Hemoglobin Pitie-Salpetriere beta 34 (B16) Val replaced by Phe. A new high oxygen affinity variant associated with familial erythrocytosis.

Hemoglobin Pitie-Salpetriere was detected by routine isoelectric focusing. It moved up on isoelectric focusing between hemoglobin A and hemoglobin F, near Hb F. On cellulose acetate strips at pH 8.6, it moved as hemoglobin A. This new variant exhibits a high oxygen affinity associated with familial erythrocytosis. The valine residue in position beta 34 has been replaced by a phenylalanine residue. This residue is involved in the alpha 1 beta 1 contact.

Posttransplant lymphoproliferative disorders not associated with Epstein-Barr virus: a distinct entity?

PURPOSE: Organ recipients are at a high risk of posttransplant lymphoproliferative disorders (PTLD) as a result of immunosuppressive therapy. Most B-cell lymphomas are associated with Epstein-Barr virus (EBV) infection. We describe a morphologically and clinically distinct group of PTLD in 11 patients that occurred late after organ transplantation and were not associated with EBV. PATIENTS AND METHODS: There were seven kidney, three heart, and one liver transplant recipients (group I). The clinical manifestations, pathologic findings, treatment, and outcome were compared with those in 21 patients with EBV-associated PTLD treated in our institution (group II). EBV was detected with at least two techniques: Epstein-Barr-encoded RNA (EBER) in situ hybridization with EBER 1 + 2 probes, Southern blotting, and detection of latent membrane protein 1 (LMP1) expression by immunohistochemistry. RESULTS: The time between transplantation and the diagnosis of lymphoma ranged from 180 to 10,220 days in group I (mean, 2,234; median, 1,800) and from 60 to 2,100 days in group II (mean, 546; median, 180), and was significantly shorter in group II (P = .02). Among 19 tumors diagnosed within 2 years after the graft, 16 were associated with EBV; among 13 tumors diagnosed after more than 2 years, only five were associated with EBV. All of the B-cell PTLDs in group I were classified as monomorphic, meeting the criteria of B diffuse large-cell lymphoma (B-DLCL) with a component of immunoblasts, and genotyping confirmed their monoclonality. Three tumors were T-cell pleomorphic lymphomas. Tumor sites were mainly bone marrow and lymph nodes. Overall median survival was 1 month in group I and 37 months in group II, with two patients still alive in group I and nine in group II. The survival time was significantly longer in group II (P < .01). CONCLUSION: EBV-negative PTLD may be a late serious complication of organ transplantation. Half the tumors observed after kidney transplantation in our center were not associated with EBV and emerged after more than 5 years, which suggests the number of EBV-negative PTLDs in organ recipients might increase with time.

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.

Adult T-cell leukemia/lymphoma revealed by a surgically cured cardiac valve lymphomatous involvement in an Iranian woman: clinical, immunopathological and viromolecular studies.

A 60-year-old woman from the town of Mashhad in northeastern Iran developed cardiac failure due to aortic and mitral regurgitations which needed cardiac valve replacement. Histopathological study of the valves revealed a T-cell non-Hodgkin's lymphoma. Blood examination showed leukemic features with 32% of abnormal white blood cells. Human T-cell leukemia/lymphoma virus type I (HTLV-I) antibodies were present in the serum and the specific env HTLV-I sequences were detected in the DNA extracted from the valves and peripheral blood mononuclear cells (PBMC) using polymerase chain reaction technique. Clonal integration of two HTLV-I copies was found in both the valves and PBMC DNA, thus the diagnosis of adult T-cell leukemia/lymphoma (ATL) was established. In contrast to the acute life-threatening cardiac localization, our case met the diagnostic criteria of chronic ATL, this was confirmed by favorable evolution without chemotherapy during the 24 months after diagnosis. According to our knowledge, this is the first report of an isolated lymphomatous cardiac valve involvement, without other cardiac abnormalities. It seems important to underline that the patient originated from Iran where endemicity of HTLV-I has only recently been discovered.

Autologous hematopoietic stem cell transplantation as salvage treatment for advanced B cell chronic lymphocytic leukemia.

