Favourable outcomes for high-risk diffuse large B-cell lymphoma (IPI 3-5) treated with front-line R-CODOX-M/R-IVAC chemotherapy: results of a phase 2 UK NCRI trial.

BACKGROUND: Outcomes for patients with high-risk diffuse large B-cell lymphoma (DLBCL) treated with R-CHOP chemotherapy are suboptimal but, to date, no alternative regimen has been shown to improve survival rates. This phase 2 trial aimed to assess the efficacy of a Burkitt-like approach for high-risk DLBCL using the dose-intense R-CODOX-M/R-IVAC regimen. PATIENTS AND METHODS: Eligible patients were aged 18-65 years with stage II-IV untreated DLBCL and an International Prognostic Index (IPI) score of 3-5. Patients received alternating cycles of CODOX-M (cyclophosphamide, vincristine, doxorubicin and high-dose methotrexate) alternating with IVAC chemotherapy (ifosfamide, etoposide and high-dose cytarabine) plus eight doses of rituximab. Response was assessed by computed tomography after completing all four cycles of chemotherapy. The primary end point was 2-year progression-free survival (PFS). RESULTS: A total of 111 eligible patients were registered; median age was 50 years, IPI score was 3 (60.4%) or 4/5 (39.6%), 54% had a performance status ≥2 and 9% had central nervous system involvement. A total of 85 patients (76.6%) completed all four cycles of chemotherapy. There were five treatment-related deaths (4.3%), all in patients with performance status of 3 and aged >50 years. Two-year PFS for the whole cohort was 67.9% [90% confidence interval (CI) 59.9-74.6] and 2-year overall survival was 76.0% (90% CI 68.5-82.0). The ability to tolerate and complete treatment was lower in patients with performance status ≥2 who were aged >50 years, where 2-year PFS was 43.5% (90% CI 27.9-58.0). CONCLUSIONS: This trial demonstrates that R-CODOX-M/R-IVAC is a feasible and effective regimen for the treatment of younger and/or fit patients with high-risk DLBCL. These encouraging survival rates demonstrate that this regimen warrants further investigation against standard of care. TRIAL REGISTRATION: ClinicalTrials.gov (NCT00974792) and EudraCT (2005-003479-19).

The influence of bone cement type on production of fretting wear on the femoral stem surface: a preliminary study.

BACKGROUND: It has been reported that bone cement correlates with survivorship of cemented total hip replacement. However, little research has been published to investigate the influence of bone cement type on production of fretting wear on the femoral stem. METHODS: In the present study, we performed six in vitro wear simulations using the same type of femoral stem (polished Exeter V40™) and three different bone cements (Simplex P, Palacos R, and CMW 3). FINDINGS: Fretting wear was consistently reproduced on the stem surface and the wear locations compared well with the results of retrieval studies. Selected 3D surface parameters were utilised to quantitatively evaluate fretting wear and no significant difference was identified in terms of fretting wear severity between these simulations. The bone cements were all badly damaged in those sites contacting the fretting wear areas on the femoral stem. Additionally, there were plenty of wear debris present on the cement surface, and the energy dispersive X-ray analysis confirmed that it was just cement particles for Simplex P bone cement, whilst it included metallic particles for Palacos R and CMW 3 bone cements. INTERPRETATION: This preliminary study shed some light on the influence of bone cement type on production of fretting wear on the femoral stem surface but further research is needed to gain a better understanding on this issue.

The significance of the micropores at the stem-cement interface in total hip replacement.

Cemented total hip replacement has been performed worldwide to treat patients with osteoarthritis and osteonecrosis, with aseptic loosening as its primary reason for revision. It has been indicated that the stem-cement interfacial porosity may contribute to the early loosening of cemented hip prosthesis. In addition, it is generally accepted that the micropores in bone cement surface and in the bulk material are detrimental to the mechanical integrity of bone cement and act as stress concentrators, resulting in generation of fatigue cracks in the cement mantle. Furthermore, it was demonstrated that the micropores also play an important part in initiation and propagation of fretting wear on polished femoral stems. Taking this into consideration, a detailed review of the potential significance of the micropores in bone cement and the methods that could be employed to reduce porosity is given in this article. It was considered that modern cementing techniques are clinically beneficial and should be applied in surgery to further improve the survivorship of cemented total hip replacement.

