Increased number of B-cells in the red pulp of the spleen in ITP.

Platelets are targeted by autoantibodies and destroyed in the reticuloendothelial system in the spleen, liver and bone marrow in patients with immune thrombocytopenia (ITP). Other mechanisms such as destruction by cytotoxic T-cells and defective production of platelets in the bone marrow also exist. Splenectomy normalizes the platelet count in 70% of ITP patients, however, precious little is known about the spleen in this disease. Our aim was therefore to investigate the splenic morphology and especially the number and localization of splenic leukocytes in patients with ITP and controls and to evaluate factors predicting outcome of splenectomy. Spleen sections from 29 ITP patients and 11 individuals splenectomized due to trauma were analyzed by immunohistochemistry. All except one of the ITP patients had a normalized platelet count 12 months after splenectomy and the platelet count was inversely correlated with age. ITP patients had an increased number of B-cells in the red pulp. The number of white pulp B-cells and number of T-cells in both compartments was unchanged. In conclusion, B-cells are increased in the red pulp of the spleen and together with cytotoxic T-cells, helper T-cells and macrophages line the sinusoids enabling the immunological attack on platelets in ITP.

The impact of peripheral blood values and bone marrow findings on prognosis for patients with essential thrombocythemia and polycythemia vera.

The Philadelphia chromosome-negative (Ph-) chronic myeloproliferative neoplasms include the three well-known clinical entities polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Over time, patients with ET and PV may develop myelofibrosis (MF), and all three entities carry a risk of transformation into acute myeloid leukemia (AML). In a population-based survey during 1983-1999, we studied a total of 358 patients who were diagnosed with ET and PV in the city of Gothenburg, Sweden. At the time of diagnosis, evaluable bone marrow biopsy material was available from 280 of these patients. The current work was aimed at investigating the impact of peripheral blood counts, spleen size, and bone marrow biopsy findings at diagnosis on long-term survival and the risk of development of AML or MF in this well-defined unselected population. The variables evaluated were venous blood hemoglobin concentration, packed cell volume, white blood cell count, platelet count, and splenic enlargement; as to bone marrow biopsies, interest was focused on reticulin content, focal or generalized collagen formation, bone marrow cellularity, and megakaryocyte profile number. Over the median observation time of 15 yr, the patients with ET did not demonstrate any significant difference as to survival compared to the normal Swedish population (hazard ratio, 1.23; 95% confidence interval, 0.97-1.51; p= 0.089). The patients with PV, on the other hand, had a significantly shorter survival compared to general population (hazard ratio, 1.66; 95% confidence interval, 1.38-1.99; p< 0.001). A lower hemoglobin concentration at diagnosis of ET predicted poorer survival (p =0.0281), whereas patients with PV with splenic enlargement at diagnosis had a shorter survival (p =0.037). In the patients with ET, the risk of transformation to either MF or AML was significantly associated with low hemoglobin concentration and high white cell count at diagnosis (p =0.0037 and 0.0306, respectively). An increased reticulin content and hypercellularity in the bone marrow at diagnosis were also independent risk factors (p =0.0359 and 0.0103, respectively). The risk of transformation in patients with PV was significantly associated with splenic enlargement and increase in bone marrow reticulin content (p =0.0028 and 0.0164, respectively).

Recruitment of T cells into bone marrow of ITP patients possibly due to elevated expression of VLA-4 and CX3CR1.

In idiopathic thrombocytopenic purpura (ITP), platelets are destroyed in the spleen, liver, and bone marrow (BM) by autoantibodies and cytotoxic T cells. In a DNA microarray screen of peripheral blood T cells, we found that VLA-4, CX3CR1, and CXCR4, involved in T-cell homing, had increased expression in ITP patients compared with controls. However, we only found increased protein expression of VLA-4 on T cells from peripheral blood by flow cytometry. To address a possible recruitment of T cells into the organs involved in platelet destruction, we analyzed T cells in BM. In BM, T-cell surface expression of VLA-4 and CX3CR1 was increased in ITP patients compared with controls. Furthermore, the number of CD3(+) T cells in BM, but not in blood, was increased in ITP patients compared with controls. This finding was confirmed by immunohistochemistry of BM biopsies. The number of regulatory T cells (CD4(+)/CD25(bright)) was decreased in the BM of ITP patients, whereas Fas expression was increased. In conclusion, ITP is associated with accumulation and activation of T cells in the BM. Recruitment of T cells into the target organ (eg, BM) is plausible and may be facilitated through increased VLA-4 and CX3CR1 expression. These molecules might serve as new treatment targets in ITP.

Expression of CD68+ tumor-associated macrophages in patients with diffuse large B-cell lymphoma and its relation to prognosis.

