R. Achten

T-Cell/Histiocyte–Rich Large B-Cell Lymphoma: A Distinct Clinicopathologic Entity

PURPOSEnAlthough it has proven difficult to delineate diagnostically reproducible and clinically relevant subgroups, the heterogeneity of diffuse large B-cell lymphomas (DLBCL) is widely acknowledged. In 1992, we reported on six cases that suggested that large B-cell lymphoma rich in stromal histiocytes and T cells may be identified as a distinct clinicopathologic entity within DLBCL.nnnPATIENTS AND METHODSnAn integrated clinicopathologic study of 40 cases of this DLBCL subtype is presented.nnnRESULTSnDistinguishing a DLBCL rich in histiocytes and reactive T cells, designated T-cell/histiocyte--rich large B-cell lymphoma (THR-BCL), may be justified from a clinical point of view. The disease typically affects middle-aged male patients who usually present with advanced-stage disease that is not adequately managed with current therapeutic strategies. Whereas proliferation fraction and p53 overexpression, in addition to the clinical variables incorporated in the International Prognostic Index (IPI), significantly correlate with response to treatment and survival in a univariate analysis, only the IPI score identifies relevant prognostic THR-BCL subpopulations in a multivariate model. The morphologic and immunophenotypic profile of the neoplastic B cells in THR-BCL suggests that they may originate from a germinal center ancestor.nnnCONCLUSIONnTHR-BCL constitutes a distinct clinicopathologic entity that is characterized by an aggressive behavior. Experimental therapeutic strategies may be indicated to obtain a more favorable response to treatment in this disease.

Additional Prognostic Value of Bone Marrow Histology in Patients Subclassified According to the International Prognostic Scoring System for Myelodysplastic Syndromes

PURPOSEnThe most recent and powerful prognostic instrument established for myelodysplastic syndromes (MDS) is the International Prognostic Scoring System (IPSS), which is primarily based on medullary blast cell count, number of cytopenias, and cytogenetics. Although this prognostic system has substantial predictive power in MDS, further refinement is necessary, especially as far as lower-risk patients are concerned. Histologic parameters, which have long proved to be associated with outcome, are promising candidates to improve the prognostic accuracy of the IPSS. Therefore, we assessed the additional predictive power of the presence of abnormally localized immature precursors (ALIPs) and CD34 immunoreactivity in bone marrow (BM) biopsies of MDS patients.nnnPATIENTS AND METHODSnCytogenetic, morphologic, and clinical data of 184 MDS patients, all from a single institution, were collected, with special emphasis on the determinants of the IPSS score. BM biopsies of 173 patients were analyzed for the presence of ALIP, and CD34 immunoreactivity was assessable in 119 patients. Forty-nine patients received intensive therapy.nnnRESULTSnThe presence of ALIP and CD34 immunoreactivity significantly improved the prognostic value of the IPSS, with respect to overall as well as leukemia-free survival, in particular within the lower-risk categories. In contrast to the IPSS, both histologic parameters also were predictive of outcome within the group of intensively treated MDS patients.nnnCONCLUSIONnOur data confirm the importance of histopathologic evaluation in MDS and indicate that determining the presence of ALIP and an increase in CD34 immunostaining in addition to the IPSS score could lead to an improved prognostic subcategorization of MDS patients.

Comparative Genomic Hybridization Pattern Distinguishes T-Cell/Histiocyte-Rich B-Cell Lymphoma from Nodular Lymphocyte Predominance Hodgkin’s Lymphoma

Several lines of evidences suggest that T cell/histiocyte-rich B-cell lymphoma (T/HRBCL) represents an aggressive variant of the clinically indolent entity nodular lymphocyte predominance Hodgkins lymphoma (LPHL). Still, this view has not yet been supported by firm genetic evidence. In this study, we analyzed 17 T/HRBCL cases using comparative genomic hybridization (CGH) combined with microdissection of single CD20+ neoplastic cells and DNA amplification by degenerate oligonucleotide primed-polymerase chain reaction, an approach we previously used in LPHL. Genomic imbalances were detected in all cases (in total, 80 changes). The most common imbalances included gain of Xq, 4q13q28, Xp21p11, and 18q21, and loss of 17p. Of note, a partial gain of 4q, a rare change in lymphoma, is also among the genomic imbalances most frequently encountered in LPHL. On the other hand, the CGH profiles of T/HRBCL and LPHL showed several distinct features, in particular with respect to the number of genomic imbalances (average of 4.7 in T/HRBCL versus 10.8 in LPHL) and their distribution (usually 1 to 5 in T/HRBCL versus 6 to 22 in LPHL). Altogether, our CGH findings of shared as well as distinctive cytogenetic features in both diseases suggest that T/HRBCL constitutes a separate lymphoma entity, possibly originating from the same precursor cell as LPHL.

Evaluation of B cell lymphoid infiltrates in bone marrow biopsies by morphology, immunohistochemistry, and molecular analysis

Aims—Morphological criteria to distinguish between reactive and neoplastic B cell lymphocytoid infiltrates in trephines have been defined but are not always reliable. Polymerase chain reaction (PCR) analysis of the CDR3 region of the immunoglobulin heavy chain (IgH) gene which, by demonstrating monoclonality, can provide additional arguments in favour of lymphoid malignancy is now frequently used for the detection and follow up of B cell lymphoma (NHL). The aim of this study was to investigate the usefulness of morphological findings in bone marrow biopsies in comparison with data obtained by PCR analysis. Methods—Eighty nine bone marrow biopsies displaying lymphoid infiltrates were evaluated by morphology and immunohistochemistry as well as by CDR3-PCR using consensus framework 3 (FRW3) and JH primers. Results—The presence of a clonal B cell proliferation was demonstrated by PCR analysis in 45 biopsies, including 21 samples considered to be positive, 17 to be suspicious, and seven to be negative by morphology. In the remaining 44 trephines we found no evidence of clonality, although 12 of these trephines were thought to be positive by morphology. Conclusions—These results, revealing an incomplete correlation between CDR3-PCR data and immunomorphological findings, indicate that molecular analysis may be more sensitive and specific in general. However, false negative PCR results do occur, which emphasises the necessity to combine both diagnostic tools in the evaluation of lymphoid infiltrates.

Validation and Application of a Custom-Designed Targeted Next-Generation Sequencing Panel for the Diagnostic Mutational Profiling of Solid Tumors.

The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting.

Targeted next-generation sequencing using a multigene panel in myeloid neoplasms: Implementation in clinical diagnostics.

Detection of mutations in patients with myeloid neoplasms (MNs) has shown great potential for diagnostic and prognostic purposes. Next‐generation sequencing (NGS) is currently implemented for the diagnostic profiling of the four major MN subgroups.