Tissue microarray in a subset of South African patients with DLBCL.

Tissue samples from 93 de novo diffuse large B-cell lymphoma patients seen between 1995 and 2009 randomly receiving either standard combination chemotherapy (CHOP, n=48) or the identical program with rituximab (n=45) were subtyped using an investigational immunohistochemical (IHC) based tissue microarray (TMA) and contrasted to the approximately corresponding categories as defined either by Hans and associates using a three marker panel into germinal or non-germinal centre subtypes or by Choi and colleagues with two additional antibodies into germinal centre (GCB) or activated B-cells (ABC). Each of these primary subdivisions was further evaluated for expression of BCL2 and LMO2 both of which are recognised to predicate response. The addition of rituximab to the uniform drug regimen did not show any significant improvement in 5 years overall (63% versus 59%, p 0.68) or event-free survival (42% versus 39%, p 0.94), for CHOP versus R-CHOP comparisons. Similarly no differences were evident in subtype analysis. Interestingly however, when segregated on the Choi criteria, cytotoxic drugs alone showed a non-significant trend in improved survival (74% versus 55%, p 0.32) as well as event-free survival (44% versus 40%, p 0.42) for the germinal centre as opposed to the activated B-cell subtype. Nevertheless not even a small difference could be demonstrated in the presence of the anti CD 20 monoclonal antibody. According to Choi, both regimens (chemotherapy or immunotherapy antibody) revealed similar results to the Hans algorithm on 5 years OS as well as 3 year EFS when comparing GCB versus ABC or non-GCB subgroups. BCL2 and LMO2 marker expression of the respective immunohistochemical (IHC) subtype, despite small sample size, revealed the following. Analysis by Choi criteria on survival for BCL2, no matter for which subsets (GCB or ABC) or treatment modality (chemotherapy with or without the addition of rituximab) showed no difference in 5 years OS or EFS. In contrast, a significant difference for better EFS (p=0.0015) in the BCL2 positive group of the ABC subgroups subtypes treated with rituximab containing chemotherapy. For LMO2 similar results on survival outcome were seen thus showing no difference in 5 years OS or EFS - regardless of subtype or treatment modality. Also here, this was contrasted by better EFS (p=0.039) in the LMO2 positive group of ABC subtypes when treated with the rituximab containing regimen. The use of the IHC based TMA methodology has shown to be a simple, cost effective and a robust alternative to gene expression profiling (GEP) which is currently regarded as the gold standard for the classification in lymphomas. It provides a useful prognostic tool in stratifying DLBCL or other entities in future, even when frozen tissue samples are not available for GEP analysis. With the current budgetary limitations in South African public hospitals chemotherapy protocols for lymphoproliferative disorders exclude agents such as rituximab. Local therapeutic drug committees consider the approximately 15% overall survival benefit seen at 5 years for DLBCL when rituximab is added to combination chemotherapy as too marginal for justifying the arising additional expenses. Accordingly, demonstration that a specific molecular subtype accounts for superior outcome, when using these regimens, is needed. Such an option would provide convincing evidence for the use of immunochemotherapy in a resource constrained setting.

Lymphoma--emerging realities in sub-Saharan Africa.

Substantial geographical differences exist for Hodgkin and other lymphoproliferative disorders with these having previously been documented in a report from the lymphoma reclassification project. In the light of rampant human immunodeficiency syndrome, largely centred in sub-Sahara, this experience is updated in a further 512 consecutive individuals treated over an 8-year period in a privately based academic centre. Median age was 55.2 years 61% were males, 10% had Hodgkin lymphoma and, overall, constitutional symptoms were present in 20%. Prior to referral 19% had received chemotherapy and a further 20% some form of irradiation. Median survival in hairy cell leukaemia (n=14), chronic lymphocytic leukaemia-small lymphocytic lymphoma (n=103), Hodgkin (n=41) and follicular lymphoma (n=59) was not reached at the time of analysis and exceeded 36 months. This was followed by 32 months for those with mantle cell (n=7), splenic (n=2) and extranodal marginal cell (n=11), 24 months for T-cell lymphomas (n=24), 20 months for diffuse large B-cell variants (n=88) but only 12 months for the aggressive tumours exemplified by Burkitt (n=7) and lymphoblastic subtypes (n=6). The remaining 36 patients had to be excluded because numbers were too small for statistical analysis or unreliable staging. Adverse factors were constitutional symptoms, prior treatment with chemotherapy, intermediate or high-risk scores as defined by the international prognostic index, histologic grading and certain anatomical sites of primary tumour. In contrast gender, staging by Rye or Rai classification, retroviral infection and prior treatment with radiotherapy were without effect. Overall survival at 3 years in each category was compared to the curve for the entire cohort and was 100% in hairy cell leukaemia receiving two chlorodeoxyadenosine and greater than 88% in Hodgkin lymphoma treated according to the German study group protocols (p=0.0004). Corresponding figures for chronic lymphocytic leukaemia-small lymphocytic lymphoma were 82% (p=0.0006), follicular lymphoma 71% (p=0.060), peripheral T-cell lymphoma 43% (p=0.0156), diffuse large B-cell lymphoma 39% (p<0.0001), aggressive tumours 25% (p=0.0002) and for the indolent categories including mantle cell, splenic and extra nodal marginal cell lymphomas 22% (p=0.2023). Outcome argues in favour of patient management by a multidisciplinary team implicit in which are standardised protocols for diagnosis, staging and treatment. Under these circumstances the well recognized centre effect applies when results approximate those from first world reference centres. Conversely any deviation from such a disciplined approach is unlikely to achieve comparable benefit and therefore to be strongly discouraged.

