Elena Sabattini

The EnVision++ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques.

AIM: To assess a newly developed immunohistochemical detection system, the EnVision++. METHODS: A large series of differently processed normal and pathological samples and 53 relevant monoclonal antibodies were chosen. A chessboard titration assay was used to compare the results provided by the EnVision++ system with those of the APAAP, CSA, LSAB, SABC, and ChemMate methods, when applied either manually or in a TechMate 500 immunostainer. RESULTS: With the vast majority of the antibodies, EnVision++ allowed two- to fivefold higher dilutions than the APAAP, LSAB, SABC, and ChemMate techniques, the staining intensity and percentage of expected positive cells being the same. With some critical antibodies (such as the anti-CD5), it turned out to be superior in that it achieved consistently reproducible results with differently fixed or overfixed samples. Only the CSA method, which includes tyramide based enhancement, allowed the same dilutions as the EnVision++ system, and in one instance (with the anti-cyclin D1 antibody) represented the gold standard. CONCLUSIONS: The EnVision++ is an easy to use system, which avoids the possibility of disturbing endogenous biotin and lowers the cost per test by increasing the dilutions of the primary antibodies. Being a two step procedure, it reduces both the assay time and the workload.

Follicular Lymphoma International Prognostic Index 2: A New Prognostic Index for Follicular Lymphoma Developed by the International Follicular Lymphoma Prognostic Factor Project

PURPOSE The aim of the F2 study was to verify whether a prospective collection of data would enable the development of a more accurate prognostic index for follicular lymphoma (FL) by using parameters which could not be retrospectively studied before, and by choosing progression-free survival (PFS) as principal end point. PATIENTS AND METHODS Between January 2003 and May 2005, 1,093 patients with a newly diagnosed FL were registered and 942 individuals receiving antilymphoma therapy were selected as the study population. The variables we used for score definition were selected by means of bootstrap resampling procedures on 832 patients with complete data. Procedures to select the model that would minimize errors were also performed. RESULTS After a median follow-up of 38 months, 261 events for PFS evaluation were recorded. beta2-microglobulin higher than the upper limit of normal, longest diameter of the largest involved node longer than 6 cm, bone marrow involvement, hemoglobin level lower than 12 g/dL, and age older than 60 years were factors independently predictive for PFS. Using these variables, a prognostic model was devised to identify three groups at different levels of risk. The 3-year PFS rate was 91%, 69%, and 51% for patients at low, intermediate, and high risk, respectively (log-rank = 64.6; P < .00001). The 3-year survival rate was 99%, 96%, and 84% for patients at low, intermediate, and high risk, respectively (P < .0001). CONCLUSION Follicular Lymphoma International Prognostic Index 2 is a simple prognostic index based on easily available clinical data and may represent a promising new tool for the identification of patients with FL at different risk in the era of immunochemotherapy.

Antigen retrieval techniques in immunohistochemistry: comparison of different methods.

Routine sections of normal and pathological samples fixed in 10 per cent buffered formalin or B5, including EDTA‐decalcified bone‐marrow biopsies, were tested with 61 antibodies following heating in three different fluids: 0·01 m citrate buffer (pH 6·0), 0·1 m Tris–HCl (pH 8·0), and 1 mm EDTA–NaOH solution (pH 8·0). The sections underwent either three cycles of microwave treatment (5 min each) or pressure cooking for 1–2 min. The alkaline phosphatase/anti‐alkaline phosphatase (APAAP) technique was used as the standard detection method; with 16 antibodies a slightly modified streptavidin–biotin complex (SABC)‐immunoperoxidase technique was applied in parallel. The results obtained were compared with those observed without any antigen retrieval (AR), or following section digestion with 0·05 per cent protease XIV at 37°C for 5 min. Chess‐board titration tests showed that all antibodies but one profited by AR. Protease XIV digestion represented the gold standard for five antibodies, while 55 produced optimal results following the application of heat‐based AR. By comparison with the other fluids, EDTA appeared to be superior in terms of both staining intensity and the number of marked cells. These results were independent of tissue processing, immunohistochemical approach, and heating device. Pressure cooking was found to be more convenient on practical grounds, as it allowed the simultaneous handling of a large number of slides and a time saving of 1 min 30 s, representing the proper time for the treatment.

