ICSH guidelines for the standardization of bone marrow immunohistochemistry.

Bone marrow (BM) tissue biopsy evaluation, including trephine biopsy and clot section, is an integral part of BM investigation and is often followed by ancillary studies, in particular immunohistochemistry (IHC). IHC provides in situ coupling of morphological assessment and immunophenotype. The number of different IHC tests that can be applied to BM trephine biopsies and the number of indications for IHC testing is increasing concurrently with the development of flow cytometry and molecular diagnostic methods. An international Working Party for the Standardization of Bone Marrow IHC was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines for the standardization of BM IHC based on currently available published evidence and modern understanding of quality assurance principles as applied to IHC in general. The guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus and represent further development of the previously published ICSH guidelines for the standardization of BM specimens handling and reports.

Call for a European programme in external quality assurance for bone marrow immunohistochemistry; report of a European Bone Marrow Working Group pilot study.

BACKGROUND AND AIMS: In diagnostic immunohistochemistry (IHC), daily quality control/quality assurance measures (QC/QA) and participation in external quality assurance programmes (EQA) are important in ensuring good laboratory practice and patient care. Bone marrow trephine biopsies (BMTB) have been generally excluded from EQA programmes for diagnostic IHC due to a lack of standards for tissue processing. The European Bone Marrow Working Group (EBMWG) has set up an EBMWG IHC Committee with the task of exploring the plausibility of an EQA programme for BMTB IHC in Europe. METHODS: 28 laboratories participated in a web-based anonymous survey; 19 laboratories submitted a total of 109 slides stained for CD34, CD117, CD20, CD3, Ki-67 and a megakaryocyte marker of choice. RESULTS: Eight different fixatives and nine different decalcification methods were used. While 93% of participants believed that they produced excellent results in BMTB IHC, only 4/19 (21%) laboratories did not have any poor results. CD117 and Ki-67, with 53% and 50% poor results, respectively, were the most problematic immunostains, while CD20 was the least problematic, with only 11% poor results. CONCLUSIONS: The EBMWG IHC Committee calls for a reduction in the tissue processing methods for BMTB and establishment of an EQA programme for BMTB IHC to help diagnostic IHC laboratories calibrate their tests according to expert recommendations. This is especially necessary in the light of recent introduction of predictive IHC tests in BMTB.

A marginal zone phenotype in follicular lymphoma with t(14;18) is associated with secondary cytogenetic aberrations typical of marginal zone lymphoma.

Marginal zone differentiation of follicular lymphomas (FL), sometimes referred to as monocytoid B-cell differentiation, is a relatively uncommon phenomenon. Recently, this type of differentiation was also linked to secondary cytogenetic aberrations of chromosome 3 in a small number of patients. We have analysed 131 primary nodal FL with t(14;18)(q32;q21) for secondary cytogenetic aberrations previously described as recurrent in marginal zone lymphomas (MZL) to identify their frequency and possible association with morphological evidence of marginal zone differentiation. We searched for trisomy of chromosomes 3, 12, and 18, gains of chromosome arm 3q, deletions of chromosome arm 7p, structural anomalies with break-points in 1q21 and 1p34, as well as the t(1;2)(p22;p12), t(1;14)(p22;q32), t(3;14)(q27;q32), t(6;14)(p21;q32), and t(11;18)(q21;q21) translocations. At least focal morphological evidence of marginal zone differentiation occurred in 35/131 (27%) FL with t(14;18)(q32;q21) as the primary chromosomal abnormality. None of the recurrent balanced translocations characteristic of extranodal MZL were seen secondarily in the nodal FLs with t(14;18)(q32;q21). However, 43/131 (33%) cases had at least one of the above secondary cytogenetic aberrations previously reported as recurrent aberrations in MZL and, when combined, these were significantly more frequent in FL with morphological evidence of marginal zone differentiation (p<0.0001, two-sided Fisher's exact test). Aberrations of chromosome 3 and, in particular, trisomy 3 occurred frequently in FL with marginal zone differentiation (p=0.002 and p<0.0001, respectively, two-sided Fisher's exact test), while chromosome 21, 22, and X chromosome aberrations, which have not been described previously as recurrent in MZL, were also significantly associated with marginal zone differentiation in FL (p=0.002, p=0.037, p=0.039, respectively, two-sided Fisher's exact test).

Prognostic significance of PU.1 in follicular lymphoma.

Very few prognostic factors are known in follicular lymphoma (FL), a common malignancy of germinal centre (GC) B-cells. The Follicular Lymphoma International Prognostic Index (FLIPI) thus far appears to be the most important predictor of clinical outcome. This study explores the predictive power of the degree of GC differentiation for outcome in FL. Samples from 73 patients with FL were evaluated by immunohistochemistry for expression of GC markers. Strong PU.1, CD20, and CD75 expression were significantly associated with longer progression-free survival (PFS) and overall survival (OS). Results for PFS were independent of the International Prognostic Index or the Italian Lymphoma Intergroup prognostic index for CD75 and PU.1, but only PU.1 expression was independent of FLIPI for PFS and OS. Oct-2 was weakly expressed overall, but more strongly in higher grades of FL; it had a trend for negative linear association with PU.1 and strong positive linear association with CD27, which possibly reflects its role in terminal B-cell differentiation. We show that the level of GC differentiation, as determined by the levels of PU.1, CD75, CD20, Bcl-6, and CD10 expression, has an association with outcome in patients with FL. While this is determined qualitatively in most studies of diffuse large B-cell lymphoma, in FL there is a quantitative positive association between a high level of expression of GC antigens and longer OS and PFS even when data are stratified by the FLIPI score.