptidase-IV). To date, the significance of circulating Ph+ LSCs in TFR is unclear. The aim of this work is to compare and examine the values obtained using the three different methods of detecting minimal residual disease (MRD) in CML at RNA (RT-qPCR and dPCR) and LSC (flowcytometry) levels among patients in TFR or exhibiting a DMR. The twenty-seven patients enrolled received treatment with either imatinib (12), dasatinib (6), nilotinib (7), bosutinib (1), or interferon (1). Twelve patients were in TFR, while the rest exhibited a DMR. The TFR patients had stopped therapy for less than 1 year (3), <3 years (2), 6 years (6), and 17 years (1). Blood samples were collected and tested using the three methods at the same time. Both d-PCR and LSCs showed higher sensitivity than RT-qPCR, exhibiting positive results in samples that were undetectable using RT-qPCR (17/27). None of the patients tested negative with d-PCR; however, 23/27 were under the threshold of 0.468 copies/µL, corresponding to a stable DMR. The results were divided into quartiles, and the lowest quartiles defined the lowest MRD. These data were strongly correlated in 15/27 patients, corresponding to almost half of the TFR patients. Indeed, the TFR patients, some lasting up to 17 years, corresponded to the lowest detectable DMR categories. To the best of our knowledge, this is the first attempt to analyze and compare DMRs in a CML population using standard (RT-qPCR) and highly sensitive (dPCR and LSCs) methods.

Chronic Myeloid Leukemia and Pregnancy: When Dreams Meet Reality. State of the Art, Management and Outcome of 41 Cases, Nilotinib Placental Transfer.

The overwhelming success of tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML) patients has opened a discussion among medical practitioners and the lay public on the real possibility of pregnancy and conception in females and males with CML. In the past 10 years this subject has acquired growing interest in the scientific community and specific knowledge has been obtained "from bench to bedside". Embryological, pharmacological, and pathophysiological studies have merged with worldwide patient databases to provide a roadmap to a successful pregnancy and birth in CML patients. Male conception does not seem to be affected by TKI therapy, since this class of drugs is neither genotoxic nor mutagenic, however, caution should be used specially with newer drugs for which little or no data are available. In contrast, female patients should avoid TKI therapy specifically during the embryonic stage of organogenesis (5-12 weeks) because TKIs can be teratogenic. In the last 15 years, 41 pregnancies have been followed in our center. A total of 11 male conceptions and 30 female pregnancies are described. TKI treatment was generally terminated as soon as the pregnancy was discovered (3-5 weeks), to avoid exposure during embryonic period and to reduce the risk of needing treatment in the first trimester. Eleven pregnancies were treated with interferon, imatinib or nilotinib during gestation. Nilotinib plasma levels in cord blood and maternal blood at delivery were studied in 2 patients and reduced or absent placental crossing of nilotinib was observed. All of the patients were managed by a multidisciplinary team of physicians with obligatory hematological and obgyn consultations. This work provides an update on the state of the art and detailed description of pregnancy management and outcomes in CML patients.

Serum or plasma? An old question looking for new answers.

Serum or plasma? An old question looking for new answers. There is a continual debate on what type of sample a clinical laboratory should use. While serum is still considered the gold standard and remains the required sample for some assays, laboratories must consider turn-around time, which is an important metric for laboratory performance and, more importantly, plays a critical role in patient care. In addition, a body of evidence emphasise the choice of plasma in order to prevent modifications of some analytes due to the coagulation process and related interferences. Advantages and disadvantages of serum and plasma are discussed on the basis of current literature and evidence. In addition, data are provided on the current utilisation of the samples (serum or plasma) in Italy and in other countries. Finally, a rationale for a possible switch from serum to plasma is provided.

Endothelin-1 in hypertensive patients with ischemic heart disease.

