Aberrant immunoarchitecture distinguishes hyperplastic germinal centres in pattern 1 angioimmunoblastic T-cell lymphoma from reactive follicles.

We compare 30 biopsies each of Pattern 1 angioimmunoblastic T-cell lymphoma (AITL1) and reactive lymphoid hyperplasia (RLH) by immunohistology, in-situ hybridization for Epstein-Barr virus-encoded RNA and T-cell receptor-γ (TRG)-clonality. AITL1 cases, more often than RLH controls, were older [median ages 61 (range 23-79) vs 46 (range 11-59) years, p < 10(-4)], non-Chinese [16/30 (53%) vs 8/28 (29%), p = 0.035], presented nodally [29/30 (97%) vs 23/30 (77%), p = 0.024], showed: pan-T cell antigen attenuation [25/29 (86%) vs 5/21 (24%), p = 1.0 × 10(-5)], CD4 predominance [25/28 (89%) vs 12/23 (52%), p = 3.4 × 10(-3)], interfollicular lymphoid CD10-positivity [16/30 (53%) vs 1/29 (3%), p = 1.5 × 10(-5)], TRG clonality [16/28 (57%) vs 1/20 (5%), p = 1.4 × 10(-4)], higher maximum number of Epstein-Barr virus-encoded RNA + nuclei per 0.5-mm high-power field [median 6 (range 0-70) vs 1 (range 0-40), p = 0.012] and interfollicular Ki-67 proliferation fraction [median 40% (range 10-80%) vs 20% (range 5-40), p < 10(-4)], whereas their germinal centres (GCs) more often showed attenuation of CD10 [30/30 (100%) vs 11/29 (38%), p = 5.3 × 10(-8)] and CD57 [18/25 (72%) vs 4/22 (18%), p = 2.4 × 10(-4)] (respectively). GC-predominant PD-1 and ICOS immunoreactivity were more often seen in RLH [20/22 and 9/19 controls (91% and 47%)] than AITL1 [9/25 and 3/19 cases (36% and 16%), p = 1.0 × 10(-4) and 0.033, respectively]. Significant independent predictors against AITL1 were: solid GC CD10 immunoreactivity {p = 0.023, odds ratio (OR) for AITL1 0.01 [95% confidence interval (CI): 0.0002-0.529]}; lower interfollicular proliferation fraction [p = 0.047, OR for AITL1 1.1 (95% CI: 1.001-1.209) per % rise in Ki-67]; younger presenting age [p = 0.028, OR for AITL1 1.136 (95% CI: 1.014-1.272) per year older]. Hence, GCs and perifollicular zones in AITL1 are distinct from those in RLH.

Comparative analysis of extra-nodal NK/T-cell lymphoma and peripheral T-cell lymphoma: significant differences in clinical characteristics and prognosis.

AIM: We aimed to compare the frequencies, clinical characteristics, and prognostic factors of peripheral T-cell lymphoma (PTCL) vs. extra-nodal natural killer (NK)/T-cell lymphoma and to characterize the subtypes of extra-nodal NK/T-cell lymphoma. METHODS: We reviewed 97 consecutive patients with PTCL and extra-nodal NKT lymphoma from 2000 to 2006. During this period, a total of 780 patients with malignant lymphomas were treated in our center. The diagnostic criteria used were based on the WHO classification system of malignant lymphomas. RESULTS: Extra-nodal-NK/T-cell lymphoma and PTCL comprised 5.0% (39/780) and 7.4% (58/780) of all cases. Of the PTCL cases, histology was PTCL-NOS in 25, anaplastic large cell in 11, angioimmunoblastic T cell in 18 and other subtypes in four patients. Compared with PTCL, extra-nodal NK/T-cell lymphoma was associated with a significantly inferior rates of complete remission (33% vs. 53%, P = 0.05) and 3 yr overall survival (29.5% vs. 47.5%, P = 0.003). On multivariate analysis, extra-nodal NK/T-cell histology was independently associated with decreased survival. Further analysis into this subtype showed the nasal variant (n = 25) differed significantly from extra-nasal variant (n = 14) in terms of stage at presentation (stages III/IV, 36% vs. 79%), international prognostic index scores (high intermediate or high IPI scores, 24% vs. 64%), complete remission rates (48% vs. 7%), and median survival (10 months vs. 1 month, P < 0.0001). CONCLUSIONS: Extra-nodal NK/T-cell lymphoma was associated with a poorer prognosis compared with PTCL and is likely to comprise two distinct variants with different clinical behavior and prognosis.