Immunohematopoietic stem cell transplantation in Cape Town: a ten-year outcome analysis in adults.

BACKGROUND AND OBJECTIVES: Immunohematopoietic stem cell transplantation has curative potential in selected hematologic disorders. Stem cell transplantation was introduced into South Africa in 1970 as a structured experimental and clinical program. In this report, we summarize the demography and outcome by disease category, gender, and type of procedure in patients older than 18 years of age who were seen from April 1995 to December 2002. PATIENTS AND METHODS: This retrospective analysis included 247 individuals over 18 years of age for whom complete data were available. These patients received grafts mostly from peripheral blood with the appropriate stem cell population recovered by apheresis. RESULTS: Patient ages ranged from 20 to 65 years with a median age of 42 years. There were 101 females and 146 males. There were no withdrawals and 63% survived to the end of the study. At 96 months of follow-up, a stable plateau was reached for each disease category. Median survival was 3.3 years (n=6, 14.6%) for acute lymphoblastic anemia, 3.1 years (n=44, 18%) for acute myeloid leukemia, 2.8 years (n=47, 19%) for chronic granulocytic leukemia, 2.8 years (n=71, 29%) for lymphoma, 1.5 years (n=23, 9%) for myeloma, 1.43 years (n=10, 4%) for aplasia, and 1.4 years (n=38, 15%) for a miscellaneous group comprising less than 10 examples each. Multivariate analysis showed that only diagnosis and age had a significant impact on survival, but these two variables might be interrelated. There was no significant difference in outcome by source of graft. CONCLUSION: The results confirm that procedures carried out in a properly constituted and dedicated unit, which meets established criteria and strictly observes treatment protocols, generate results comparable to those in a First World referral center. Low rates of transplant-related mortality, rejection and graft-versus-host disease are confirmed, but the benefits cannot be extrapolated outside of academically oriented and supervised facilities.

The use of short tandem repeat polymorphisms for monitoring chimerism following bone marrow transplantation: a short report.

Following immunohaematopoietic stem cell transplantation, it is of importance to determine whether the new blood forming system is of recipient or donor origin and such phenotypic characterisation is called chimerism analysis. This is a dynamic process, which may be complete, mixed or split between compartments and in this way, plays an increasingly important role in predicting outcome for engraftment, rejection or residual disease predicating the need for pre-emptive immunotherapy. Based on recent workshop recommendations, peripheral blood cells have been used in the short tandem repeat (STR) assay to serially characterise the haematologic course and so evaluate the usefulness of this system. Forty-six patients from a single centre were followed serially for periods ranging between 3 and 60 months. The analysis was initially performed using the Applied Biosystems Profiler Plus Kit; currently, the Promega Powerplex 16 system is used. The overlap between the two assays has allowed for continuous comparison. The initial analysis was performed at 14 days post-transplant and repeated monthly. Stored DNA from the patient and donor was used to establish the pre-transplant profile. All post-transplant analyses were performed using peripheral blood. The results obtained were expressed as a percentage of the donor profile. To illustrate the ability of this technology, three representative profiles are described. In the first, stable engraftment is confirmed at 20 months with only donor pattern present. The second is intermediate, and while the patient is clinically disease free, there exists stable mixed chimerism at about 75% of donor cells. The third patient initially engrafted but the reappearance of recipient alleles presaged a haematological relapse; the latter is an indication for salvage with donor lymphocyte infusion and here this assay will be used to show the effectiveness of the intervention. These preliminary results show this to be a useful additional tool in monitoring post-transplant engraftment. As a basis for pro-active therapy, a larger study integrating the results of haematological and cytogenetic markers is planned.

