Pitfalls in TRAP assay in routine detection of malignancy in effusions.

Telomerase has been found to be reactivated in a majority of cancers but is inactive in most somatic cells. Our principal goal was to determine the potential use of the telomeric repeat amplification protocol (TRAP) assay as marker for malignancy in cytological effusions. The simple selection criterion was the cytological diagnosis, and routine samples were classified into malignant (58 samples) and nonmalignant (233 samples). Of the malignant samples, 44/58 (76%) were positive by TRAP assay. Of the 14 telomerase-negative cytology-positive samples, RNA integrity was poor in 9, indicating suboptimal sample conservation for molecular analysis. In 3 of the remaining 5 samples with a negative TRAP assay, a high number of malignant cells was observed, and these cells might have been telomerase-negative. Thus, the sensitivity of TRAP assay for the presence of malignant cells was about 76%. In the cytologically nonmalignant effusions, the presence of telomerase activity was observed in 24% (55/233). Of these, 6% were highly suspicious for malignancy, 9% were doubtful, and 9% were cytologically nonmalignant effusions confirmed by a follow-up of 12 mo or more. According to these data, the specificity of the TRAP assay to detect tumor cells in effusions ranged only between 82-91%. Our results indicate that, although the TRAP assay is positive in 6-15% of putative malignant effusions, the relatively high number of TRAP false-negative and false-positive cases renders this test unsuitable for routine diagnostic purposes.

Detection of malignant effusions: comparison of a telomerase assay and cytologic examination.

Telomerase is inactive in most somatic cells, but has been found to be reactivated in a majority of cancers. Our principal goal was to test whether the presence of telomerase activity concurred with positive cytology, and was thus of potential use in detecting cancer cells in effusions. The telomeric repeat amplification protocol (TRAP) assay and cytological examination were performed in a blinded fashion on 91 unselected effusions, for which laboratory processing was done according to standard procedures. In our series, 30% (27/91) of samples were found to be malignant by cytology. Of these, 19 (70%) were also positive in the TRAP assay. Of the 8 telomerase-negative cytology-positive samples, RNA integrity was generally poor, indicating suboptimal sample conservation for molecular analysis. Negative cytology in the presence of telomerase activity was observed in 17 effusions. Of these, 11 were from patients with advanced cancer, and thus a diagnosis of malignant effusion should be suspected. The TRAP assay for telomerase activity holds promise in the analysis of effusions, but its routine use as an adjunct to cytology awaits further confirmation of its positive predictive value.

Polymerase chain reaction in the diagnosis of T-cell lymphoma in paraffin-embedded bone marrow biopsies: a comparative study.

AIMS: In routine histological analysis of bone marrow biopsies, the distinction between reactive T-cell infiltrates and T-cell lymphoma can be difficult, even with the use of extensive immunohistochemistry. The aim of this study was to evaluate the diagnostic contribution of TCR-gamma gene rearrangement analysed by PCR. METHODS AND RESULTS: The samples studied consisted of 46 paraffin-embedded bone marrow biopsies (diagnosis, staging and follow-up) from 26 patients with T-cell lymphoma. The bone marrow biopsies were categorized into three groups according to the morphological and immunohistochemical results. Group 1, positive for T-cell lymphoma (24 bone marrow biopsies), group 2, suspicion of T-cell lymphoma (15 bone marrow biopsies) and group 3, negative for T-cell lymphoma (seven bone marrow biopsies). DNA could be amplified in 45/46 bone marrow biopsies (98%). Clonal rearrangement was detected in 30/45 bone marrow biopsies tested (67%) including 15/24 bone marrow biopsies (62.5%) of group 1, 11/14 (78.5%) of group 2 and 4/7 (57%) of group 3. In total, PCR analysis supported a diagnosis of T-cell lymphoma in 15/45 bone marrow biopsies (33%), in which histological and/or immunohistochemical examination provided inconclusive evidence of malignancy. CONCLUSIONS: TCR-gamma PCR is a complementary tool for the assessment of T-cell lymphoma in bone marrow biopsies. Optimal evaluation of bone marrow biopsies requires an integrative approach of all available results from morphology, immunohistochemistry, molecular biology and clinical data.