This study was aimed at evaluating whether transient dipyridamole-induced myocardial ischemia in hypertensive patients reflects on endothelin-1 plasma levels by comparing normotensives and hypertensives with or without stable angina. Endothelin-1 plasma levels were assessed in baseline conditions and after provocative stress test by dipyridamole. Four groups of ten age- and sex-matched subjects were retrospectively considered among patients referred for chest pain evaluation and submitted to high-dose Dipyridamole Echocardiographic-Scintigraphic combined test (DES). On the basis of DES results we considered: (1) control normotensives subjects; (2) essential hypertensives (for both groups negative result of DES); (3) essential hypertensives with stable angina; and (4) normotensives with stable angina (for both groups concordant DES detection of myocardial ischemia). Our data showed a marked post-DES increase of endothelin-1 plasma levels in hypertensives with stable angina (mean levels = 16.50 ± 4.19 pg/ml p < 0.001 vs. baseline = 9.05 ± 1.37 pg/ml) and a minor increase in stable angina pts (mean levels = 8.3 ± 1.75 pg/ml p < 0.01 vs. baseline = 6.74 ± 0.61 pg/ml) whereas non significant increase was observed both in control (mean levels = 5.09 ± 0.83 pg/ml p = n.s. vs. baseline = 4.91 ± 1.04 pg/ml) and hypertensives groups (mean levels = 6.34 ± 1.72 pg/ml p = n.s. vs. baseline = 5.95 ± 1.04 pg/ml). ET-1 involvement in hypertension-related ischemic heart disease patho-physiology appears to be considered.

Early systemic sclerosis: analysis of the disease course in patients with marker autoantibody and/or capillaroscopic positivity.

OBJECTIVE: To investigate whether patients affected by 1 of the 3 subsets of early systemic sclerosis (SSc; scleroderma), i.e., subset I, Raynaud's phenomenon with SSc marker autoantibodies and typical capillaroscopic findings; subset II, autoantibody positive only; and subset III, capillaroscopy positive only and not satisfying the 2013 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria for SSc at admission, differ from each other in the time to satisfy the criteria. METHODS: Early SSc patients subdivided into the 3 subsets indicated above consecutively admitted to a rheumatology/angiology center were monitored for 12-102 months (median 36 months). Patients were reevaluated twice yearly to assess whether and when each patient satisfied the new ACR/EULAR classification criteria for SSc. Patients with undifferentiated connective tissue disease (UCTD) served as the comparator group. RESULTS: During followup, 11 (52.3%) of 21 subset I, 10 (66.6%) of 15 subset II, 0 of 24 subset III, and 0 of 44 UCTD patients satisfied the criteria (P = 0.0001). The difference was significant between early SSc and UCTD patients (P = 0.0001) and, within the group of early SSc patients, between each of the 2 autoantibody-positive subsets (subsets I and II) and the capillaroscopic-positive/autoantibody-negative subset (subset I versus III: P = 0.0001; subset II versus III: P = 0.0009). There was no difference between the 2 autoantibody-positive subsets (P = 0.454). In addition to marker autoantibody positivity, preclinical lung or heart involvement was associated with an increased risk to satisfy the criteria during followup. CONCLUSION: Our data demonstrated faster progression of SSc in autoantibody-positive patients, particularly in those with preclinical internal organ involvement at baseline, than in autoantibody-negative patients.

Bivalirudin or heparin in primary angioplasty performed through the transradial approach: results from a multicentre registry.

BACKGROUND: Data on the effect of bivalirudin therapy in primary percutaneous coronary interventions (PCI) performed through the transradial approach are limited. The aim of our study was to evaluate bleeding complications and clinical outcomes in primary PCI performed through the transradial approach with bivalirudin therapy. METHODS: We retrospectively evaluated primary PCI performed through the transradial approach from January 2008 to June 2013. Patients were divided in two groups according to the use (group 1) or not (group 2) of bivalirudin. The primary end points were major bleedings and major adverse cardiac events (MACE) within 30 days. RESULTS: During the 5 years analysed, 1009 patients underwent primary PCI through the transradial approach: 154 patients were treated with bivalirudin (males 79%, age 65 ± 14 years) and 855 with heparin (males 82%, 63 ± 12 years). In group 1, the use of glycoprotein IIb/IIIa inhibitors was only 4%, compared to 55% (p<0.001) in group 2. There were no significant differences between the two groups for major bleedings (0.65% in group 1 and 1.17% in group 2, p=0.88) nor for minor bleedings (1.3% in group 1 and 1.5% in group 2, p=0.83). There were also no significant differences in MACE (7.1% in group 1 and 10.4% in group 2, p=0.27). The 30-day mortality rate was 3.9% in group 1 and 5.4% in group 2 (p=0.56). CONCLUSIONS: In this registry of primary PCI performed through the transradial approach, bivalirudin was not associated with a significant reduction in major bleeding or MACE compared to heparin and provisional glycoprotein IIb/IIIa inhibitors.

