The "Burkitt-like" immunophenotype and genotype is rarely encountered in diffuse large B cell lymphoma and high-grade B cell lymphoma, NOS.

Burkitt lymphoma (BL) is a B cell lymphoma composed of monomorphic medium-sized blastic cells with basophilic cytoplasm and a high proliferation index. BL has a characteristic immunophenotype of CD10 and BCL6 positive and BCL2 negative and harbours MYC gene rearrangements (MYCR) in >90% of the cases. Owing to its highly aggressive nature, intensified chemotherapy regimens are usually administered, requiring an exact diagnosis. Since the diagnosis usually warrants an integration of morphologic, immunophenotypic and genetic findings and because there is a morphologic overlap with the new WHO category of high-grade B cell lymphoma, not otherwise specified (HGBL, NOS) and some cases of diffuse large B cell lymphoma (DLBCL), we wanted to test the distinctiveness of the CD10+, BCL6+, BCL2- and MYCR positive immunopheno-genotype in a large cohort of >1000 DLBCL and HGBL. Only 9/982 DLBCL classified by an expert panel of haematopathologists (0.9%) displayed a single MYCR and were CD10+, BCL6+ and BCL2-. In a similar fashion, only one out of 32 HGBL, NOS (3%) displayed the "Burkitt-like" genetic/immunophenotypic constitution. The samples of non-BL showing the BL-typic immunopheno-genotype, interestingly, harboured higher copy number variations (CNV) by OncoScan analysis (mean 7.3 CNVs/sample; range: 2-13 vs. 2.4; range 0-6) and were also distinct from pleomorphic BL cases regarding their mutational spectrum by NGS analysis. This implies that the characteristic immunophenotype of BL, in concert with a single MYCR, is uncommon in these aggressive lymphomas, and that this constellation favours BL.

Diffuse large B-cell lymphomas of immunoblastic type are a major reservoir for MYC-IGH translocations.

The immunoblastic variant of diffuse large B-cell lymphoma (IB-DLBCL) has recently been recognized as an aggressive lymphoma type with inferior prognosis as compared with other DLBCL variants. At the same time, the presence of MYC rearrangements in DLBCL has been shown to indicate shorter survival in R-CHOP-treated patients. In this study, we investigated the occurrence of MYC gene rearrangements in IB-DLBCL versus non-IB-DLBCL in a large series. Using fluorescence in situ hybridization with an MYC break-apart and MYC-IGH fusion probe, we found that 13/39 evaluable IB-DLBCLs (33%) harbor translocations involving MYC, in contrast with only 5/68 (7%) in the non-IB-DLBCL group (P<0.01). The immunoglobulin heavy chain gene (IGH) was the translocation partner in all rearrangements (100%) involving MYC in IB-DLBCL, which is in contrast to what has been reported for DLBCL in the literature (50% to 70%). Moreover, MYC rearrangements occurred as the sole translocation in the majority of cases (77%), whereas across all DLBCLs the majority of MYC-rearranged cases carry additional rearrangements of either BCL2 and/or BCL6 genes (between 58% and 83% of cases). Finally, MYC-rearranged IB-DLBCLs were CD10 positive in 62% (8/13), whereas this was an uncommon feature in MYC germline IB-DLBCLs (15%). In conclusion, IB-DLBCLs are genetically characterized by frequent MYC-IGH translocations that often occur without additional BCL2 and/or BCL6 translocations. The activation of MYC, therefore, may be an important pathogenetic feature in IB-DLBCL.

Fluorescence in situ analysis of soft tissue tumor associated genetic alterations in formalin-fixed paraffin-embedded tissue.

No prospective studies are available to date evaluating the combined analysis of chromosomal alterations via interphase FISH in different soft tissue sarcoma (STS) subtypes. We tested 64 consecutive sarcoma specimens with FISH probes to detect aberrations specific for a given STS subtype. We first determined the translocation frequency in the specific STS subtypes in 48 tumors, with the primary pathological diagnosis as the gold standard. Subsequently, to evaluate sensitivity and specificity, all FISH probes were hybridized to 16 STS of hitherto unknown diagnosis. DDIT3 translocations occurred in 8/10 (80%) of myxoid liposarcomas. FOXO1 translocations were noted in 4/4 (100%) of alveolar but in none of 7 embryonal rhabdomyosarcomas. All 15 (100%) Ewing sarcomas/PNET and 4 clear cell sarcomas (4/4) harbored EWSR1 translocations. SS18 rearrangements were demonstrated in 8/9 (89%) synovial sarcomas. MDM2 amplification was noted in 7/8 (88%) atypical lipomatous tumors/well-differentiated and 3/3 (100%) dedifferentiated liposarcomas, respectively, but not in four pleomorphic liposarcomas. Sensitivities and specificities ranged from 80% to 100% and from 93% to 100%, respectively, with the highest values observed for FOXO1 (100% each). We conclude, therefore, that is possible to accurately predict the STS subtype using a panel of different subtype-specific FISH probes, thereby greatly facilitating the differential diagnosis of these tumors.

Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.

Follicular lymphoma grade 3B is a distinct neoplasm according to cytogenetic and immunohistochemical profiles.

BACKGROUND: According to the current World Health Organization Classification of Lymphoid Tumours, follicular lymphoma is subclassified into three grades according to the number of centroblasts. Follicular lymphoma grade 3 can be further divided into types A and B. Almost all available genetic data on grade 3B follicular lymphomas have been generated from tumors with an additional diffuse large B-cell lymphoma component. The purely follicular type of follicular lymphoma grade 3B is a rare neoplasm. DESIGN AND METHODS: We performed a detailed immunohistochemical (CD10, IRF4/MUM1, BCL2, Ig light chains) and genetic (translocations of BCL2, BCL6, MYC, IRF4) characterization of the largest series of purely follicular cases of grade 3B follicular lymphoma available to date, comprising 23 tumor samples. We also included 25 typical grade 1 or 2 follicular lymphomas, 9 follicular lymphomas with large centrocytes and/or high proliferation indices (FL/LCC), 12 cases of follicular lymphoma grade 3A, 16 cases of diffuse large B-cell lymphoma/follicular lymphoma grade 3B and 15 follicular lymphomas in which a straightforward distinction between grades 3A and 3B was not possible. RESULTS: Translocations affecting BCL2 and BCL6 genes are rare in grade 3B follicular lymphomas (2/23, 9% and 4/23, 17%) when compared with grade 1 or 2 follicular lymphomas (22/25, 88%, P<0.001 and 0/25, P<0.05), FL/LCC (7/9, 78%, P<0.001 and 2/9, 22%), grade 3A follicular lymphomas (7/12, 58%, P<0.01 and 2/12, 17%), unclassified grade 3 follicular lymphomas (6/15, 40% and 2/15, 13%) and diffuse large B-cell lymphoma/follicular lymphoma grade 3B (2/16, 13% and 8/16, 50%, P<0.05). MYC translocations were detected occasionally in FL/LCC, follicular lymphoma grade 3B, and diffuse large B-cell lymphoma/follicular lymphoma grade 3B (13%-22%), but not in grade 1, 2 or 3A follicular lymphomas (P<0.05 when compared with follicular lymphoma grade 3B). Both follicular lymphoma grade 3B and diffuse large B-cell lymphoma/follicular lymphoma grade 3B were enriched in samples with a CD10(-)IRF4/MUM1(+) immunophenotype (8/19, 42% and 7/16, 44%), with the vast majority of them lacking BCL2 translocations. In contrast, 42/46 grade 1 or 2 follicular lymphomas, FL/LCC and grade 3A follicular lymphomas were CD10(+) (91%) while 0/46 expressed IRF4/MUM1. None of the tumor samples tested with increased IRF4/MUM1-expression harbored a translocation of the IRF4 gene locus. CONCLUSIONS: Our results show that grade 3B follicular lymphomas form a distinct category of follicular lymphomas with infrequent BCL2 and BCL6 translocations, while grades 1, 2 and 3A follicular lymphomas and FL/LCC display homogeneous features with frequent BCL2 translocations and a CD10(+)IRF4/MUM1(-) immunophenotype.

A distinctive subtype of t(14;18)-negative nodal follicular non-Hodgkin lymphoma characterized by a predominantly diffuse growth pattern and deletions in the chromosomal region 1p36.

Follicular lymphoma (FL) is a morphologically and genetically well-characterized B-cell non-Hodgkin lymphoma that can show predominantly follicular, combined follicular and diffuse, or predominantly diffuse growth patterns. Although approximately 85% of FLs harbor the translocation t(14;18)(q32;q21) and consistently display a follicular growth pattern, predominantly diffuse FLs are less well characterized on the phenotypical, molecular, and clinical level. We studied 35 predominantly diffuse FL by immunohistochemistry, classical chromosome banding analysis, fluorescence in situ hybridization (FISH), and gene expression profiling. A total of 28 of 29 analyzable cases lacked t(14;18), and 27 of 29 cases revealed a unifying chromosomal aberration, a deletion in 1p36. Morphologically, 12 FLs were grade 1 and 23 were grade 2, and the immunophenotype with frequent expression of CD10, BCL6, and CD23 was in line with a germinal center B-cell phenotype. The gene expression profiles of 4 predominantly diffuse FLs fell into the spectrum of typical FL, with a unique enrichment of specific gene signatures. Remarkably, patients with diffuse FL frequently presented with low clinical stage and large but localized inguinal tumors. These results suggest that predominantly diffuse FL represent a distinct subtype of t(14;18)-negative nodal FL with a unifying genetic alteration (deletion of 1p36) and characteristic clinical features.