[Primary bilateral adrenal lymphoma]. / Lymphome surrénalien primaire bilatéral.

When abdominal imaging reveals the existence of unsuspected adrenal masses, a diagnostic strategy is necessary. We report the case of a woman presenting with pulmonary embolism, in whom abdominal ultrasound revealed voluminous masses in both adrenals without clinical or biological signs of hormone hypersecretion, but with mild primary adrenal failure. From a CT scan-directed needle biopsy of the right adrenal mass and subsequent staging we were able to diagnose a primary bilateral adrenal lymphoma, diffuse large B-cell type (REAL/WHO). On CHOP chemotherapy both adrenal masses decreased dramatically and the patient is in remission 18 months later. Primary adrenal lymphoma is a rare condition, since 65 cases have been reported to date. Histological diagnosis is nevertheless important, in view of the excellent response to specific therapy observed in some cases.

CD34/QBEND10 immunostaining in bone marrow biopsies: an additional parameter for the diagnosis and classification of myelodysplastic syndromes.

CD34/QBEND10 immunostaining has been assessed in 150 bone marrow biopsies (BMB) including 91 myelodysplastic syndromes (MDS), 16 MDS-related AML, 25 reactive BMB, and 18 cases where RA could neither be established nor ruled out. All cases were reviewed and classified according to the clinical and morphological FAB criteria. The percentage of CD34-positive (CD34 +) hematopoietic cells and the number of clusters of CD34+ cells in 10 HPF were determined. In most cases the CD34+ cell count was similar to the blast percentage determined morphologically. In RA, however, not only typical blasts but also less immature hemopoietic cells lying morphologically between blasts and promyelocytes were stained with CD34. The CD34+ cell count and cluster values were significantly higher in RA than in BMB with reactive changes (p<0.0001 for both), in RAEB than in RA (p=0.0006 and p=0.0189, respectively), in RAEBt than in RAEB (p=0.0001 and p=0.0038), and in MDS-AML than in RAEBt (p<0.0001 and p=0.0007). Presence of CD34+ cell clusters in RA correlated with increased risk of progression of the disease. We conclude that CD34 immunostaining in BMB is a useful tool for distinguishing RA from other anemias, assessing blast percentage in MDS cases, classifying them according to FAB, and following their evolution.

Frequent methylation silencing of p15(INK4b) (MTS2) and p16(INK4a) (MTS1) in B-cell and T-cell lymphomas.

The methylation status of p15(INK4b) (MTS2), p16(INK4a) (MTS1) and p14(ARF) (p16beta) was analyzed in 56 lymphomas by restriction-enzyme related polymerase chain reaction (PCR) (REP), methylation-specific PCR (MSP), and bisulfite genomic sequencing (BGS). Methylation of the p15 and p16 genes was detected, respectively, in 64% and 32% of the B-cell lymphomas, in 44% and 22% of the T-cell lymphomas, and in none of the 5 reactive lymph nodes analyzed. Both p15 and p16 genes were methylated more often in the high-grade (78% and 50%, respectively) than in the low-grade B-cell lymphomas (55% and 21%, respectively). For 5 cases, mapping of the methylated CpGs of the p16 promoter region confirmed the results of REP and MSP. In addition, a large variation in the methylation patterns of p16 exon 1 was observed, not only from one lymphoma to another, but also within a given tumor. Methylation of p15 and p16 was associated with an absence of gene expression, as assessed by reverse transcription-PCR. The p14 gene was unmethylated and normally expressed in all 56 tumors. We found no mutations of p15, p16, or p14 in any of the 56 lymphomas. Our results suggest a role for p15 and p16 gene methylation during lymphomagenesis and a possible association between p15 and p16 inactivation and aggressive transformation in B-cell and T-cell lymphomas.

The role of Helicobacter pylori in primary gastric MALT lymphoma.