Given the generally poor outcome of advanced B cell chronic lymphocytic leukemia, experimental approaches are warranted, especially for younger patients in whom classical treatments have failed. We therefore conducted a prospective single-center study, using polychemotherapy (ESHAP) to prepare patients for hematopoietic stem cell collection and autologous stem cell transplantation as consolidation therapy. Twenty patients entered the study. An adequate response to ESHAP was obtained in 13 patients, and sufficient stem cells for grafting were obtained in eight of the 12 patients who underwent the collection procedure. Six of these grafted patients are alive in complete clinical remission a median of 30 months after transplantation. It should be noted that we were only able to graft 40% of the patients enrolled in this study, either because a new remission could not be obtained or because not enough hematopoietic stem cells could be collected. This argues for stem cell collection as soon as a first remission is obtained, even if the autograft is done later in the course of the disease.

T-cell depletion of bone marrow transplants: assessment of standard immunological methods of quantification.

The efficacy of bone marrow transplant (BMT) T-cell depletion for the prevention of acute graft-versus-host disease (GVHD) has been demonstrated in animal models and in clinical studies. The importance of T-cell depletion has to be evaluated with standardized methods suitable for routine purposes. We report herein an in vitro mature T-cell depletion using a cocktail of three monoclonal antibodies (CD2, CD5, and CD7) and baby rabbit complement in 38 histocompatibility leucocyte antigen (HLA)-identical BMT with no more than grade II acute GVHD. The T-cell depletion was quantified using three prestandardized immunological methods: immunofluorescence (IF) analysis, SRBC-rosetting assay, and PHA proliferation assay. A mean of 97.5% IF-assessed T-cell depletion was achieved in the 38 BMT. The immediate IF analysis using three distinct sets of anti-T-cell monoclonal antibodies allowed us to detect a mean of 1.2% residual T cells. The SRBC-rosetting assay was not useful to quantify T-cell depletion because no residual SRBC-rosette-forming cells could be detected in every case. The results of a prestandardized PHA-induced proliferation assay gave a mean 96.7% inhibition of proliferation, and they were correlated with the IF results although the IF threshold of detection was higher. From these data we conclude that our in vitro T-cell-depletion procedure is reproducible and that standardized simple immunological methods such as immediate immunofluorescence analysis and PHA proliferation assay provide good tools to assess a T-cell depletion effective in the prevention of acute GVHD.

Investigation of a new parameter in chronic lymphocytic leukemia: the percentage of large peripheral lymphocytes determined by the Hemalog D. Prognostic significance.

The number of large peripheral lymphoid cells and the ratio of these large unstained cells to the total number of peripheral lymphocytes was determined by means of the Hemalog D in 57 patients with chronic lymphocytic leukemia (CLL) and in 100 control subjects. Although the absolute number of large unstained cells/mm3 is simply a reflection of peripheral lymphocytosis, the ratio large unstained cells to total lymphocyte count was shown to correlate with clinical staging. In control subjects, this ratio ranged from 3.2 per cent to 11¿per cent. In those with CLL it was less than 11.2 per cent in 43 patients and greater than 11.2 per cent in 14 patients. These 14 patients corresponded statistically to patients with advanced disease in our clinical staging system (stages III and IV). An increase in the large unstained cells to total lymphocyte ratio is therefore a statistical criterion of poor prognosis.

Synthesis of a series of compounds related to betaxolol, a new beta 1-adrenoceptor antagonist with a pharmacological and pharmacokinetic profile optimized for the treatment of chronic cardiovascular diseases.

A series of para-substituted phenoxypropanolamines has been synthesized and tested for beta-adrenoceptor blocking activity. Some derivatives (8, 11, 12, 20, 21) exhibited greater in vitro potency than the reference drugs metoprolol and propranolol. This series, in contrast to propranolol but similar to metoprolol, possesses cardioselectivity. The 3-[p-[(cycloalkylmethoxy)ethyl]phenoxy]-1-substituted-amino-2-prop anol derivatives 8 (cyclopropylmethoxyethyl: betaxolol) and 11 (cyclobutylmethoxyethyl) produced antihypertensive effects in spontaneously hypertensive rats. Betaxolol (Kerlon, 8) was found to exhibit an appropriate preclinical pharmacological and human pharmacokinetic profile (elevated oral bioavailability and prolonged plasma half-life) for the treatment of chronic cardiovascular diseases such as hypertension and angina.

Synthesis and antihypertensive activity of a series of 4-amino-6,7-dimethoxyquinazoline derivatives.