Pediatric immunohematopoietic stem cell transplantation at a tertiary care center in Cape Town.

UNLABELLED: INTRODUCTION AND STUDY DESIGN: We conducted a retrospective analysis of consecutive referrals of patients under 18 years of age undergoing immunohematopoietic stem cell transplantation to assess the influence of age, diagnosis, graft type and gender on survival. We also contrasted program activity and outcome to that reported from a state hospital in the same geographical area over a comparable period. METHODS: Conditioning employed either a sequential combination of fractionated 12Gy whole body and 6Gy total nodal irradiation separated by 120mg/kg of cyclophosphamide in patients over 15 years of age. Alternatively, the latter agent was combined initially with oral busulphan and later the intravenous equivalent. Neuroblastoma cases were prepared using a different regimen. In allografts the harvested product underwent ex vivo T-cell depletion with the humanized version of anti-CD 52 monoclonal antibody designated Campath 1H. No additional immunosuppression was given except where matched unrelated volunteer donors were employed. RESULTS: Sixty-eight procedures were carried out in 61 patients over a 6-year period. Of 11 with acute myeloid leukemia, 8 are alive and well whereas 8 of the 14 with the lymphoblastic variant have died. Of the remaining 12 with hematologic malignancy, all but 2 are alive. Ten of the 17 with aplasia are alive as are all with thalassemia or sickle cell disease. None of the four variables tested affected survival. CONCLUSION: Our analysis indicates that the standardized preparative regimen, coupled with a now well-established immunosuppressive regimen, is as effective in patients under 18 years of age as in adults. Our analysis also indicates that in a resource-scarce or developing country, it is mandatory to limit high-risk and relatively expensive procedures to active teams that enjoy international accreditation, whether these be in the state or private sector.

A single unit lymphoma experience: outcome in a Cape Town academic centre.

To document outcome in Hodgkin and other lymphomas from a privately based academic centre the clinical records from 253 consecutive referrals were analysed. Diagnosis was according to World Health Organization criteria, prognosis assigned by the international index and therapy risk-stratified with results subject to appropriate statistical methodology. None of these patients underwent transplantation. For the cohort the median age was 55 years (range 11-94) and 63% were male. Constitutional symptoms were present in 22%; a quarter had previous chemotherapy and a third some form of irradiation prior to referral. Fifty-seven percent were stage I or II and 21% had nodal disease above and below the diaphragm whilst in the remainder cells were present in the circulation and this included the subset of chronic lymphocytic leukaemia -- small lymphocytic lymphoma. Positron emission scanning was not available for these studies. Median survival for the cohort is 3.2 years and reduced to 1.3 years by the presence of unexplained fever, sweating or inappropriate weight loss. Further adverse factors included any prior treatment, intermediate or high-grade histopathology, risk factors defined by the International Prognostic Index as well as late Rai stages. Analysed by disease category Hodgkin lymphoma (n=17) when managed according to the German Study Group protocols and hairy cell leukaemia (n=10) treated with two chlorodeoxyadenosine -- both had a stable plateau in excess of 90%. The corresponding figures for follicular variants (n=31) was 72% in the low risk and 58% in the remainder when treated with cyclophosphamide, vincristine and prednisone. Curves for the aggressive or diffuse large B-cell lymphoma (n=44) fell initially to 48%, but relapse continued in stages III and IV to the current level of 18% when receiving cyclophosphamide, hydroxydaunorubicin, vincristine and prednisone on the 21-day schedule. Chronic lymphocytic leukaemia -- small lymphocytic lymphoma (n=58) were initially given pulsed chlorambucil and sustained response was over 90% with low bulk, but declined to reach 30% as prognostic score rose. The miscellaneous categories (n<5 each) managed variably, but using the same criteria, were pooled and are presently at 62% and 30% for high and low grades. It is concluded that precise diagnosis, accurate staging and therapy on standardised risk-stratified programmes, delivered uniformly by a single multidisciplinary group, creates the all-important centre effect; matching figures are unlikely to apply outside these disciplined circumstances. The expectation from patients and referring physicians alike is that, since lymphomas are potentially curable, such an approach to comprehensive management will be regarded as standard even in an under resourced or Third World country. It follows that late referral and prior therapy will adversely affect performance status and compromise life span: These alternative approaches are inappropriate and strongly discouraged.