Early systemic sclerosis: marker autoantibodies and videocapillaroscopy patterns are each associated with distinct clinical, functional and cellular activation markers.

INTRODUCTION: Early systemic sclerosis (SSc) is characterized by Raynaud's phenomenon together with scleroderma marker autoantibodies and/or a scleroderma pattern at capillaroscopy and no other distinctive feature of SSc. Patients presenting with marker autoantibodies plus a capillaroscopic scleroderma pattern seem to evolve into definite SSc more frequently than patients with either feature. Whether early SSc patients with only marker autoantibodies or capillaroscopic positivity differ in any aspect at presentation is unclear. METHODS: Seventy-one consecutive early SSc patients were investigated for preclinical cardiopulmonary alterations. Out of these, 44 patients and 25 controls affected by osteoarthritis or primary fibromyalgia syndrome were also investigated for serum markers of fibroblast (carboxyterminal propeptide of collagen I), endothelial (soluble E-selectin) and T-cell (soluble IL-2 receptor alpha) activation. RESULTS: Thirty-two of the 71 patients (45.1%) had both a marker autoantibody and a capillaroscopic scleroderma pattern (subset 1), 16 patients (22.5%) had only a marker autoantibody (subset 2), and 23 patients (32.4%) had only a capillaroscopic scleroderma pattern (subset 3). Patients with marker autoantibodies (n = 48, 67.6%) had a higher prevalence of impaired diffusing lung capacity for carbon monoxide (P = 0.0217) and increased serum levels of carboxyterminal propeptide of collagen I (P = 0.0037), regardless of capillaroscopic alterations. Patients with a capillaroscopic scleroderma pattern (n = 55, 77.5%) had a higher prevalence of puffy fingers (P = 0.0001) and increased serum levels of soluble E-selectin (P = 0.0003) regardless of marker autoantibodies. CONCLUSION: These results suggest that the autoantibody and microvascular patterns in early SSc may each be related to different clinical-preclinical features and circulating activation markers at presentation. Longitudinal studies are warranted to investigate whether these subsets undergo a different disease course over time.

The Management of Membranous Glomerulopathy in Allogeneic Stem Cells Transplantation: Updated Literature

BACKGROUND: membranous glomerulopathy (MG) is an immunomediated disorder which accounts for the most common cause of nephrotic syndrome (NS) following allogeneic hematopoietic stem cell transplantation (HSCT). OBJECTIVE AND METHODS: to provide an update on the issue by reviewing pertinent literature on the MEDLINE database. RESULTS: sixty-nine post allogenic HSCT patients (42 male) with MG were identified. The median age was 43 (5 to 68) years. Time interval from allogenic HSCT to MG diagnosis ranged from 3 to 134 months (median 17). Most MG patients had a history of acute (70%) or chronic (84%) graft versus host disease (GVHD). Corticosteroids and cyclosporine were the most common therapeutic agents used in this setting; alternative therapies, including rituximab, were given to a lower number of patients. Outcome data were available in 64 out of 69 MG patients; 38 (59%) and 18 (28%) patients achieved a complete and a partial response respectively, whereas treatment failure was recorded in the remaining 8 (13%). CONCLUSION: MG after allogenic HSCT appears to be associated with a sub clinical or overt cGVHD, which follows the discontinuation of immunosuppressive prophylaxis. Although a standard therapeutic approach has not been established, the application of available measures can induce favorable response in more than 80% of affected patients, but treatment-failure and progressive deterioration of the renal function may occur in about one fifth of cases.

Pain management in hematological patients with major organ dysfunctions and comorbid illnesses.