Marker Expression in Peripheral T-Cell Lymphoma: A Proposed Clinical-Pathologic Prognostic Score

PURPOSE Although peripheral T-cell lymphoma, unspecified (PTCL/U), is the most common T-cell tumor in Western countries, no study to date has been based on the application of a wide panel of markers to a large series of patients and assessed the impact of phenotype on survival. We evaluated the expression of 19 markers in 148 PTCLs/U and 45 PTCLs of the angioimmunoblastic type (AILD). PATIENTS AND METHODS The analysis was performed on tissue microarrays by immunohistochemistry and in situ hybridization. Clinical data were available in 93 PTCL/U patients, most of whom had been included in a previous study proposing a prognostic index (PIT). RESULTS An aberrant phenotype with frequent loss of CD5 and/or CD7 was typical for PTCLs, irrespective of whether they were U or AILD. Aberrantly expressed proteins rarely included CD20, CD15, and CD30. Positivity for Epstein-Barr virus-associated small RNAs and CD15 expression emerged as adverse prognostic factors. Among PTCLs/U, the proliferation-associated protein Ki-67 turned out to be prognostically relevant and was integrated in a new predictive score, incorporating age (> 60 years), high lactate dehydrogenase, poor performance status, and Ki-67 > or = 80%. This score was associated with the patient outcome (P < .0001) and was found to be more robust than PIT (P = .0043) in the present series. CONCLUSION Our retrospective analysis shows a wide range of protein expression in PTCLs and proposes a new prognostic index. The latter represents one of the first examples of mixed score (including patient- and tumor-specific factors) applied to malignant lymphomas and may be the basis for future prospective therapeutic trials.

Response of refractory Hodgkin's disease to monoclonal anti-CD30 immunotoxin

In Hodgkins disease, Hodgkin and Reed-Sternberg cells consistently express the antigen CD30. We investigated the possible therapeutic role of an immunotoxin prepared by covalent linking of an anti-CD30 monoclonal antibody (Ber-H2) to saporin (SO6), a type-1 ribosome-inactivating protein. The immunotoxin (0.8 mg/kg in one or two doses) was given to four patients with advanced refractory Hodgkins disease. In three, there was rapid and substantial reduction in tumour mass (50% to greater than 75%). Clinical responses were transient (6-10 weeks). In-vivo binding of the immunotoxin to tumour cells was shown by immunohistology in two patients. Antibodies to both parts of the immunotoxin developed in all patients.

Phase II Trial of Proteasome Inhibitor Bortezomib in Patients With Relapsed or Refractory Cutaneous T-Cell Lymphoma

PURPOSE To determine the antitumor activity of the proteasome inhibitor bortezomib in patients with cutaneous T-cell lymphoma (CTCL) or peripheral T-cell lymphoma unspecified (PTCLU) with isolated skin involvement. PATIENTS AND METHODS From May 2005 to June 2006 at our institute, we treated patients with previously pretreated CTCL or PTCLU using bortezomib as a single agent, at a dose of 1.3 mg/m2 intravenously on days 1, 4, 8, and 11, every 21 days for a total of six cycles. RESULTS Fifteen patients were registered, of whom 12 (10 CTCL, all mycosis fungoides, and two PTCLU with isolated skin involvement) were assessable. The overall response rate was 67%, with two (17%) complete remissions and six (50%) partial remissions. The remaining four patients had disease progression. Histologically, the responder patients were seven with CTCL and one with PTCLU with isolated skin involvement. All responses were durable, lasting from 7 to 14 or more months. Overall, the drug was well tolerated, with no grade 4 toxicity. The most common grade 3 toxicities were neutropenia (n = 2), thrombocytopenia (n = 2), and sensory neuropathy (n = 2). CONCLUSION This study suggests that bortezomib was well tolerated and has significant single-agent activity in patients with cutaneous T-cell lymphoma.

Simple diagnostic assay for hairy cell leukaemia by immunocytochemical detection of annexin A1 (ANXA1)

A marker capable of distinguishing with certainty between hairy cell leukaemia and other B-cell malignant disease would be of great diagnostic value. Through gene expression profiling, annexin A1 (ANXA1) has been identified as a gene that is upregulated in hairy cell leukaemia. We did immunostaining of 500 B-cell tumours with a specific anti-ANXA1 monoclonal antibody and showed that ANXA1 protein expression is specific to hairy cell leukaemia. Immunocytochemical detection of ANXA1 represents a simple, inexpensive, highly sensitive and specific (100%) assay for diagnosis of hairy cell leukaemia. This assay will be especially useful in distinguishing hairy cell leukaemia from splenic lymphoma with villous lymphocytes and variant hairy cell leukaemia, both of which usually respond poorly to treatments that are effective in hairy cell leukaemia.