Investigation of relative micromotion at the stem-cement interface in total hip replacement.

Cemented total hip replacement has become a standard surgical technique to treat patients with osteoarthritis and osteonecrosis. The stem-cement interface experiences fretting wear in vivo due to low-amplitude oscillatory micromotion under physiological loading, and this wear is currently becoming important as a potential mechanism for the overall wear of cemented total hip replacements. However, the relative micromotion at the stem-cement interface has not been widely reported. In the present study, a new micromotion sensor is developed that is based on the deformation of a strain gauge, and this sensor is used to probe the migration of a polished Exeter stem within a Simplex P cement mantle through an in vitro wear simulation. It is demonstrated that the stem migration value generally increases with an increase in the number of loading cycles, with a gradual decrease of migration rate. Additionally, fretting wear is successfully replicated on the stem surface, and the micropores in the cement surface are considered to contribute to initiation and propagation of the fretting damage on the stem. This is confirmed by the observation that no evidence of fretting wear is detected on the stem where the surface is in contact with the pore-free areas on the cement. This study allows a deep insight into the micromotion at the stem-cement interface, and provides evidence highlighting the significance of the micropores in the cement surface in the generation of fretting wear on a polished femoral stem.

Femoral stem wear in cemented total hip replacement.

The great success of cemented total hip replacement to treat patients with endstage osteoarthritis and osteonecrosis has been well documented. However, its long-term survivorship has been compromised by progressive development of aseptic loosening, and few hip prostheses could survive beyond 25 years. Aseptic loosening is mainly attributed to bone resorption which is activated by an in-vivo macrophage response to particulate debris generated by wear of the hip prosthesis. Theoretically, wear can occur not only at the articulating head-cup interface but also at other load-bearing surfaces, such as the stem-cement interface. Recently, great progress has been made in reducing wear at the head-cup interface through the introduction of new materials and improved manufacture; consequently femoral stem wear is considered to be playing an increasingly significant role in the overall wear of cemented total hip replacement. In this review article, the clinical incidences of femoral stem wear are comprehensively introduced, and its significance is highlighted as a source of generation of wear debris and corrosion products. Additionally, the relationship between femoral stem surface finish and femoral stem wear is discussed and the primary attempts to reproduce femoral stem wear through in-vitro wear testing are summarized. Furthermore, the initiation and propagation processes of femoral stem wear are also proposed and a better understanding of the issue is considered to be essential to reduce femoral stem wear and to improve the functionality of cemented total hip replacement.

Influence of femoral stem surface finish on the apparent static shear strength at the stem-cement interface.

The stem-cement interface has long been implicated in failure of cemented total hip replacement. Much research has been performed to study the factors affecting the bond strength between the femoral stem and the bone cement. The present study aims to further investigate the influence of femoral stem surface finish on the apparent static shear strength at the stem-cement interface through a series of pull out tests, where stainless steel rods are employed to represent the femoral stem. The results demonstrated that there was a general tendency for the apparent static shear strength to be increased with the rise of surface roughness. The polished and glass bead-blasted rods illustrated a slip-stick-slip failure whereas the shot-blasted and grit-blasted rods displayed gross interface failure. Following pull out test, cement transfer films were detected on the polished rods, and there was cement debris adhered to the surface of the grit-blasted rods. Micropores, typically 120 mum in diameter, were prevalent in the cement surface interfaced with the polished rods, and the cement surfaces in contact with the shot-blasted and grit-blasted rods were greatly damaged. There was also evidence of metal debris embedding within the cement mantle originating from the tests of the grit-blasted rods, indicating an extremely strong mechanical interlocking at the interface. In summary, this present research demonstrated that the grit-blasted rods with the highest surface roughness were the best in terms of apparent static shear strength. However, it seemed to be most applicable only to the stem designs in which mechanical interlocking of the stem in the initial fixed position was essential.