Tumor-associated macrophages (TAM) have been ascribed both pro- and anti-tumor properties, but the majority of clinical cancer studies have shown that the presence of a high number of TAM is related to poor prognosis, suggesting that TAM predominantly exert pro-tumoral activity. The prognostic role of TAM in patients with diffuse large B-cell lymphoma (DLBCL), however, is so far unknown. Therefore, TAM were immunohistochemically stained with a CD68 antibody in a retrospective, population-based study including 176 DLBCL patients treated with curative intent. With the exception that patients >60 years of age had a larger number of CD68+ cells (1143 vs 1018 cells/mm2; P = 0.05), no significant differences were found between the number of CD68+ cells and other clinical factors. Similarly, germinal center B-cell (GCB)/non-GCB immunophenotype or low/high Ki-67 percentage were not associated with CD68 expression. Finally, no significant correlation was found between the number of CD68+ cells and progression-free survival (P = 0.34) or overall survival (P = 0.94). These data indicate that the pro-tumor effect of TAM has limited clinical relevance in DLBCL patients, which could imply that therapeutic strategies aimed at enhancing their anti-tumor activity are of continuous clinical interest.

The impact of gender, age and patient selection on prognosis and outcome in diffuse large B-cell lymphoma - a population-based study.

Most studies concerning therapy and prognosis in diffuse large B-cell lymphoma (DLBCL) are based on highly selected patient material. To evaluate treatment, clinical prognostic factors, and outcome in a population-based cohort, we performed a retrospective study comprising 535 de novo DLBCL patients in western Sweden, diagnosed between 1995 and 2000. The median age was 73 years. Treatment with a curative intent was administered to 376 (70%) patients. The International Prognostic Index (IPI) strongly predicted overall (OS) and progression-free (PFS) survival, but high age (>68 vs < or = 68 years) had no significant influence on response rate (p = 0.86) or PFS (p = 0.14). Male sex had a negative impact on both OS (p < 0.001) and PFS (p < 0.001), independent of IPI. In conclusion, a considerable proportion of the patients did not receive curative treatment, but among those treated, the response and PFS were not influenced by age. As men had lower PFS and OS than women, it seems important that gender perspective be taken into account in future studies.

Tumour-type-specific capillary endothelial cell stainability in malignant B-cell lymphomas using antibodies against CD31, CD34 and Factor VIII.

The microvessel density (MVD) was assessed in lymph nodes infiltrated by diffuse large B-cell lymphomas, mantle cell lymphomas, chronic lymphatic leukemia and follicular lymphomas, and in lymphadenitis. Serial sections of formalin-fixed and paraffin-embedded tissue were stained with antibodies against CD31, CD34 or Factor VIII. Using light microscopy and computerised image analysis, the number and size of individual immunostained vessel profiles within a preselected area size range corresponding to capillaries, postcapillary venules, small collecting venules and small arterioles were determined. A significantly larger number of vessels were registered following staining with anti-CD34 than with anti-CD31 or anti-Factor VIII. Moreover, among the smallest capillary-sized vessel profiles in all lesion types, there was a selective relative loss of stainability of anti-CD31 and anti-Factor VIII, resulting in a substantial total loss of visualised capillary-sized vessels compared with anti-CD34. In fact, the number of non-detected capillaries following staining with anti-CD31 and anti-Factor VIII was significantly tumour type specific. These findings influence how we evaluate MVD data in B-cell lymphomas and possibly also other tumour types, as well as data relating to capillary endothelium-related functional variables of proliferation, apoptosis and maturation when different double-labelling immunohistochemical techniques are used and different tumour types are analysed.

Comparison of reverse transcription quantitative real-time PCR, flow cytometry, and immunohistochemistry for detection of monoclonality in lymphomas.

In healthy humans, 60-70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription quantitative real-time PCR analysis has complementary values. In a considerable number of cases tumor cells produced both kappa and lambda light chain transcripts, but only one type of light chain peptide was produced.

Low rather than high Ki-67 protein expression is an adverse prognostic factor in diffuse large B-cell lymphoma.

The prognostic role of overexpression of Ki-67 protein in diffuse large B-cell lymphoma (DLBCL) is still unclear. Furthermore, immunohistochemical studies have suggested a correlation between markers of proliferation, B-cell differentiation and apoptosis, but the prognostic relevance of these findings has not been clarified. To investigate the prognostic impact of Ki-67, in the context of this correlation, a retrospective immunohistochemical study was performed on 199 DLBCL patients treated with curative intent. Patients with low Ki-67 expression (<49%) had significantly worse progression-free (PFS) and overall (OS) survival, independent of clinical risk factors. In addition, low Ki-67 correlated to bcl-2 expression but not to non-germinal centre B-cell-like (non-GCB) phenotype. Each of these factors had negative impact on PFS and OS, but low Ki-67 expression also remained as an adverse prognostic factor independent of non-GCB phenotype and bcl-2 expression. Together, these results suggest that low rather than high Ki-67 protein expression is of prognostic importance in DLBCL.

The number of tumour-infiltrating TIA-1+ cytotoxic T cells but not FOXP3+ regulatory T cells predicts outcome in diffuse large B-cell lymphoma.