A cytomorphological and immunohistochemical profile of aggressive B-cell lymphoma: high clinical impact of a cumulative immunohistochemical outcome predictor score.

We analyzed morphological and immunohistochemical features in 174 aggressive B-cell lymphomas of nodal and extranodal origin. Morphological features included presence or absence of a follicular component and cytologic criteria according to the Kiel classification, whereas immunohistochemical studies included expression of CD10, BCL-2, BCL-6, IRF4/MUM1, HLA-DR, p53, Ki-67 and the assessment of plasmacytoid differentiation. Patients were treated with a CHOP-like regimen. While the presence or absence of either CD10, BCL-6 and IRF4/MUM1 reactivity or plasmacytoid differentiation did not identify particular cytomorphologic or site-specific subtypes, we found that expression of CD10 and BCL-6, and a low reactivity for IRF4/MUM1 were favourable prognostic indicators. In contrast, BCL-2 expression and presence of a monotypic cytoplasmic immunoglobulin expression was associated with an unfavourable prognosis in univariate analyses. Meta-analysis of these data resulted in the development of a cumulative immunohistochemical outcome predictor score (CIOPS) enabling the recognition of four distinct prognostic groups. Multivariate analysis proved this score to be independent of the international prognostic index. Such a cumulative immunohistochemical scoring approach might provide a valuable alternative in the recognition of defined risk types of aggressive B-cell lymphomas.

Delineation of distinct tumour profiles in mantle cell lymphoma by detailed cytogenetic, interphase genetic and morphological analysis.

Mantle cell lymphoma (MCL) is an aggressive lymphoid tumour characterized by the translocation t(11;14)(q13;q32) and a poor clinical outcome (median survival: 3-4 years). Recent studies revealed that increased proliferation of the tumour cells and certain chromosomal aberrations, such as deletions of 17p13 and 9p21 represent major adverse biological markers in this disease, although the molecular targets of chromosomal imbalances in MCL have not been identified for the large majority of loci affected. To correlate histomorphological and proliferation features of MCL with genetic findings, we investigated 223 MCL by fluorescence in situ hybridization (FISH) (n = 157) and/or classical cytogenetic banding analysis (n = 129). FISH analysis turned out to be distinctly more sensitive in the delineation of aberrations. Complex karyotypic alterations were associated with higher proliferation indices and inferior prognosis. A comprehensive analysis of biological features including genetic alterations in MCL by hierarchical clustering resulted in the delineation of four tumour subgroups differing with respect to their genetic constitution and suggesting different transformation or progression pathways. Moreover, in one of the groups identified, a more indolent clinical behaviour was associated with few secondary aberrations and fewer known high-risk chromosomal aberrations, which points to the importance of the quality of karyotypic evolution in MCL tumours.

Presence of preserved reactive germinal centers in follicular lymphoma is a strong histopathologic indicator of limited disease stage.

Follicular lymphoma (FL) typically presents as a systemic disease (stages III/IV). We repeatedly observed in cases with conserved reactive follicle structures (so-called partial infiltration) an association with a limited clinical stage (I/II). In this study, we analyzed 53 lymph node biopsies of FL with conserved reactive follicle structures. In 44 cases (83%) of the patients with partial infiltration, a limited stage of disease (Ann Arbor stage I/II) was found, whereas only 9 of 53 cases (17%) suffered from a systemic disease. In those cases with at least one follicle totally spared by lymphoma, 95% of the patients (38 of 40 cases) presented with a limited stage (I/II) of disease, compared with only 20% (10 of 49 cases) in a control group with full-blown infiltration (P < 0.001). Analyzing systematically all 321 FL cases sent to the Reference Center Würzburg in the year 2001, reactive follicle remnants were detected in 34 of 321 (10.6%) cases with 26 of 34 (76%) tumors showing limited stage (I/II) disease, including all 18 cases with at least one totally spared follicle. Our results therefore show a clear association between the occurrence of preserved reactive follicles in FL and limited disease stage.

Cytogenetic alterations affecting BCL6 are predominantly found in follicular lymphomas grade 3B with a diffuse large B-cell component.