A series of N2-[(acylamino)alkyl]-6,7-dimethoxy-2,4-quinazolinediamines was synthesized as potential alpha 1-adrenoceptor antagonists. When administered to spontaneously hypertensive rats at 10 mg/kg po, a number of propanediamine derivatives showed good antihypertensive activity, whereas the ethanediamine derivatives, albeit being structurally more closely related to prazosin, were devoid of this property. The most active derivative, N-[3-[(4-amino-6, 7-dimethoxy-2-quinazolinyl)methylamino]propyl]tetrahydro-2-furancarbo xamide hydrochloride, alfuzosin (12), showed high selectivity for peripheral alpha 1-postjunctional adrenoceptors. At equiactive antihypertensive doses, its effect on the pressor response to postural changes in conscious dog was less marked than that shown by prazosin. In the light of these results, alfuzosin was selected for clinical evaluation.

Structural and stereoelectronic requirements for the inhibition of mammalian 2,3-oxidosqualene cyclase by substituted isoquinoline derivatives.

2,3-Oxidosqualene lanosterol-cyclase (OSC; EC 5.4.99.7) is an attractive target for the design of compounds that block hepatic cholesterol biosynthesis. (4a alpha, 5 alpha, 6 beta, 8a beta)-Decahydro-5,8a-dimethyl-2-(1,5,9-trimethyldecyl)-6- isoquinolinol (1) and simplified analogs have been devised to inhibit this enzyme by mimicking the postulated pro-C-8 high-energy intermediary carbocation occurring during the cyclization-rearrangement pathway. In order to gain an understanding into the mechanism by which these types of molecules inhibit OSC, we have synthesized a series of substituted isoquinoline derivatives 3 and investigated the structural and stereoelectronic requirements, and their stringency, that make 3 potential high-energy intermediate analogs of OSC. Determination of the IC50 values of the different compounds with rat liver microsomal cyclase, allowed the study of the relative importance of (i) the nature and the stereochemistry of the nitrogen side chain, (ii) the presence of methyl groups at C-5 and C-8a (ring junction), (iii) the presence and stereochemistry of the C-6 hydroxyl group, (iv) the nature of the ring junction, and (v) the absolute configuration of the bicyclic system. The resulting structure-activity relationships seem to validate the mechanism of action of these inhibitors as analogs of a pro-C-8 high-energy intermediate and delineate the minimal requirements for the design of efficient isoquinoline-based, or simplified, OSC inhibitors.

Liver dysfunction in allogeneic bone marrow transplantation recipients.

Liver dysfunction is common in allogeneic bone marrow graft recipients, but no systematic studies of pre- and posttransplantation liver biopsies have been performed to identify and compare hepatic lesions. This study involved 25 consecutive patients who had undergone serial viral screening tests, liver tests, and pre- and posttransplantation liver biopsy. The aims were to ascertain the origin of liver disorders prior to bone marrow transplantation, to determine the mechanism and severity of liver dysfunction occurring early after transplantation, and to identify a possible relationship between pre-existing liver lesions and the frequency and nature of early liver dysfunction after transplantation. Pretransplantation biochemical liver tests were abnormal in 72% of patients, despite the absence of clinical liver disease. Eleven patients had chronic viral hepatitis B or C. Mild or moderate histological lesions were present in all the patients, with bile duct abnormalities in 48%, central vein abnormalities in 24%, sinusoidal fibrosis in 52%, portal fibrosis in 88%, portal necrosis in 52%, and parenchymal siderosis in 76%. After transplantation, fatal veno-occlusive disease occurred in two patients and biochemical abnormalities occurred in 24. Coded review of needle biopsy specimens failed to provide a single diagnosis. Histological lesions differed between pre- and posttransplantation biopsy specimens only by increased iron overload (96%, P<0.01). We conclude that pretransplant liver lesions contribute to hepatic dysfunction early after bone marrow transplantation, being very similar in nature and degree to lesions observed posttransplantation.

5th International Workshop on Chronic Lymphocytic Leukemia.

In the past few years important advances have been made in our understanding of the biology and natural history of CLL; also, new strategies and agents offer promise in the treatment of this form of leukemia. Now, patients can be treated on a more rationale basis and with real prospects for a sustained control of their disease, and improved quality of life. Hopefully, progresses in CLL will continue to accumulate in the coming years.

Transmission of Plasmodium falciparum by allogeneic bone marrow transplantation.