The prognostic significance of tumour-infiltrating lymphocytes (TILs) in patients with diffuse large B-cell lymphoma (DLBCL) remains controversial. Furthermore, the possible impact of regulatory T cells (T(regs)) on survival in DLBCL is still unknown. We performed a retrospective study on the immunohistochemical expression of cytotoxic cells and T(regs), and their correlation with survival in 195 DLBCL patients. Patients with a small number of cytotoxic T-cell intracytoplasmic antigen-1 (TIA-1)+ T cells (< or =260 cells/mm(2) tumour area; n = 52) had significantly better outcome than patients with a large number (>260 cells/mm(2); n = 143); progression-free survival (PFS) at 5 years was 67% vs. 50% (P = 0.03) and overall survival (OS) was 73% vs. 57% (P = 0.03). In multivariate analysis, the low TIA-1+ group still had a better PFS (relative risk 0.75, 95% confidence interval 0.31-0.99; P = 0.05). The number of forkhead box protein 3 (FOXP3)+ T(regs) had no influence on PFS (P = 0.89) or OS (P = 0.75). These results suggest that immunohistochemical analysis of cytotoxic T cells at time of diagnosis could provide additional prognostic information. The lack of correlation between the number of FOXP3+ cells and survival could possibly indicate that tumour-infiltrating T(regs) are of less clinical importance in DLBCL. However, these findings need to be explored in functional studies.

Testicular lymphoma--a retrospective, population-based, clinical and immunohistochemical study.

From a population-based registry, 35 patients with histologically verified testicular lymphomas were identified: diffuse large B-cell lymphomas (DLBCL) in 33 and peripheral T-cell lymphomas in two cases. Twenty-two patients had localized disease (Pe stage I and II). Twenty-eight patients received systemic chemotherapy, 17 of whom also received intrathecal prophylaxis, and 12 out of these 17 also received radiotherapy to the contralateral testis. In the Pe stage I/II group, 7 out of 21 patients in complete remission (CR) relapsed. In 5 of them the CNS was involved (isolated CNS relapse in three). Remarkably late relapses occurred (up to 127 months). Intrathecal prophylaxis seemed to reduce the frequency of relapses involving the CNS, but the relatively short follow-up (median 45 months, range 34-88, for censored patients) prevents firm conclusions regarding efficacy. The outcome for the stage IV patients was poor, with only 1 out of 11 patients in continuous CR. Immunohistochemical analysis of the DLBCL tumours revealed that 31% had the germinal centre B-cell-like phenotype. CD44 was expressed in all the tumours of stage IV patients but in less than half of the Pe stage I/II patients. A high intratumoural microvessel density was correlated with a high degree of Ki-67 positive tumour cells and an inferior overall survival.

Quantitative real-time PCR method for detection of B-lymphocyte monoclonality by comparison of kappa and lambda immunoglobulin light chain expression.

BACKGROUND: An abnormal IgLkappa:IgLlambda ratio has long been used as a clinical criterion for non-Hodgkin B-cell lymphomas. As a first step toward a quantitative real-time PCR-based multimarker diagnostic analysis of lymphomas, we have developed a method for determination of IgLkappa:IgLlambda ratio in clinical samples. METHODS: Light-up probe-based real-time PCR was used to quantify IgLkappa and IgLlambda cDNA from 32 clinical samples. The samples were also investigated by routine immunohistochemical analysis and flow cytometry. RESULTS: Of 32 suspected non-Hodgkin lymphoma samples analyzed, 28 were correctly assigned from real-time PCR measurements assuming invariant PCR efficiencies in the biological samples. Four samples were false negatives. One was a T-cell lymphoma, one was a diffuse large B-cell lymphoma, and one was reanalyzed and found lymphoma-positive by in situ calibration, which takes into account sample-specific PCR inhibition. Twelve of the samples were fine-needle aspirates, and these were all correctly assigned. CONCLUSIONS: This work is a first step toward analyzing clinical samples by quantitative light-up probe-based real-time PCR. Quantitative real-time PCR appears suitable for high-throughput testing of cancers by measuring expression of tumor markers in fine-needle aspirates.

Philadelphia (Ph) chromosome-positive thrombocythemia without features of chronic myeloid leukemia in peripheral blood: natural history and diagnostic differentiation from Ph-negative essential thrombocythemia.

We have evaluated the clinical symptoms, hematological features, and natural history of 3 cases and 20 reported cases described as Philadelphia chromosome-positive (Ph+) essential thrombocythemia (ET). The presence of increased small mononuclear megakaryocytes in bone marrow smears and biopsy material in patients with pronounced thrombocytosis and no evidence of chronic myeloid leukemia (CML) in peripheral blood appeared to be a diagnostic clue to the diagnosis of Ph+ (essential) thrombocythemia. As compared to cases of reactive thrombocytosis, the megakaryocytes in Ph+ thrombocythemia are smaller than normal ones and typically have hypolobulated round nuclei. This contrasts with the finding of clustered mature and enlarged megakaryocytes in Ph-negative true ET. Patients diagnosed as Ph+ ET may progress to CML and show a high tendency to myelofibrosis and blastic transformation. These observations indicate that both Ph+ ET and Ph+ thrombocythemia associated with CML can be regarded as early manifestations of the chronic stable phase of CML.