AIMS: Helicobacter pylori has been claimed to be an important aetiological factor which raises the risk of mucosa-associated tissue lymphoid (MALT) lymphoma. However, some studies on gastric MALT lymphoma revealed a low rate of H. pylori infection suggesting that not all gastric lymphomas are related to H. pylori infection. The aim of this study was to verify the H. pylori infection frequency in a series of patients with primary gastric MALT lymphomas and to examine the relationship between H. pylori and the pathological features of those lymphomas. METHODS AND RESULTS: Thirty-one cases of resected gastric lymphoma were analysed: 10 cases (32%) were low-grade MALT lymphomas and 21 cases (68%) were high-grade MALT lymphomas. Helicobacter pylori was found in only 18 of 31 (58%) cases. Helicobacter pylori infection was significantly correlated with the grade and depth of invasion of MALT lymphoma since 63% of superficial low-grade MALT lymphomas were positive for H. pylori compared with 38% of advanced high-grade MALT lymphomas (P = 0.02). CONCLUSION: We confirmed the relationship between H. pylori infection and a subset of gastric MALT lymphoma. Our results also showed that not all low- and high-grade gastric MALT lymphomas are H. pylori-dependent. This suggests that H. pylori infection may play a promoter role in the development of MALT lymphoma, but its presence is not mandatory for the progression of the lymphoma in view of its low frequency in advanced high-grade MALT lymphoma.

Prognostic value of follicular dendritic cells in nodular sclerosing Hodgkin's disease.

AIMS: In nodular sclerosing Hodgkin's disease (NSHD), the prognostic relevance of the histopathological grading in two subtypes NSI (low-grade) and NSII (high-grade) remains controversial. Analysis of follicular dendritic cells (FDC) may provide new prognostic parameters. METHODS AND RESULTS: Tumours from 59 patients with NSHD were studied. Mean follow-up time was 8 years. Forty-one cases were classified as NSI and 18 as NSII. FDC were immunostained with the paraffin-resistant monoclonal antibodies CD21 and CNA.42. We distinguished three patterns in the neoplastic tissue: FDC1, the presence of well-defined follicle-like structures (n = 20); FDC2, the presence of largely destroyed FDC networks (n = 25); and FDC3, no or a few isolated FDC (n = 14). The three groups differed clearly regarding the frequency of relapse and the survival. The longest survival was seen in the FDC1 group, the shortest in the FDC3 group, the FDC2 group being intermediate (P = 0.0025). FDC status was a discriminating prognostic factor for all patients, and within various age and stage categories. Combining the FDC status and the NSI-NSII grading defined the best survival group as FDC1-NSI. CONCLUSIONS: Assessment of FDC pattern, associated with histological subtyping, brings valuable data for predicting survival and outcome in NSHD.

Systemic mastocytosis following a malignant ovarian germ cell tumour.

Cases of mediastinal germ cell tumours associated with haematological disorders (two cases of systemic mastocytosis included) have been reported previously. This combination is more frequent than would be expected by chance alone. We report the case of a 30-year-old woman, who presented with a systemic mastocytosis following a malignant ovarian germ cell tumour which was treated by chemo- and radiotherapy. The patient predominantly complained of skeletal pains, which led to an erroneous radiological diagnosis of fibrous dysplasia for years. An aggressive variant of systemic mastocytosis was diagnosed on bone marrow examination. Systemic mastocytosis was confirmed by splenectomy, liver biopsy and finally autopsy. The present case is unique because of the ovarian location of the germ cell tumour. We suggest our observation could be related to the broad group of haematological malignancies associated with germ cell tumours.

Follicular dendritic cells in bone marrow lymphoproliferative diseases: an immunohistochemical study including a new paraffin-resistant monoclonal antibody, DR53.