Amyloidosis: a changing clinical perspective.

Primary amyloidosis is a plasma cell dyscrasia characterised by excess production of abnormal immunoglobulin light chains with their subsequent accumulation in kidneys, heart, liver as well as gastrointestinal tract and bone marrow 1-21, 2. These tissue deposits take the form of a fibrillar protein which damages the involved organ in proportion to the extent of the infiltration and roughly parallels the duration of the disease. Most cases have evidence of the underlying lymphoplasmacytoid neoplasm recognisable in two ways. Firstly, the monoclone appears in the serum [2]. Secondly is a morphologically and immunohistochemically distinctive cellular infiltrate in the bone marrow [3] that has a specific microscopic and ultrastructural pattern 4-54, 5. Interestingly occasional patients, who survive long enough, may progress to multiple myeloma [6] but the correlation is variable [7].

Homocysteine in vascular disease: an emerging clinical perspective.

Traditionally, arterial and venous thrombo-embolism has been attributed to pathophysiological lesions in the vessel wall, or altered blood contents or flow through the lumen. Logically these fall into the category of a high-shear system, as opposed to the rather different circumstances in slowly moving columns of blood. The re-evaluation of this belief has been prompted by acknowledgement that there is currently a shift in emphasis. Recently we have recognised a common central factor in the internal dysfunction of the vessel that precedes and so initiates thrombocyte adhesion.

Silent angiodysplasia and occult haemobilia as rare but treatable causes of refractory iron deficiency--two case reports.

Once menorrhagia has been excluded in females then, in both sexes, the gastrointestinal tract remains the commonest site for haemorrhage. This may be of surprisingly large volume but intermittent and therefore not universally demonstrated on stool testing. However, if loss is persistent it may nevertheless culminate in absolute iron deficiency and thus, even when occult blood is not present on repeated examinations, quantitation using chromium labelled red cells becomes invaluable. In this situation, endoscopy or contrast radiology of the small and large bowel may fail to reveal any lesion even when these procedures are repeated or used in combination. Modifications by direct inspection or camera study may be helpful in improving diagnostic accuracy. It is nevertheless practical, as illustrated by these two cases, to more widely recognise the value of radionuclide scanning methods. In one this was due to unsuspected haemobilia and the second to major duodenal vascular malformation although it could be reasonably argued that initial recourse to angiography might have demonstrated this. The principle is that when precisely defined anatomically surgery can be elective and limited as a result of careful proactive planning and operations likely to have a high initial rate of success. The role of nuclear medicine in the investigative algorithm of such patient is re-emphasised. Thus, in any individual with unexplained but proven absolute iron deficiency failure to reveal the cause by first screening with gastroscopy and colonoscopy or barium studies including the small bowel should not automatically be repeated. Rather, the blood loss needs to be documented and, if possible, subsequent evaluation moved to advancement or push enteroscopy, capsule endoscopy or the more invasive angiography only once quantity a pattern of bleeding are defined by radioisoptic imaging.

Splenectomy in haematology--a 5-year single centre experience.

OBJECTIVE: To define indications and outcome in haematologic cases undergoing splenectomy. STUDY DESIGN: A retrospective review of clinical records from consecutive patients having open or laparoscopic removal of the spleen in an academic centre in the private sector. Endpoints were survival, operating time, spleen size, histopathology, requirements for blood or related products complications and average costs. RESULTS: In the total group (n = 69) there were two deaths. Referrals were for immune thrombocytopaenia (41%), acquired haemolytic anaemia (10%), myeloproliferative syndrome (9%), acute or chronic leukaemia (19%), lymphoma (13%) and a miscellaneous group (8%), comprising cholelithiasis, aplasia or as a diagnostic procedure for otherwise unexplained splenomegaly. An open midline approach was predicated by spleens greater than twice normal size and a history of any bleeding disorder. Here the mean operating time was 83 min (range 40-295) whereas for laparoscopy this was 251 min (range 181-272). SUMMARY: Careful stratification between the two options facilitated optimum haemostasis and consequently reduced requirement for packed red cells and platelets. Neither underlying pathology nor the choice of treatment influenced morbidity or mortality. Overall local experience is consistent with published international standards of surgical practice. Outcome is directly proportional to the number of each procedure carried out by a single team, observance of consistent protocols for preoperative evaluation and standardized proactive management through the recovery period.

Clonogenic growth patterns correlate with chemotherapy response in acute myeloid leukaemia.