Recently, classical banding cytogenetic studies suggested that follicular lymphomas (FLs) grade 3 with preserved maturation to centrocytes (FL3A) are closely related to FL grades 1 and 2 and frequently harbor the t(14;18), whereas FL grade 3B, consisting of centroblasts exclusively, do frequently show 3q27 alterations. To clarify the prevalence of BCL6 and BCL2 rearrangements in FL and diffuse large B-cell lymphomas (DLBLs), we performed a large scale bicolor interphase cytogenetic (fluorescence in situ hybridization) study on 188 well-characterized B-NHLs classified according to the World Health Organization Classification of Tumors of the Lymphoid Tissues. BCL6 rearrangements were detected in a significantly higher number of FL3B with a DLBL component (12 of 22, 55%) compared with purely diffuse nodal DLBLs (19 of 77, 25%) and DLBLs with a well-documented primary extranodal origin (2 of 27, 7%) (P < 0.001). Five FL3B without a DLBL component were negative for both t(14;18) and 3q27 aberrations. FL grades 1/2 and FL3A were t(14;18)-positive in 88% and 64% of cases, respectively, but 3q27 alterations were identified in only four FL3A. These data exemplify different genetic pathways in the genesis of FLs with a high content of centroblasts and suggest that 3q27 rearrangements are predominantly associated with FL grade 3B harboring a DLBL component.

Genetic analysis of de novo CD5+ diffuse large B-cell lymphomas suggests an origin from a somatically mutated CD5+ progenitor B cell.

CD5(+) diffuse large B-cell lymphomas (DLBLs) have recently been described as a particular subgroup of DLBLs. Classical banding and interphase cytogenetic analyses targeting ATM, TP53, and P16(INK4a) genes and the D13S25 locus from 13 CD5(+) DLBLs were compared with 55 CD5(-) DLBLs. Additionally, analysis of somatic mutations of the immunoglobulin heavy chain variable region (IgVH) genes were performed in CD5(+) DLBLs. CD5(+) DLBLs were somatically mutated (7 of 8 cases) and were negative for t(11;14)(q13;q32) and t(14;18)(q32;q21), whereas t(3;14)(q27;q32) was found in only one tumor. Trisomy 3 and gains on chromosomes 16/16p and 18/18q were significantly overrepresented in CD5(+) DLBLs. No ATM deletions were detected. The prevalence of deletions at the D13S25 locus was significantly higher in CD5(+) DLBLs (4 of 12 [33%]) compared with CD5(-) DLBLs (4 of 42 [10%]), as were p16(INK4a) deletions (33% versus 8%). On the basis of these findings, CD5(+) DLBLs are likely to arise from the same progenitor cell as the mutated variant of CD5(+) lymphocytic lymphoma/B-cell chronic lymphocytic leukemia (B-CLL).

Cytomorphologic, immunohistochemical, and cytogenetic profiles of follicular lymphoma: 2 types of follicular lymphoma grade 3.

Follicular lymphoma (FL) grades 1 and 2 are regarded as a distinct disease entity, whereas data suggest that FL grade 3 might be an inhomogeneous tumor category. To define the biologic spectrum of FL, 89 follicular lymphomas were studied for their cytologic composition, antigen expression, mitotic and proliferation indices, cytogenetics, and clinical data. In contrast to the homogeneous appearance of FL grades 1 and 2 (29 and 33 cases, respectively), 2 types of FL grade 3 were recognized. Eleven cases of FL 3a displayed structural features similar to those of FL 1 and 2 and were composed of centroblasts and centrocytes, whereas 16 cases of FL 3b, with (n = 4) or without (n = 12) a diffuse large B-cell lymphoma component (DLBL) (FL 3b +/- DLBL), consisted exclusively of blasts. In contrast to FL 3a, FL 3b +/- DLBL were CD10(+) in only 50% of cases and displayed plasmacytoid differentiation in 44% of cases. Although FL3a was t(14;18)+ in 8 of 11 (73%) cases, only 2 of 16 (13%) FL3b +/- DLBLs harbored this translocation. In contrast, chromosomal breaks at 3q27 were encountered in 7 of 16 (44%) FL 3b +/- DLBL in contrast to only 2 of 11 (18%) FL 3a, and the spectrum of secondary aberrations in FL 3b +/- DLBL was similar to that of diffuse large B-cell lymphoma. We conclude, therefore, that FL grade 3 is a heterogeneous disease group and that the distinction proposed in the new World Health Organization classification between FL 3a (with centrocytes) and FL3b (without centrocytes) is of biologic, and possibly clinical, importance.