Comparison of AlphaLISA and RIA assays for measurement of wool cortisol concentrations.

Radioimmunoassay (RIA) methods have always represented a technique of choice for the determination of steroids in biological samples. The Amplified Luminescent Proximity Homogenous Assay-Linked Immunosorbent Assay (AlphaLISA) is now emerging as the new-generation immunoassay technology that does not require washing/separation steps. The aim of this study was to adapt the Perkin-Elmer's AlphaLISA kit for wool cortisol and compare it with a RIA wool cortisol assay. Wool from lambs, 35 at birth (A0) and 54 at two months old (A2), was collected and each extract was evaluated for wool cortisol concentrations (HCC) both by RIA and AlphaLISA immunoassay. The two methods showed good precision, sensitivity and specificity for determining HCC. Both methods were able to detect significant differences between the high and the low HCC assessed in lambs at A0 and A2 (P < 0.01). The HCC assessed with RIA were significantly higher than those assessed with AlphaLISA (P < 0.01). Moreover, the correlation between HCC measured using the AlphaLISA and RIA methods was strong (r = 0.878). The regression analyses show a constant and not proportional error. This could be due to the diversity in the dosage steps and to the diversity of the molecules used in the two methods. Results support the validity of using AlphaLISA as an alternative method to RIA for the quantification of cortisol in sheep wool and considering the performances showed it has a great potential to be further applied as an excellent tool to evaluate HCC in samples derived from animal species.

Refractory and 17p-deleted chronic lymphocytic leukemia: improving survival with pathway inhibitors and allogeneic stem cell transplantation.

Refractory/early relapsed and 17p deletion/p53 mutation (del(17p)/TP53mut)-positive chronic lymphocytic leukemia (CLL) has been conventionally considered a high-risk disease, potentially eligible for treatment with allogeneic stem cell transplantation (alloSCT). In this multicenter retrospective analysis of 157 patients, we compared the outcomes of patients with high-risk CLL treated with alloSCT, a B-cell receptor pathway inhibitor (BCRi), and both. Seventy-one patients were treated with BCRis, 67 patients underwent reduced-intensity conditioning alloSCT, and 19 received alloSCT with a BCRi before and/or after transplantation. Inverse probability of treatment weighting analyses were performed to compare the alloSCT and no-alloSCT groups; in the 2 groups, 5-year OS, PFS, and cumulative incidence of nonrelapse mortality (NRM) and relapse were 40% versus 60% (P = .096), 34% versus 17% (P = .638), 28% versus 5% (P = .016), and 38% versus 83% (P = .005), respectively. Patients treated with alloSCT plus BCRi had a 3-year OS of 83%. The 3-year OS and NRM by year of alloSCT, including patients treated with BCRi, were 53% and 17% in 2000 to 2007, 55% and 30% in 2008 to 2012, and 72% and 18% in 2013 to 2018. In conclusion, the combination of pathway inhibitors and alloSCT is feasible and may further improve the outcome of high-risk CLL patients.

Extended follow-up and impact of high-risk prognostic factors from the phase 3 RESONATE study in patients with previously treated CLL/SLL.

In the phase 3 RESONATE study, ibrutinib demonstrated superior progression-free survival (PFS), overall survival (OS) and overall response rate (ORR) compared with ofatumumab in relapsed/refractory CLL patients with high-risk prognostic factors. We report updated results from RESONATE in these traditionally chemotherapy resistant high-risk genomic subgroups at a median follow-up of 19 months. Mutations were detected by Foundation One Heme Panel. Baseline mutations in the ibrutinib arm included TP53 (51%), SF3B1 (31%), NOTCH1 (28%), ATM (19%) and BIRC3 (14%). Median PFS was not reached, with 74% of patients randomized to ibrutinib alive and progression-free at 24 months. The improved efficacy of ibrutinib vs ofatumumab continues in all prognostic subgroups including del17p and del11q. No significant difference within the ibrutinib arm was observed for PFS across most genomic subtypes, although a subset carrying both TP53 mutation and del17p had reduced PFS compared with patients with neither abnormality. Reduced PFS or OS was not evident in patients with only del17p. PFS was significantly better for ibrutinib-treated patients in second-line vs later lines of therapy. The robust clinical activity of ibrutinib continues to show ongoing efficacy and acceptable safety consistent with prior reports, independent of various known high-risk mutations.