Multi-lineage interrogation of the performance characteristics of a split-signal fluorescence in situ hybridization probe for anaplastic lymphoma kinase gene rearrangements: a study of 101 cases characterized by immunohistomorphology on fixed archival tissue.

BACKGROUND: Fluorescence in situ hybridization (FISH) can identify chromosomal translocations on fixed archival tissue, but studies cross-validating the utility of FISH on lesions of different cell lineages that harbor similar translocations (e.g. those involving anaplastic lymphoma kinase [ALK]) have not been published. AIM: Our objective was to define the diagnostic utility, performance characteristics, and limitations of a commercially available, split-signal, FISH probe for ALK gene rearrangements on fixed, archived tissue from lesions of diverse cell lineage. STUDY DESIGN: The sensitivity, specificity, and positive and negative predictive values of the Vysis ALK FISH probe were compared with those of the ALK-1 antibody (Dako) in a series of 101 cases, comprising 43 hematolymphoid neoplasms, 4 reactive lymphoid controls, 50 non-hematolymphoid (including neuroectodermal, epithelial, myofibroblastic, and germ cell) lesions, and 4 early-trimester aborted fetuses that served as neuroblastic controls. METHODS: The study involved a predominantly (72%) Singaporean Chinese population aged between 9 months and 88 years (excluding the aborted fetal controls). All cases were reviewed both histologically and immunohistochemically with a wide panel of antibodies using the standard protocols in order to diagnose them according to the latest WHO classification systems. A positive cut-off value was determined, both by comparison with diagnostic categories with and without ALK translocations, as well as with negative controls. RESULTS: The ALK FISH probe suffered a 33% non-informative rate, but in informative cases it showed 94% concordance with the ALK-1 immunostain. A minimum cut-off value of 5 in 200 informative cells was adopted to make a positive call in each case. Of the ALK-1 immunoreactive lesions, nine lymphomas were concordantly ALK translocation-positive but one vesical inflammatory myofibroblastic tumor was discordantly FISH-negative. Among the ALK-1-immunonegative lesions, one case each of anaplastic lymphoma and pulmonary mycobacterial spindle cell pseudotumor were discordantly ALK FISH-positive, while a case each of intestinal myeloblastic tumor and ganglioglioma showed initial--but not reproducible--positive FISH readings. The remaining cases were concordantly negative. DISCUSSION: The discrepancies between ALK FISH results and well-established immunomorphological parameters indicate that interpretation is not always straightforward. Notably, the derivation of threshold cut-off values for positive calls on FISH assays has seldom been addressed in the literature, and has raised issues in interpreting cases with borderline positivity in this study. The factors that may influence such cut-off values are extensively reviewed. CONCLUSIONS: We propose the term 'conditional threshold positivity' to encourage the adoption of different cut-off values for making positive calls in lesions of different origin.

Detection of ALK gene rearrangements in formalin-fixed, paraffin-embedded tissue using a fluorescence in situ hybridization (FISH) probe: a search for optimum conditions of tissue archiving and preparation for FISH.