Cytosine arabinoside and anthracycline-containing regimens induce remission in upwards of 60% of previously untreated patients with adult acute myeloid leukaemia (AML). Despite this, in addition to primary drug resistance, the majority of these patients relapse. Reliable methods for uniformly recognising these two subgroups at presentation do not exist and therefore a further attempt has been made to relate in vitro toxicity, using a clonogenic assay, to clinical outcome. In 10 normal controls and 12 chemotherapy naïve cases, mononuclear cells harvested by density gradient separation were re-suspended at a concentration of 2 x 10(5)/ml and quadruplicates of 250 microl per well cultured in methycellulose containing foetal calf serum and phytohaemagglutinin stimulated leucocyte conditioned medium. Cell kill was determined for cytosine arabinoside, daunorubicin and etoposide either singly or in combination using both a pulsed and continuous exposure. Aggregates were scored after seven days and three distinct patterns recognised. The patients all received the same drugs in a standard protocol and achievement of complete remission correlated with growth pattern. The survival of normal marrow colony-forming cells or GM-CFUc and the leukemic equivalent designated L-CFUc were assessed and a sensitivity index (SI) determined as a ratio of these two values in which more reproducible results were found when the drug was continuously present. It is concluded that the microculture technique is feasible and clearly demonstrates chemotherapy effect but no correlation was demonstrated with clinical outcome. This is a negative pilot study and, as a means of recognising drug sensitivity or resistance, should be discarded in favour of currently available molecular techniques.

The effect of peptide stimulation on haematopoietic stem cell mobilisation including engraftment characteristics and a note on donor side effects.

Aplasia or irreversible bone marrow failure and a variety of haematologic malignancies, as well as an increasing number of solid tumours, currently include various forms of marrow or equivalent transplantation in routine management. In both allogeneic and autologous procedures stable recipient immunohaematopoietic reconstitution depends upon infusing the requisite population harvested at a precise time following commencement of a stimulatory peptide. In a first step this prospective study documented the safety of apheresis, defined side effects and enumerated mononuclear, CD34+ and CD3+ cells obtained. In the second stage delivery of the graft, characterised in this way and with the additional measurement of in vitro growth in clonogenic assay, to the suitably conditioned patient was correlated with recovery of neutrophil and platelet numbers appearing in the circulation. In a third and ongoing analysis the influence of passenger T-lymphocytes is being evaluated for impact on infection and a potential anti-tumour effect. The conclusion is that this technology is reliable, has a high degree of patient acceptability without untoward complications, and that local results correspond to international experience thereby providing an important and relevant measure of quality control.

Estimation and predictive use of the corrected count increment--a proposed clinical guideline.

BACKGROUND AND OBJECTIVE: Platelets are frequently given inappropriately. The accepted indication is symptomatic thrombocytopaenia due to bone marrow failure. In contrast replacement therapy is contraindicated in immune mediated peripheral sequestration because it is almost always ineffective and also aggravates the already rapid rate of the clearance due to superimposed isoimmunisation. Furthermore circulating levels, rather than the clinical situation, customarily trigger the request and would typically be at 20 x 10(9)/l. Usage is under evaluated and, accordingly, current practice has been scrutinised and a guideline proposed that is applicable to user and vendor alike. DESIGN AND METHODOLOGY: Prospectively a consecutive series of eligible cases were selected and, with informed consent, all relevant information recorded. Specific observations were haematologic status, quality of the product and the impact of previous exposure to blood fractions on outcome. Additionally, factors that influence response were documented and included disseminated intravascular coagulation, splenomegaly and concurrent use of intravenous Amphotericin B. The absolute numbers infused, the increase in peripheral value recorded and this ratio which is designated as corrected count index generated in each instance. RESULTS: The CCI was computed for each of the 85 megaunits given to 29 individuals and subsequently estimated for the entire sample population using regression analysis. Each of the variables was used to test the hypothesis that such a figure might differ depending on patient sub-population. This approach was accurate in 60% of the time and predicates a more rational use of donor characteristics. The cardinal measurement is that supplied by the transfusion service which can be used as a basis for clinically important predictors of anticipated benefit. CONCLUSION: This data, in keeping with international practice, leads to the recommendation that commercial and other services routinely measure and specify absolute numbers. The clinicians, reciprocally, should always confirm this figure and then match anticipated to observed outcome. Failure to carry out these simple procedures lead to gross over utilisation of this expensive intervention, risks unnecessary sensitisation and furthers persistence with sub-optimum administration.