We describe a case of Plasmodium falciparum infection in a Comorian patient undergoing BMT. The patient's last visit to an endemic area was 1 year prior to BMT. The donor left the Comoro Islands 2 months before marrow harvesting. They had both had previous episodes of malaria and were seropositive for Plasmodium falciparum. At the time of BMT, blood smears were negative in both the donor and recipient. On day 12 post-BMT the patient was asymptomatic but a blood smear revealed 12.5% parasitemia. We consider that donors and recipients at risk pre-BMT should routinely be given specific treatment before marrow harvesting and conditioning, independent of the appearance of blood smears.

Alterations in natural anticoagulant levels during allogeneic bone marrow transplantation: a prospective study in 27 patients.

The natural anticoagulants (antithrombin III, protein C, protein S), plasminogen and tissue plasminogen activator antigen (t-PA ag), were measured in 27 consecutive patients following allogeneic BMT. Thrombosis and veno-occlusive disease were not seen in this study. Changes in the levels of these proteins occurred mainly during acute GVHD. There were 14 patients who had no acute GVHD (group I) and 13 patients who had acute GVHD (group II). No changes in antithrombin III (ATIII), protein C, protein S and t-PA levels were found in group II before the appearance of acute GVHD when compared with group I. However, we noted a significant rise in protein S (p = 0.01), antithrombin III (p = 0.001) and t-PA ag (p = 0.0004) levels during acute GVHD. In contrast, protein C levels decreased early in GVHD (p = 0.005), and then increased progressively over the course of a month post-GVHD. No changes in plasminogen levels were observed. These results might reflect activation of and/or damage to endothelial cells during GVHD.

Autologous bone marrow transplantation in relapsed HIV-related non-Hodgkin's lymphoma.

There are few reports of salvage chemotherapy for HIV-related non-Hodgkin's lymphoma (NHL). We report a relapsed HIV-related high-grade NHL which was treated successfully with ESHAP chemotherapy followed by high-dose chemotherapy and autologous bone marrow transplantation (ABMT). ABMT may later have triggered opportunistic infections in this patient.

Proposals for a Phenotypic Classification of B-Chronic Lymphocytic Leukemia, Relationship with Prognostic Factors.

Immunophenotypic analysis was performed in 53 cases of B chronic lymphocytic leukemia using a large panel of monoclonal antibodies recognizing B, T, activation and myeloid antigens. Our results showed four patterns of reactivity: (a) several molecules were constantly expressed: CD19, CD20, CD24, CD37, HLA-DR, mu heavy chain, CD5, CD23, B5, CD32; (b) one antigen, CD11b, was found in 50 to 80% of the cases; (c) some markers were detected in less than 50% of the cases: CD25, CD38, CD71, CD11a, c, CD14b-c; (d) CD2 and CD16 were never detected. From these results, a phenotypic classification in three groups has been proposed and these groups were correlated with the progression of the disease, mainly with the lymphocyte doubling time of less than one year. We hypothesized that the leukemia cells could be at various stages of differentiation and/or activation according to their expression of activation and myeloid markers.

Skewed rearrangement of the VH4-21 gene during pre-B acute lymphoblastic leukemia.

Thirty-six pre-B acute lymphoblastic leukemias (ALL) were studied for VH family expression. Among the 35 detected rearrangements, VH1 family genes were expressed in 7, VH2 in 1, VH3 in 18, VH4 in 6 and VH6 in 3. This expression is close to that expected according to the complexity of the system. The complete sequence of the 6 VH4 genes was examined in order to determine whether there is a skewed rearrangement of individual genes in this family. Our results indicate rearrangement of VH4-21 in 3 cases, 71-4 in one, 58P2 in one case and probably of a new germinal VH4 gene for the sixth case. All the genes were displaying an almost complete homology with their germinal VH counterparts. The 6 sequenced genes associated with 6 different D gene segments displaying a close homology with their germinal counterpart. JH4 segment was expressed in 3 cases and JH6 in the remaining 3. These results associated with previous results obtained by others indicate that there is skewed rearrangement of the VH4-21 gene in pre-B ALL. It is presently unknown whether this phenomenon is the consequence of a selective process or whether it reflects what normally occurs in the normal human functional repertoire, which could be more limited than the germline repertoire.

VH gene expression in CD5 positive and CD5 negative B cell chronic lymphoid malignancies.