BACKGROUND: It is widely known that the efficiency of fluorescence in situ hybridization (FISH) probes applied to formalin-fixed, paraffin-embedded tissues is affected by the conditions under which the tissues are fixed and embedded. However, relatively few studies address exactly how tissue archiving conditions affect the performance of FISH probes. We report our experience based on use of an ALK FISH probe, during the validation of its diagnostic utility. METHODS: We applied the probe to 77 formalin-fixed, paraffin-embedded tissue blocks archived from 1991 through to 2000, and studied the interrelationship between the archival age (which ranged up to 10 years), type and condition of tissue, duration required for optimum hydrolysis, and obtainability of hybridization signals. RESULTS: We found that as archival age and tissue collagen content increased, not only did hydrolysis times have to be prolonged in order to yield interpretable hybridization signals, but also the likelihood of blocks becoming non-signaling increased. The most striking positive correlations were seen between the archival age of signaling lymphoid blocks and their requisite hydrolysis times. CONCLUSIONS: The difficulty in applying FISH on archival tissue increases with its archival age and collagen content, and may necessitate changes in laboratory protocol accordingly.

Relationship between atypical T- and B-cell size predicts survival in peripheral T-cell lymphomas with large B-cells.

BACKGROUND: Pathological prognostication for peripheral T-cell lymphomas (PTCLs) complicated by large B-cell (LBC) proliferations has not been previously devised. METHODS: Forty-six cases of PTCL with LBCs were reviewed immunohistologically, by in situ hybridisation for Epstein-Barr virus encoded RNA and polymerase chain reaction analyses for T-cell receptor (TCR) clonality. Follow-up intervals ranged from 1 to 149 months (mean 40 months). RESULTS: Cases with atypical T-cell size equal to or exceeding that of interspersed LBCs (ATEB+, n = 12, including an ALK negative anaplastic large cell lymphoma) had significantly inferior disease-specific survival compared to ATEB negative (ATEB-, n = 34) cases (p = 0.002 for all cases and p = 0.014 when restricted to TCR clonal cases, n = 30) [hazard ratio 11.2; 95% confidence interval (CI) 0.94-85.0, p = 0.019]. All recorded deaths amongst ATEB+ cases occurred in 26 months, while TCR polyclonal ATEB- cases (n = 9) had none; TCR clonal ATEB- cases (n = 22) had 62% 5-year survival (95% CI 33-91%, p = 0.001). There was no survival separation between angioimmunoblastic (n = 25) and unspecified (n = 20) subsets of PTCL (p = 0.957). CONCLUSION: In PTCLs, cytological grading using harboured LBCs as internal yardsticks, as well as molecular genotypic measures of lymphoma cell burden, have prognostic value.

A practical approach to the understanding and diagnosis of lymphoma: an assessment of the WHO classification based on immunoarchitecture and immuno-ontogenic principles.

This review aims to interrelate the major lymphoma types in the current World Health Organization (WHO) classification to construct a framework for understanding and diagnostic application. Multiple morphological, phenotypical and molecular genotypical data are assessed in order to categorise lymphomas into germinal centre (GC) and extracentric (EC) subgroups. GC entities [lymphocyte-predominant Hodgkin, follicular, Burkitt's, angioimmunoblastic T-cell and diffuse large B-cell lymphoma (DLBCL) with GC profile] express bcl-6, CD10 and/or the GC-homing chemokine CXCL13, and harbour ongoing somatic hypermutations (SHM), but not Epstein-Barr virus (EBV) in its higher latency states. Post-GC entities [classical Hodgkin, marginal zone and lymphoplasmacytic lymphomas, half of chronic lymphocytic leukaemia (CLL)/small lymphocytic lymphoma (SLL), DLBCL with 'activated' or post-GC profile, primary effusion lymphoma, plasmacytoma and myeloma] express, instead, MUM.1 and/or CD138, harbour static rather than ongoing SHM, and may harbour EBV in higher latency states. The remainder of CLL/SLL and the majority of mantle cell lymphoma without SHM constitute the pre-GC ('naive') category, with coexpression of IgD and CD5. Lymphomas can be categorised across lineage (B- or T-cell) and relationship against host immune response (Hodgkin or non-Hodgkin) into GC and EC groups, affording leverage in their differential diagnosis.

An Epstein-Barr virus positive natural killer lymphoma xenograft derived for drug testing.