Two-hundred and twenty-one bone marrow biopsies with lymphoid infiltrates were studied histologically and immunohistochemically, to assess the incidence and the pattern of follicular dendritic cells. Three monoclonal antibodies selective for follicular dendritic cells were used: CD21, CD35 and DR53, all reactive on paraffin-embedded material. Follicular dendritic cells were present in two of 38 benign lymphoid aggregates, 92 of 134 low grade B-cell lymphomas (45 of 62 lymphocytic, 16 of 27 lymphoplasmacytoid, 0 of six hairy cell leukaemias, five of six centrocytic, 19 of 21 centroblastic-centrocytic, seven of 12 low grade NOS), one of 23 high grade B-cell lymphomas, 0 of 10 T-cell lymphomas, 0 of three Hodgkin's disease and four of 13 suspicious infiltrates. Follicular dendritic cells were found in lymphomatous involvement with nodular, patchy and massive growth pattern, but not in interstitial ones. They formed follicle-like networks, whose number and size were directly correlated to the tumour mass. The origin and frequency distribution of follicular dendritic cells in bone marrow biopsy lymphomas is discussed and the diagnostic relevance of follicular dendritic cell immunostaining in routine bone marrow biopsy lymphoid infiltrates is assessed.

[Polyarthralgia, fever and purpura in a patient known to have splenomegaly]. / Polyarthralgies, fièvre et purpura chez un patient connu pour une splénomégalie.

A 43-year-old patient was admitted for swollen and painful ankles, knees and elbows, palpable purpura of the lower extremities and fever. Splenomegaly had been diagnosed 5 years previously. At clinical workup, spleen and liver size were increased and purplish papular skin lesions were noted above the ankles. Erythrocyte sedimentation rate was 2 mm/h. ASAT and ALAT as well as rheumatoid factor were moderately elevated. Serology was positive for hepatitis C. Differential diagnosis of palpable purpura and complementary diagnostic procedures are discussed.

Improved polymerase chain reaction detection of clonal T-cell lymphoid neoplasms.

We have developed and tested a rapid and sensitive method of detecting expansion of T-cell clones using the polymerase chain reaction (PCR) and a single set of consensus primers for the V and J regions to amplify rearranged T-cell receptor-gamma (TCR-gamma) genes. Monoclonality was continued in all of the 18 cases of T-cell neoplasms tested, but not in reactive lymphadenopathy, non-Hodgkin's B lymphomas, and Hodgkin's disease. PCR analysis, using the primer sequence outlined in this study, had an overall specificity of 100% when compared with Southern blot analysis. No false-negative results were observed, certainly owing to the choice of consensus primers and to the control of PCR reactions on agarose gels before testing for clonality by separation of PCR products on polyacrylamide gels. This method for the detection of T-cell monoclonality can be especially useful in cases that are diagnostically problematic with standard histological and immunological analysis and in cases where the material available is limited.

[Immunophenotyping of B lymphoma in bone marrow biopsies. Contribution of 4 monoclonal antibodies used on paraffin-embedded tissues:DDB.42,DNA.7,DNA.44 and DND.53]. / Immunophénotype des lymphomes B dans les biopsies médullaires. Apport de 4 anticorps monoclonaux utilisables en paraffine: DBB.42, DNA.7, DBA.44 et DND.53.

An immunohistochemical study was performed on 132 routinely processed trephine biopsies of the bone marrow (40 reactive lymphocytosis, 80 B-cell lymphomas, 7 T-cell lymphomas, 5 Hodgkin's diseases), using 4 monoclonal antibodies, DBB.42, DNA.7, DBA.44, DND.53, which are directed against B-lymphocyte associated antigens, not destroyed by fixation. DBB.42, DNA.7 and DBA.44 are formalin and Bouin resistant. They do not stain myeloid, erythroblastic, histiomonocytic and endothelial cells, and are particularly suitable for bone marrow biopsies. DBB.42 and DNA.7 in association, identify all B-cell lymphomas and no T-cell lymphoma. DBB.42 recognizes respectively 65/65 and 13/15 low and high grade malignancy B-cell lymphomas, and DNA.7, 54/65 and 15/15. In our hands, these 2 antibodies are more reliable than L26 and MB2 on bone marrow biopsies. DBA.44 identifies 15/15 hairy-cell leukemias, and interestingly stains only 10% of other B-cell lymphomas, and no T-cell lymphoma. DND.53 is less specific, but reveals Reed-Sternberg cells. Combined immunostaining with anti T-lymphocyte antibody CD3, let us appreciate the contribution and the limits of immunohistochemistry in the diagnosis of bone marrow lymphoproliferative diseases, especially for interstitial and nodular lymphoid infiltrates of undetermined significance. These results point out the value of these 4 monoclonal antibodies in routinely processed bone marrow biopsies, particularly of DBB.42 and DBA.44.