Luteal activity and effect of dietary energy restriction on follicular development in lactating cows.

The aim of this research has been to evaluate the presence of anomalies in the ovarian cycle activity during postpartum and to verify whether 72-hr dietary fasting during the dominance phase, the phase before ovulation, might modify the ovarian follicle population. The presence of anomalies in ovarian cycle activity has been evaluated in 30 Italian Friesian cows starting from 20 days postpartum until 211 days of lactation. Long oestrus and brief dioestrus or scarce luteal activity have been the main anomalies found through measuring progesterone concentrations in the whey. Until 100 days of lactation, the BCS values of the problematic animals have been significantly lower than those in animals with normal ovarian activity. After 100 days of lactation, the ovarian anomalies continued to appear despite the fact that all the animals have reached comparable BCS values. Starting from the results of this trial, the effect of 72-hr dietary fasting on dominant follicles has been studied in six cows. Ultrasonography revealed that the diameter of the follicles at 71 days postpartum has been significantly lower than at 181 days. A 72-hr dietary restriction at 101 and 211 days postpartum did not affect the size of the dominant follicle. However, at 101 days postpartum, half of the animals presented follicular cysts. The effect of fasting differed if the animal has been in early postpartum or 211 days of lactation. Further researches are necessary to understand how different metabolic conditions can modify the follicular population but on the other hand the study shows the utility for farmers and field veterinarians of monitoring the resumption of the ovarian cycle postpartum through the whey progesterone concentrations.

Short communication: hair cortisol concentrations in Holstein-Friesian and crossbreed F1 heifers.

The aim of this study was to evaluate the hypothalamic-pituitary-adrenal (HPA) axis activity of Holstein-Friesian and crossbreed F1 heifers by analysis of the cortisol concentrations in hair samples. Cortisol, the primary hormone of the HPA axis, is the biological endpoint for the investigation of the HPA response. The study was conducted on 290 prepubertal heifers; 142 heifers were pure Holstein-Friesian and 148 were crossbreed F1 heifers obtained from the 3-way rotational system with Swedish Red and Montbéliarde breeds. Extraction was performed on the hair using methanol, and cortisol concentrations were determined by a radioimmunoassay method. Cortisol concentrations measured in regrown hair of crossbreed F1 heifers were significantly lower than those in hair of Holstein-Friesian heifers. This result helps us to better understand the differences in HPA activity and allostatic load between Holstein-Friesian and crossbreed F1 heifers and allows us to better assess the adaptability of these animals to the environment and the importance of crossbreed traits for profitability in dairy farming.

Chronic lymphocytic leukemia: treatment of relapse.

Despite significant advances in the frontline treatment of chronic lymphocytic leukemia (CLL), patients eventually experience disease progression. Treatment selection of relapsed disease depends upon a variety of factors, including patient age, performance status, duration of response to initial therapy, type of prior therapy, disease-related manifestations and genetic abnormalities within the CLL cells. This presentation offers synthetic overview of the options in this field.

The impact of advanced age according to IPSSWM cut-off on the outcome of symptomatic and asymptomatic Waldenström's macroglobulinemia at diagnosis.