Reproduction of fretting wear at the stem-cement interface in total hip replacement.

The stem-cement interface experiences fretting wear in vivo due to low-amplitude oscillatory micromotion under physiological loading, as a consequence it is considered to play an important part in the overall wear of cemented total hip replacement. Despite its potential significance, in-vitro simulation to reproduce fretting wear has seldom been attempted and even then with only limited success. In the present study, fretting wear was successfully reproduced at the stem-cement interface through an in-vitro wear simulation, which was performed in part with reference to ISO 7206-4: 2002. The wear locations compared well with the results of retrieval studies. There was no evidence of bone cement transfer films on the stem surface and no fatigue cracks in the cement mantle. The cement surface was severely damaged in those areas in contact with the fretting zones on the stem surface, with retention of cement debris in the micropores. Furthermore, it was suggested that these micropores contributed to initiation and propagation of fretting wear. This study gave scope for further comparative study of the influence of stem geometry, stem surface finish, and bone cement brand on generation of fretting wear.

Cutaneous B-cell lymphoma: pathological spectrum and clinical outcome in 51 consecutive patients.

The clinical behavior and optimal treatment of patients presenting with skin infiltration by B-cell lymphoma have not been established. To clarify this we assessed the clinical and laboratory features of 51 patients presenting with cutaneous infiltration by B-cell lymphoma. Follow-up data was available for 46 patients with a median age of 68 years (range 16-89 years) and a median follow-up of 32.5 months (range 5-123 months). Thirty-three of 51 (65%) patients had diffuse large B-cell lymphoma (DLBCL), and 15/51 (29%) had marginal zone lymphoma (MZL). The remaining 3 patients had follicular lymphoma, CLL, and post-transplant lymphoproliferative disease. Of the 33 patients with DLBCL, follow-up was available in 29; 24/29 (83%) had primary cutaneous disease, which was unifocal in 17/24 (71%). Following treatment, 8/24 (33%) of the primary cases relapsed. Of the 8 who relapsed, 7 had received local forms of treatment only. Follow-up data was available in 14/15 patients with MZL. 11/14 (79%) had primary cutaneous disease, which was unifocal in 8 (73%). Following treatment, 4 of these cases relapsed (36%); all had received local therapy only. It is evident from this study that a significant proportion ( reverse similar 20%) of patients who present with cutaneous infiltration by B-cell lymphoma have systemic disease. Staging is therefore mandatory in these patients. Approximately 1/3 patients with primary cutaneous DLBCL or MZL ultimately relapse, and relapse rates appear higher in those patients receiving local therapy only. Systemic or combined modality therapy may therefore be the most appropriate treatment at presentation. This should be assessed prospectively in randomized trials.

Waldenström macroglobulinemia. Development of diagnostic criteria and identification of prognostic factors.

To establish whether a combination of morphologic and immunophenotypic criteria could be developed to more precisely define Waldenström macroglobulinemia (WM) and prognostic factors, we retrospectively assessed the clinical and laboratory features of 111 cases of WM. Bone marrow infiltration by small lymphocytes was documented in each case; and diffuse, interstitial, nodular, and paratrabecular patterns of infiltration were documented in 58%, 32%, 6%, and 4% of cases, respectively. Ninety percent were characterized by a surface immunoglobulin-positive, CD19+CD20+CD5-CD10-CD23- immunophenotype. The median overall survival from diagnosis was 60 months; univariate analysis revealed the following adverse prognostic factors: older than 60 years, performance status more than 1, platelet count less than 100 x 10(3)/microL (< 100 x 10(9)/L), pancytopenia, and diffuse bone marrow infiltration. Associated median survival was 40, 38, 46, 28, and 59 months, respectively. Multivariate analysis revealed age, performance status, and platelet count as prognostically significant, but stratification of patients according to the International Prognostic Index had limited value. We suggest defining WM by the following criteria: IgM monoclonal gammopathy; bone marrow infiltration by small lymphocytes, plasmacytoid cells, and plasma cells in a diffuse, interstitial, or nodular pattern; and a surface immunoglobulin-positive, CD19+CD20+CD5-CD10-CD23- immunophenotype.