Distribution and localization of Epstein-Barr virus subtypes A and B in AIDS-related lymphomas and lymphatic tissue of HIV-positive patients.

Tissue samples of 21 HIV-positive patients have been studied for the presence and distribution of Epstein-Barr virus (EBV) subtypes A and B. This was done by PCR, EBER in situ hybridization, and immunohistochemical detection of EBV latent membrane protein (LMP) in AIDS-associated malignant lymphomas (16 cases) and lymphatic organs of patients without lymphoma (5 cases). Eleven cases were considered to be EBV-positive, with type A in four, and type B virus in four other cases. In patients with malignant lymphoma, EBV was localized in tumour tissue exclusively. One patient without lymphoma presented with multiple EBV genome type B-positive cells in all the lymphoid tissue samples examined. In HIV-positive patients, both subtypes of EBV, A and B, may play a role in the pathogenesis of lymphoproliferative lesions.

Current concepts in pediatric burn care: management of burn wounds with cultured epidermal autografts.

Our experience with CEA is based on 21 patients operated on from 1986 to 1991. The areas covered with CEA measured 500 cm2 to 3160 cm2. At one setting no more than 40 sheets of 40 cm2 CEA have been transplanted. The take of CEA is over 75% when applied to dermis. The same holds true when covering "deepithelialised" skin homografts on immunosuppressed patients. Scar formation has not been a problem, and the overall results have been good.

Genotypic and phenotypic evidence of T-cell leukemia in a patient successfully treated by interferon-alpha for typical hairy cell leukemia.

A 53-year-old man was diagnosed to have typical hairy cell leukemia. Immunophenotyping of frozen splenic tissue showed clonality of hairy cells for mu lambda, confirmed by the corresponding immunoglobulin gene rearrangements. The patient was successfully treated with interferon-alpha (IF-alpha). In the fifth year of treatment with IF-alpha the morphology of peripheral blood mononuclear cells (PBMC) and of bone marrow infiltration changed with the appearance of numerous small to intermediate shaped lymphocytes of a T-helper phenotype. Frank leukemia, resistant to IF-alpha treatment and ultimately aggressive chemotherapy, developed. Emergence of this second clonal disease was confirmed by rearrangement studies performed on PBMC; rearrangements of both alleles of the TCR beta were identified, whereas the JH and lambda IVS genes were in germline configuration. The outgrowth of a second, malignant T-cell clone paralleled by the disappearance (down-regulation?) of the initial B-cell clone while under cytokine treatment is consistent with the possibility that IF-alpha favoured the emergence of this second clone.

Dysmegakaryopoiesis predicting response to therapy in acute myeloid leukaemia. A histologic and clinical study.

With standard induction therapy between 50 to 85% of patients with Acute Myeloid Leukaemia (AML) achieve Complete Remission (CR). We investigated whether any morphological feature of bone marrow (BM) plastic embedded biopsies could predict failure of therapy. We reviewed BM plastic embedded biopsies from 54 adult patients presenting with untreated AML. The main histologic parameters analysed were cellularity, dysmegakaryopoiesis (DysM), percentage of marrow blasts and fibrosis. CR was obtained in 34 of 49 treated patients (69%). The rate of CR was significantly lower in the group of patients presenting with DysM: CR was achieved in 54% of the 28 treated patients with DysM and in 90% of the 21 treated patients without DysM (p less than 0.02). Patients with DysM had a significantly lower blood count and bone marrow blasts at presentation. Median age was not significantly different in the 2 groups. Cellularity and fibrosis were not predictive. DysM may be the hallmark of an AML subgroup with distinct clinical behaviour and lower rate of CR with conventional therapy. DysM should be carefully looked for on BM marrow biopsies and aspirate from AML patients at diagnosis.