Advanced age is one of the variables more frequently considered to be associated with an adverse prognosis in Waldenström's macroglobulinemia (WM). In a series of 238 symptomatic and asymptomatic WM patients, we retrospectively identified an age cut-off distinguishing two groups of patients with different outcome in terms of overall survival (OS), disease-specific survival (DSS) and treatment-free survival (TFS). Although for the OS the best cut-off was identified at 65 years with shorter OS for elderly patients, no difference was detected in terms of DSS between the two groups. Furthermore, patients over 65 years showed a longer TFS compared with patients under 65 years. Clinical and laboratory disease characteristics did not significantly differ between the two groups of patients except for ß2M level. Therefore, the poorer survival of patients over 65 years at diagnosis should probably be attributed to the higher number of no disease-related deaths and is independent from WM.

Management guidelines for the use of alemtuzumab in chronic lymphocytic leukemia.

The consensus views of an expert roundtable meeting are presented as updated management guidelines for using alemtuzumab in chronic lymphocytic leukemia. Since the publication of previous management guidelines in 2004, clinical experience with alemtuzumab has grown significantly, especially regarding its efficacy and safety, management of cytomegalovirus (CMV) reactivation, identification of patient subgroups likely to benefit from alemtuzumab therapy and subcutaneous administration of alemtuzumab. The updated recommendations include (1) alemtuzumab monotherapy can be safely used as first-line therapy; (2) suitable patient subgroups for alemtuzumab therapy include elderly patients, patients with 17p deletion, patients with refractory autoimmune cytopenias and patients with profound pancytopenia at baseline due to heavily infiltrated bone marrow; (3) alemtuzumab treatment should be continued for 12 weeks (36 doses) whenever possible, and bone marrow examination may be considered at week 12 to evaluate response; (4) monitoring CMV reactivation by weekly PCR is mandated during therapy; when CMV reactivation becomes symptomatic or viremia increases, alemtuzumab therapy should be interrupted and anti-CMV therapy started; (5) subcutaneous administration is safe, easy to perform and appears equally effective compared with intravenous infusion and (6) our strong recommendation is that alemtuzumab combination therapy and consolidation therapy shall not be used outside carefully controlled clinical studies.

Characteristics and stage of the underlying diseases could determine the risk of opportunistic infections in patients receiving alemtuzumab.

Alemtuzumab is usually associated with opportunistic infections. We have treated 67 patients, 8 non-Hodgkin's lymphoma and 59 chronic lymphocytic leukemia (CLL) with campath. Among CLL patients, 6 used alemtuzumab in first line, alone or with chemotherapy, 41 as consolidation therapy and 11 as salvage therapy, 3 alone and 8 with chemotherapy. In our series opportunistic infections were prevalently found in patients submitted to alemtuzumab salvage therapy (33.3%), with or without chemotherapy; in particular 1 pulmonary nocardiosis, 1 tubercolosis. Also during the first line alemtuzumab therapy one case of lysteriosis and one case of HBV reactivation were found (33.3%). No opportunistic infections were diagnosed to our CLL patients in consolidation therapy, when the underlying hematologic disease was reduced or present only as minimal residual disease. A good response of malignancy, namely CLL, to induction therapy, such as a less aggressive schedule of therapy, determine a lower risk of immunosuppression and therefore a low number of opportunistic infections.

A new sensitive enzyme-linked immunosorbent assay (ELISA) for Alemtuzumab determination: development, validation and application.

Alemtuzumab is a humanized (IgG(1)) rat monoclonal antibody to CD52 antigen and is currently used in the treatment of chronic lymphocytic leukemia (CLL) and other CD52-positive lymphoproliferative disorders. Various techniques have been developed to measure Alemtuzumab levels in human serum/plasma. The authors report on the validation of a very sensitive enzyme-linked immunosorbent assay (ELISA) to measure serum concentrations of the humanized IgG(1) using a rabbit polyclonal antibody specifically produced against the rat sequence of Alemtuzumab after papain digestion. The assay was successfully applied to test the serum samples of patients with B-lymphocyte CLL who received Alemtuzumab subcutaneously. This ELISA assay could be easily used to determine human serum levels of Alemtuzumab pre- and post-treatment to optimize dosing and scheduling and to study the relationship between dose and clinical response.