Hodgkin's Disease with Monoclonal and Polyclonal Populations of Reed–Sternberg Cells

BACKGROUND There is strong evidence that Reed-Sternberg cells have a lymphoid phenotype, but clonally rearranged genes for B-cell and T-cell antigen receptors have not been demonstrable in tumor tissue from most patients with Hodgkins disease. To elucidate this issue, we assayed single Reed-Sternberg cells from 12 patients with classic Hodgkins disease of a B-cell immunophenotype to detect rearranged immunoglobulin variable-region heavy-chain (VH) genes. METHODS We isolated single Reed-Sternberg cells from frozen sections that had been immunostained for CD30. The rearranged VH genes of these cells were amplified by the polymerase chain reaction and analyzed by gel electrophoresis and nucleotide sequencing. RESULTS In all 12 patients, the Reed-Sternberg cells studied contained rearranged VH genes. Three patterns were observed: in three patients the rearrangements in each patient were identical, in six patients all the rearrangements were unrelated and unique, and in three patients both identical and unrelated rearrangements were detected. Apparently somatic mutations of VH genes were present in some Reed-Sternberg cells but absent in others. CONCLUSIONS Reed-Sternberg cells with B-cell phenotypes have rearranged VH genes; therefore, these cells arise from B cells. The pattern of VH gene mutations suggests that Reed-Sternberg cells can correspond to either immunologically naive or memory B cells. In half our patients the population of Reed-Sternberg cells was polyclonal; in the other half, monoclonal or mixed cell populations were found. Correlation with the clinical stage suggests that polyclonal Hodgkins disease can present as a widespread lymphoma.

Primary Mediastinal B-Cell Lymphoma: High Frequency of BCL-6 Mutations and Consistent Expression of the Transcription Factors OCT-2, BOB.1, and PU.1 in the Absence of Immunoglobulins

Although primary mediastinal (thymic) large B-cell lymphoma has been primarily studied, its precise phenotype, molecular characteristics, and histogenesis are still a matter of debate. The International Extranodal Lymphoma Study Group collected 137 such cases for extensive pathological review. Histologically, the lymphomatous growth was predominantly diffuse with fibrosis that induced compartmentalized cell aggregation. It consisted of large cells with varying degrees of nuclear polymorphism and clear to basophilic cytoplasm. On immunohistochemistry, the following phenotype was observed: CD45(+), CD20(+), CD79a(+), PAX5/BSAP(+), BOB.1(+), Oct-2(+), PU.1(+), Bcl-2(+), CD30(+), HLA-DR(+), MAL protein(+/-), Bcl-6(+/-), MUM1/IRF4(+/-), CD10(-/+), CD21(-), CD15(-), CD138(-), CD68(-), and CD3(-). Immunoglobulins were negative both at immunohistochemistry and in situ hybridization. Molecular analysis, performed in 45 cases, showed novel findings. More than half of the cases displayed BCL-6 gene mutations, which usually occurred along with functioning somatic IgV(H) gene mutations and Bcl-6 and/or MUM1/IRF4 expression. The present study supports the concept that a sizable fraction of cases of this lymphoma are from activated germinal center or postgerminal center cells. However, it differs from other aggressive B-cell lymphomas in that it shows defective immunoglobulin production despite the expression of OCT-2, BOB.1, and PU.1 transcription factors and the lack of IgV(H) gene crippling mutations.

Peripheral T-cell lymphomas. Clinico-pathologic study of 168 cases diagnosed according to the R.E.A.L. Classification

BACKGROUND One hundred sixty-eight peripheral T-cell lymphomas (PTCLs) were reviewed according to the Revised European-American Lymphoma (R.E.A.L.) Classification. PATIENTS AND METHODS The cases, originally diagnosed on the basis of the Updated Kiel Classification (UKC), were all provided with histological preparations, immunophenotype, clinical information, and follow-up data. The slides were reclassified by five observers, who integrated the R.E.A.L. criteria with cell size measurements. The prognostic value of clinical and pathologic findings was assessed by univariate and multivariate analysis. RESULTS The R.E.A.L. Classification was reproducibly applied by all of the observers. Clinically, anaplastic large cell lymphomas (ALCLs) differed from the remaining PTCLs by mean age (29.5 vs. 52.9 years), bulky disease (52.3% vs. 11.3%; P = 0.000), mediastinal mass (52.7% vs. 32%; P = 0.004), and disease-free survival (68.0% vs. 38.2%; P = 0.0001). Although each histological type displayed specific clinical aspects, PTCLs other than ALCL were basically characterised by a poor clinical outcome which was not influenced by the UKC malignancy grade. At multivariate analysis, the risk of a lower complete remission rate was related to bulky disease (P = 0.001), histologic group (non-ALCL) (P = 0.01), and advanced stage (III-IV) (P = 0.0002). CONCLUSIONS The present study supports the classification of T-cell lymphomas proposed by the R.E.A.L. scheme.