In this review, we report analyses of VH genes in mature B cell malignancies generally or occasionally bearing CD5 antigen such as B CLL, MCL, SLVL and PLL. In the majority of cases, B CLL and MCL use VH genes in germline configuration. However in some cases a higher rate of random mutations is observed. These differences are not related to CD5 expression but are accounted by Ig phenotype, since less mutations are observed in CLL cases expressing membrane mu delta, when compared to forms exclusively expressing membrane mu. PLL and SLVL cases display mutated V genes independently of CD5 expression. Although there is some evidence that CD5+ B cells constitute a separate lineage, the possibility that CD5 constitutes an activation marker cannot be ruled out. Indeed, CD5- B cells can be induced to differentiate into CD5+ B cells and VH gene analyses showed no significative differences between CD5+ and CD5- B cell lymphoproliferative disorders. In this review we have tried to examine B cell chronic malignancies on the basis of phenotype and VH gene usage. Thus we propose a tentative classification where these disorders are allocated according to these characteristics.

Evaluation of residual disease in B-cell chronic lymphocytic leukemia patients in clinical and bone-marrow remission using CD5-CD19 markers and PCR study of gene rearrangements.

We evaluated minimal residual disease (MRD) in 23 CD5 + B-chronic lymphocytic leukemia (CLL) patients who achieved clinico-hematological remission confirmed by bone-marrow biopsy. MRD was evaluated by dual marker analysis flow-cytometry using CD5 and CD19 markers, and by the study of Ig heavy chain gene rearrangements using the fast polymerase chain reaction (PCR). According to our laboratory conditions patients were considered to be in complete phenotypic remission when total CD19+ cells were < 25% and the ratio of CD5 + CD19 + /CD19 + cells was < 25%. According to these strict criteria only 9 of the 23 patients were in complete phenotypic remission. In order to evaluate the sensitivity of the above method, PCR analysis of the configuration of the Ig heavy chain gene region was performed in 12 of these patients. Five of 7 patients in complete phenotypic remission retained a detectable monoclonal rearrangement of the Ig heavy chain gene. For the remaining 5 patients in partial phenotypic remission, only one failed to show a monoclonal band and this is probably explained by the presence of an unusual gene rearrangement. In conclusion, this study suggests that PCR is more sensitive than dual marker flow-cytometry for evaluation of residual disease and that it is indeed possible to achieve complete remission at the molecular level, in B-CLL. Nevertheless, we suggest a word of caution as this was a retrospective study, and samples were not assessed before treatment. Thus the possibility that apparent molecular remission might correspond to unusual gene rearrangements cannot be completely excluded in these cases.

A single-center study of 11 patients with intraocular lymphoma treated with conventional chemotherapy followed by high-dose chemotherapy and autologous bone marrow transplantation in 5 cases.

Intraocular lymphoma (IOL) is a rare form of non Hodgkin lymphoma (NHL); it has a poor prognosis and is frequently associated with central nervous system (CNS) infiltration. We report the results of a prospective study of 11 patients with IOL who received conventional chemotherapy (CT), followed by salvage high-dose (HD) CT with autologous bone marrow transplantation (ABMT) in five cases. All 11 patients had abnormal funduscopic findings and six had CNS involvement at diagnosis. The diagnosis was based on vitrectomy in 10 cases and cerebral stereotaxic biopsy in one. Pathologic studies showed large-cell NHL in all cases. These large-cell NHL were of the B-cell type in 8 cases and of the T-cell type in two. First-line therapy consisted of a combination of cisplatin 25 mg/m2 as a 24-hour IV infusion on 4 consecutive days, VP-16 40 mg/m2 for 4 days, aracytine 2 g/m2 IV on day 5, and methylprednisolone 500 mg IV daily for 5 days (ESHAP) in 5 cases; alternating courses of ESHAP and HD methotrexate (MTX) in 4 cases; and HD MTX in 2 cases. Three patients underwent ocular and whole-brain radiation therapy. Five refractory patients subsequently received intensive CT with thiotepa 750 mg/m2, busulfan 10 mg/kg and cyclophosphamide 120 mg/kg, followed by ABMT. First-line treatment failed in 10 evaluable cases. One patient died of CNS progression at 12 months. All the patients who underwent intensive CT and ABMT entered CR; two relapsed at 6 months and three are alive in CR 15, 15 and 14 months after ABMT. Six patients are alive with persistent disease at 8, 13, 14, 15, 18 and 24 months. It seems in conclusion that, high-dose thiotepa, busulfan and cyclophosphamide followed by ABMT is effective in some cases of refractory IOL.