Efficacy and safety of oral fludarabine phosphate in previously untreated patients with chronic lymphocytic leukemia.

PURPOSE: A prospective, multicenter, open-label, phase II clinical trial to assess oral fludarabine phosphate treatment in terms of safety, efficacy, and quality of life. Reference to a historical group of patients treated with the intravenous (IV) formulation allowed the two formulations to be compared. PATIENTS AND METHODS: Patients with previously untreated B-cell chronic lymphocytic leukemia received 10-mg tablets of fludarabine phosphate to a dose of 40 mg/m(2)/d for 5 days, repeated every 4 weeks, for a total of six to eight cycles. Efficacy was assessed using International Workshop on Chronic Lymphocytic Leukemia and National Cancer Institute criteria for response. Safety monitoring included WHO toxicity grading for adverse events. Quality of life was also assessed. RESULTS: Eighty-one patients received treatment. According to International Workshop on Chronic Lymphocytic Leukemia criteria, the overall response rate was 71.6% (complete remission, 37.0%; partial remission, 34.6%). The response rate using National Cancer Institute criteria was 80.2% (complete remission, 12.3%; partial remission, 67.9%). Median time to progression was 841 days (range, 28 to 1,146 days). The most frequently reported grade 3/4 toxicity was myelosuppression. WHO grade 3/4 hematological toxicities included granulocytopenia (32.1%), anemia (9.9%), and thrombocytopenia (4.9%). Gastrointestinal toxicity was more common with the oral formulation than with IV fludarabine phosphate, but was generally mild to moderate and did not require treatment. Statistically significant improvements in mean emotional and insomnia quality-of-life scores were seen after treatment. CONCLUSION: This study demonstrates that oral fludarabine phosphate is clinically effective and generally well tolerated by patients with previously untreated B-cell chronic lymphocytic leukemia. Oral fludarabine phosphate has a similar clinical efficacy and safety profile to the IV formulation. Oral fludarabine phosphate does not adversely affect quality of life and may improve emotional and insomnia scores.

Successful CD34+ cell mobilization by intermediate-dose Ara-C in chronic lymphocytic leukemia patients treated with sequential fludarabine and Campath-1H.

Chronic lymphocytic leukemia (CLL) cells could be undetectable by flow cytometry or polymerase chain reaction after sequential treatment with fludarabine and Campath-1H. Concern has been raised regarding the ability to mobilize sufficient peripheral blood progenitor cells (PBPCs) for autografting after purine analogues, and there are few data about PBPC collection after Campath-1H. In all, 16 CLL patients responding to sequential chemo-immunotherapy entered the study. In 10, mobilization regimen consisted of granulocyte colony-stimulating factor (G-CSF) 5-10 microg/kg/die. Patients failing mobilization or not achieving the target of 2.5 x 10(6) CD34+ cells/kg underwent a second attempt using intermediate-dose (ID) Ara-C, 800 mg/m(2) every 12 h for six doses+G-CSF. PBPC collection after G-CSF alone was successful in two out of 10 patients. An adequate number of CD34+ cells were collected after ID Ara-C+G-CSF in eight patients failing the mobilization with G-CSF alone and in five out of six who did not receive G-CSF before. Greater yields of PBPCs were collected with Ara-C+G-CSF compared with G-CSF alone (13.8 vs 3.3). The extrahematologic toxicity was manageable. In conclusion, PBPC collection is feasible in CLL patients treated with sequential therapy including fludarabine and Campath-1H. Excellent yields were obtained in 92.8% of patients primed with ID Ara-C+G-CSF.

Prior invasive pulmonary and cerebellar mucormycosis is not a primary contraindication to perform an autologous stem cell transplatation in leukemia.