Natural killer (NK) lymphomas occurring more frequently in the Far East and South America respond poorly to anthracycline-based regimens. Here we report an in vivo NK lymphoma xenograft (NK-S1) derived from the testicular metastasis of a patient with an extranodal NK lymphoma (nasal type). The NK-S1 xenograft, established in severe combined immune deficient (SCID) mice retained the same imunophenotypic features as the original tumor. NK-S1 disseminated intra-abdominally to the testis, intestine and liver. Although doxorubicin, rapamycin, bevacizumab, rapamycin-doxorubicin, and bevacizumab-doxorubicin had no effects on the growth of subcutaneous NK-S1 xenografts, intraperitoneal (IP) delivery of cyclophosphamide caused complete tumor regression; this tumor regression was associated with apoptosis, upregulation of activated caspase-3, and cleaved Poly(ADP-ribose) polymerase (PARP). In an IP model of NK lymphoma, cyclophosphamide also prolonged the survival of mice and potently inhibited tumor dissemination and ascites formation. Our data suggest that the NK-S1 xenograft is a useful tool for screening preclinical drugs, and cyclophosphamide may be a useful drug for the treatment of this disease.

Diagnostic impact of molecular lineage analysis on paraffin-embedded tissue in hematolymphoid neoplasia reclassified by current WHO criteria.

BACKGROUND AND OBJECTIVE: By current WHO criteria, most - though not all - cases of hematolymphoid neoplasm can be diagnosed immunomorphologically, diminishing the role of molecular tests for lymphoid antigen receptor clonality in lymphoma diagnosis. Hence, our objective was to glean immunomorphological and molecular correlates from hematolymphoid neoplasms that had remained unresolvable without diagnostic molecular input. METHODS: Thirty-five such cases were reviewed histologically and with standard immunoperoxidases. In situ hybridization for Epstein-Barr virus (EBV)-encoded RNAs (EBER) was performed on selected cases. PCR amplification of genes encoding T-cell receptors (TcR) and immunoglobulin heavy chains (IgH) [TR and IGH genes, respectively] was performed on whole tissue in all cases, and on microdissected cells in two cases. RESULTS: Twenty-five cases (71%) requiring diagnostic molecular genotyping had some form of peripheral T-cell lymphoma (PTCL). Twenty (80%) of these were complicated by a proliferation of B-lineage cells, either within the same tissue ('syntopic') as large B cells (LBC) or Reed-Sternberg (RS)-like cells (17 cases), florid lymphoid hyperplasia (two cases, one also with syntopic LBC) or monotypic plasma cells (one case), or at a separate ('metatopic') site as a B-cell lymphoma (two cases, one of which also had syntopic LBC) or Hodgkin lymphoma (HL; one case, also showing syntopic LBC). Fifteen (75%) of these 20 PTCLs with B-lineage proliferation yielded monoclonal TR gene rearrangements, and only two (10%) showed IGH monoclonality, which was transient in one case. Three (18%) of the PTCLs with LBC had originally been misinterpreted as some form of HL. Conversely, of the remaining cases, three of four (75%) that had been diagnosed initially as some form of large cell non-HL (NHL), including two of three that were called 'anaplastic', had to be revised to grade II/syncytial nodular sclerosing (NS) HL, yielding polyclonal TcRgamma gene (TRG) rearrangements, with one case, in addition, disclosing a biallelic clonal IGH gene rearrangement that excluded anaplastic large cell lymphoma. DISCUSSION/CONCLUSION: Paradoxically, monoclonality of TR rather than IGH gene rearrangement may more often be detectable in a predominantly dispersed ('hodgkinoid'), large B-lineage cell proliferation, consistent with release from immune regulation in the milieu of impaired immunosurveillance within a PTCL. This is compounded by the difficulty in ascertaining clonal IGH gene rearrangements resulting from (1) poor consensus primer hybridization due to somatic hypermutations, and (2) 'dilution' in a T-cell-rich milieu. These same difficulties also account for the long-elusive identification of the RS cell lineage. Conversely, anaplastic lymphoma, which is of non-B lineage, may be mimicked by NSHL, which is of B lineage.