In multiple myeloma, only a single stage of neoplastic plasma cell differentiation can be identified by VLA-5 and CD45 expression.

The nature of the proliferating fraction in myeloma is still not known and understanding the characteristics of this fraction is central to the development of effective novel therapies. However, myeloma plasma cells typically show a very low rate of proliferation and this complicates accurate analysis. Although the level of CD45 and/or VLA-5 has been reported to identify proliferating 'precursor' plasma cells, there are discrepancies between these studies. We have therefore used a rigorous sequential gating strategy to simultaneously analyse cycle status and immunophenotype with respect to CD45, VLA-5 and a range of other integrin molecules. In 11 presentation myeloma patients, the proliferative fraction was distributed evenly between CD45+ and CD45- cells, however, cycling plasma cells were consistently VLA-5-. There was close correlation between the expression of VLA-5 and a range of other integrin molecules (CD11a, CD11c, CD103), as well as the immunoglobulin-associated molecules CD79a/b (Spearman, n = 10, P < 0.0001). In short-term culture, cells that were initially VLA-5-showed increasing VLA-5 expression with time. However, simultaneous analysis of the DNA-binding dye 7-amino-actinomycin D demonstrated that this was not as a result of differentiation, as VLA-5+ plasma cells were all non-viable. This was confirmed in freshly explanted plasma cells from nine patients. Discrete stages of plasma cell differentiation could not be distinguished by the level of CD45 or VLA-5 expression. The results indicate that there is a single stage of plasma cell differentiation, with the phenotype CD38+CD138+VLA-5-. These findings support the hypothesis that neoplastic bone marrow plasma cells represent an independent, self-replenishing population.

Distribution of myeloma plasma cells in peripheral blood and bone marrow correlates with CD56 expression.

There is a wide variation in the degree of marrow and blood involvement between patients with multiple myeloma. Both of these parameters are known to be highly significant prognostic factors, and the differences between patients may be due to variable expression of adhesion molecules. To test this we used three-colour flow cytometry to study three adhesion molecules associated with myeloma, namely CD38, CD56 and CD138. The level of expression of these molecules was compared with the distribution of myeloma plasma cells in bone marrow (n=59) and peripheral blood (n=26) in patients at presentation or relapse. The extent of marrow infiltration on the trephine biopsy correlated inversely with CD56 expression (Mann-Whitney U Test, P=0.022); there was no difference in CD38 or in CD138 expression. CD56 expression also correlated inversely with the number of circulating plasma cells (linear regression, R2=0.4268, slope=-0.58, P=0.0003). Peripheral blood plasma cell numbers correlated weakly with bone marrow plasmacytosis, and inversely with CD38 expression. The level of CD56 expression by neoplastic plasma cells was assessed in 37 patients over a median of 11 months (range 6-25). There was no significant change in expression (Wilcoxon Signed Rank, P=0.6271). We conclude that plasma cell CD56 expression is constant over the course of the disease; unlike CD138 expression, it is significantly linked to the degree of both bone marrow and peripheral blood involvement.

Use of methyl methacrylate resin for embedding bone marrow trephine biopsy specimens.

AIMS: To evaluate the use of methyl methacrylate resin as an embedding medium for undecalcified bone marrow trephine biopsy specimens. METHODS: About 2500 undecalcified bone marrow trephine biopsy specimens were processed, and embedded in methyl methacrylate resin. Semithin sections (2-3 microns) were stained by routine tinctorial and immunocytochemical staining methods with a wide range of antibodies using a standard streptavidin biotin horseradish peroxidase technique. Different antigen retrieval pretreatments were evaluated. RESULTS: Bone marrow trephine biopsy specimens are embedded routinely in methyl methacrylate at the Haematological Malignancy Diagnostic Service at The Leeds General Infirmary. Over 50 different primary antibodies are in current use; for the majority of these, microwave antigen retrieval or trypsin digestion, or both, is either essential or greatly enhances the results. CONCLUSIONS: Embedding bone marrow trephine biopsy specimens in methyl methacrylate resin retains morphology and permits reliable, high quality immunocytochemistry. This is particularly desirable for the demonstration of neoplastic cells in regenerative marrow after chemotherapy, and in the detection of residual disease after treatment. The use of methyl methacrylate for routine use on bone marrow trephine biopsy specimens is advocated.