Mucormycosis infections, caused by fungi of the families Rhizopus, Mucor or Absidia, are typically rapidly progressive and often fatal. We report a 27-year-old male with acute myeloid leukemia (AML) developing an invasive pulmonary-CNS mucormycosis during the neutropenic period after salvage induction chemotherapy; the infection was successfully controlled with surgery and antifungal therapy. The patient received two courses of consolidation chemotherapy and underwent autologous stem cells transplantation (ASCT) while receiving secondary antifungal systemic prophylaxis with liposomal Amphotericin B (L-AMB, Ambisome). There was no clinical, radiological or microbiological evidence of mycotic reactivation during the bone marrow transplantation (BMT) procedure.

Activity of oral fludarabine phosphate in previously treated chronic lymphocytic leukemia.

PURPOSE: A prospective, multicenter, open-label phase II clinical trial was conducted to assess the efficacy and safety of oral fludarabine phosphate. Reference to an historical group of patients treated with the intravenous (IV) formulation allowed the investigators to compare the two formulations. PATIENTS AND METHODS: Efficacy was assessed using the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) and National Cancer Institute (NCI) criteria for complete remission (CR), partial remission (PR), stable disease, or disease progression. Safety monitoring included World Health Organization (WHO) toxicity grading for all adverse events. RESULTS: Seventy-eight (96.3%) of 81 recruited patients with previously treated B-cell chronic lymphocytic leukemia (CLL) received 10-mg tablets of fludarabine phosphate to a dose of 40 mg/m(2)/d for 5 days, repeated every 4 weeks, for a total of six to eight cycles. According to IWCLL criteria, the overall remission rate was 46.2% (CR, 20.5%; PR, 25.6%). The comparative figures using NCI criteria were 51.3% (CR, 17.9%; PR, 33.3%). Overall, 30 incidents of severe adverse events were reported for 22 patients. WHO grade 3 or grade 4 hematologic toxicities included granulocytopenia (53.8%), leukocytopenia (28.2%), thrombocytopenia (25.6%), and anemia (24.4%). Gastrointestinal adverse events were more common with the oral formulation than previously reported with IV fludarabine phosphate. However, these events were generally mild to moderate. CONCLUSION: This study demonstrates that oral fludarabine phosphate has similar clinical efficacy to the IV formulation and a safety profile that is both predictable and essentially similar to that of the IV formulation.

Response to fludarabine in B-cell chronic lymphocytic leukemia patients previously treated with chlorambucil as up-front therapy and a CHOP-like regimen as second line therapy.

BACKGROUND AND OBJECTIVES: Fludarabine (FAMP) is the most active single agent in relapsed and refractory patients with B-cell chronic lymphocytic leukemia (B-CLL). However, it is not clear whether it should be used immediatly after failure of chlorambucil (CLB). We addressed such an issue retrospectively analyzing a series of patients in whom FAMP was used as third-line therapy after a sequential use of CLB and CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or CHOP-like regimen, respectively. DESIGN AND METHODS: On a retrospective basis, 57 B-CLL patients fulfilling the above mentioned criteria and followed-up in seven different hematologic institutions, were evaluated. RESULTS: Of 57 patients who were evaluated for response, 3 (5.2%) achieved a complete response (CR), 30 (52.6%) had a partial response (PR) and the remaining 24 (42.1%) failed to respond to FAMP. Overall median survival from the start of FAMP therapy was 30 months. Survival by tumor response did not show any difference between responders and non-responders (p = 0.536). The survival was significantly shorter in the group of patients with progressive disease than in all other patients included in our study (p = 0.05). Using each patient as his own control (McNemar test) we attempted to evaluate the value of FAMP in inducing a therapeutic response after failure of previous therapies. Among the 37 patients resistant to CLB the response rate was 40.3% with FAMP (p = 0.037) and only 17.5% with CHOP (p = 1.0). Among 35 patients resistant to a CHOP-like regimen, the response rate was 29.8% (p = 0.066) after FAMP therapy. INTERPRETATION AND CONCLUSIONS: From our results, it seems that FAMP works better than a CHOP-like regimen in patients resistant to CLB although results do not translate into a survival advantage for responders.