BACKGROUND: Organ dysfunctions and medical complications, such as renal failure, liver impairment, coagulation disorders, cardiovascular and respiratory illnesses, may hamper an adequate pain management in haematological patients. AIM: To summarize current knowledge on pain management in hematological patients presenting major organ dysfunctions and comorbidity. We also attempted to provide recommendations to optimize analgesia and to minimize side effects in the setting of medically compromised and frail haematological patients. METHODS: A systematic search of the literature, using relevant key words, was conducted in PubMed. RESULTS AND CONCLUSIONS: Pain in hematological patients is a common symptom and is often multi-factorial. Most pharmacotherapeutic measures, including causal therapies, analgesics and adjuvant agents routinely applied in pain management, may also be used in the setting of clinical frailty and medical comorbidities; however, comprehensive clinical and functional patient's evaluations and a careful consideration of expected benefits and potential adverse events are required.

Anticoagulant and Anti-thrombotic Treatments in the Management of Hematological Malignancies in a Home Care Program.

AIM: Anticoagulants (AC) and anti-platelet (AP) agents are widely administered to patients with hematological malignancies (HM). However, HM patients may be at high risk of bleeding and hemorrhagic complications, because of different form of coagulopathies and several degrees of thrombocytopenia. MATERIALS AND METHODS: A prospective evaluation of the use of anticoagulant and anti-thrombotic agents as well as of bleeding and thrombotic complications in a consecutive cohort of patients, which were followed during the first semester of 2010 by our home care service, was performed. In this regard, three pharmacological class of agents, such as oral anticoagulants (warfarin and acenocumarine), low molecular weight heparin (LMWH) and anti-platelet (AP) drugs were considered. RESULTS: Out of 129 patients, 26 (20%) were treated with AC/AP drugs. Warfarin, acenocumarine, LMWH as well as AP were used in 7, 11 and 12 patients, respectively. Adverse events (bleeding) were observed in 3 patients (11.5%), 2 cases being on warfarin (replaced by LMWH) and 1 being AP (suspension without replacement); out of the 3 patients with bleeding, none presented thrombocytopenia. CONCLUSIONS: Despite the frequent findings of hemostatic disorders in a population of frail patients managed in a home care setting, our experience demonstrated that the use of AC/AP drugs has been very rarely responsible for significant complications.

[Opioid analgesics in patients with chronic renal failure: principles for use and current guidelines]. / Analgesici oppioidi in pazienti con insufficienza renale cronica: principi d'impiego e raccomandazioni correnti.

The treatment of pain in patients with impaired renal function may be problematic, especially when opioid drugs need to be used. In the presence of renal failure, significant changes occur in the metabolism and pharmacokinetics of these drugs, which can lead to adverse reactions due to the accumulation of parental compounds and active metabolites. This paper presents and discusses the available evidence concerning the optimal use of the most frequently employed opioid analgesics to treat pain in patients with renal impairment.

Management of hematological malignancies in patients affected by renal failure.

The management of hematological malignancies (HM) in renally impaired patients may be a difficult task. Indeed, the kidney represents a major elimination pathway for many chemotherapeutic agents and their metabolites, whose serum levels are not usually measured in daily clinical practice. In addition, many antineoplastic drugs have a narrow therapeutic index for which they require dose adjustment when administered to patients with renal failure. Only limited data regarding the use of chemotherapy in patients with renal impairment and in those on dialysis are available. Indeed, renal patients with HM are often excluded from most clinical trials. Thus far, in order to provide recommendations, we have reviewed the pertinent literature, gathering information from published guidelines regarding chemotherapy in patients with kidney dysfunction and from articles describing the use of individual agents in renal patients with HM.

Pain in malignant hematology.

Pain is frequently experienced by patients with hematological malignancies, although it often receives little attention. Different underlying causes and mechanisms may sustain several pain syndromes in hematological malignant patients. Pain may be due to disease itself, to disease-related complications, to iatrogenic causes or may be associated with unrelated medical conditions. The management of pain in this setting requires a multidisciplinary approach, integrating analgesics and causal interventions. An accurate diagnostic assessment and the identification of the underlying causes and pathogenetic mechanisms may dictate the treatment approach. For most pain patients, the WHO's three-step analgesic scale for cancer pain relief can provide adequate relief with oral options, although difficult-to-treat pain syndromes, requiring a more complex treatment approach, may also be observed.