Fluorescent polymerase chain reaction of a panel of CA repeats on chromosome 6 in the indolent phase of follicular centre cell lymphoma.

Twenty-four cases of histologically defined follicle centre cell (FCC) lymphoma have been examined for allele imbalance at 19 microsatellite loci spanning the length of chromosome 6, including six markers within the major histocompatibility complex (MHC), using fluorescent polymerase chain reaction (PCR) to amplify microsatellites. Nineteen cases were observed in which imbalance of one or more markers on chromosome 6 had occurred (79%). The frequency of allele imbalance was significantly higher on 6p than 6q, and two regions of deletions, 6p24-25 and 6p21.3-23, were identified in which the loci showed a significantly high allele imbalance frequency.

Allele imbalance at tumour suppressor loci during the indolent phase of follicle centre cell lymphoma.

We have examined 41 cases of follicle centre cell lymphoma with fluorescent PCR of microsatellite repeats closely linked to or within six tumour suppressor gene loci (APC, DCC, P53, RB1, WT1 and NM23). These probes are highly informative with heterozygousity rates in the range of 57%-90%. In addition we have used four loci from chromosome 6 (D6S260, TNFa, D6S281 and D6S262) as control loci which are unlikely to be involved in the pathogenesis of lymphoma. Of 369 informative PCR reactions allele imbalance was identified in 38 (10%) and this was seen in 23 of the 41 cases. Looking at individual loci allele imbalance was seen in APC(1) 11%, APC(2) 12%, P53(1) 5%, P53 (2) 7%, WT1 5%, RB1 13%, DCC 18% and NM23 0%. This frequency of change was no different from that seen at the control loci D6S260 16%, TNFa 20%, D6S281 4% and D6S262 9%. In the indolent phase of germinal centre cell lymphoma there is therefore quite a high rate of allele imbalance at all loci but this is no higher in those loci linked to tumour suppressor genes.

Microsatellite instability in follicle centre cell lymphoma.

Fluorescent polymerase chain reaction (PCR) was used to assay 12 microsatellite markers (APC x 2, DCC, P53 x 2, RB1, NM23, WT1, D6S260, D6S262, D6S281 and TNFa) to look for evidence of microsatellite instability in 40 cases of follicle centre cell lymphoma (FCC). Evidence of novel alleles seen in the tumour tissue but not the normal uninvolved tissue was seen in seven cases (17%). In only two of these cases (5%) was more than one locus involved but in these cases multiple affected loci were seen (4/12 and 7/12 respectively). The detection of microsatellite instability indicates a DNA repair defect such as that which would be predicted to occur in cells with mutated mismatch repair genes, a novel finding in FCC lymphoma.

Heterogeneity in cell proliferation and expression of p53 and bcl-2 during the indolent phase of germinal centre cell lymphoma: an explanation for clinical variability.

Germinal centre cell lymphomas (GCCL) show a wide range of clinical outcomes from persistent indolent disease to large cell transformation. To investigate possible mechanisms of this heterogeneity, a combined morphometric and immunohistological study of p53, bcl-2 and cell proliferation was carried out. There was wide variation in p53 expression between biopsies and between individual follicles in the same tumour. A similar pattern of variation was seen using the cell-cycle marker MIB1, but this did not correlate with p53 expression. Even in cases in which a t(14;18) was demonstrated by PCR, variation occurred in the number of cells expressing bcl-2. On the basis of these results, we suggest that the probability of the clonal expansion of GCCL tumour cells carrying additional genetic abnormalities depends on a complex interaction of cell proliferation with p53 and bcl-2 expression, and that this may account for variation seen in the clinical behaviour seen in this group of tumours.