Search

Myelin and lymphocyte (MAL) protein has been previously reported as a highly specific marker for distinguishing primary mediastinal large B-cell lymphoma (PMBL) from diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS). However, there has not been a commercially available MAL antibody for immunohistochemistry. We identified a commercially available MAL monoclonal antibody and evaluated it by immunohistochemistry on 43 cases of PMBL and 63 cases of DLBCL, NOS. We also compared this with a CD200 antibody that was previously reported useful in distinguishing PMBL and DLBCL, NOS. A threshold of 10% positive tumor cells was used to determine positive protein expression. MAL was expressed in 72% cases of PMBL and 0% of cases of DLBCL, NOS (sensitivity=72%, specificity=100%). CD200 was expressed in 81% of PMBL cases and 13% of DLBCL, NOS cases (sensitivity=81%, specificity=87%). To our knowledge, this is the first report on the utility of a commercially available MAL monoclonal antibody in the diagnosis of PMBL. There is a high specificity with good sensitivity in distinguishing PMBL from DLBCL, NOS, similar to previous studies with a noncommercial source. This antibody will likely prove useful in identifying cases of PMBL in routine practice.

Angioimmunoblastic T-cell lymphoma (AITL) is a nodal-based mature T-cell lymphoma with distinctive clinical symptomatology and histology. Research into its pathogenesis supports a cellular derivation from follicular helper T cells and overexpression of genes related to B cells, follicular dendritic cells, and vascular growth. Recently, a novel recurring somatic mutation in RHOA, encoding p.Gly17Val, was discovered in nearly 70% of AITLs and in a smaller proportion of peripheral T-cell lymphomas, not otherwise specified (PTCL-NOS). We investigated a series of AITLs to compare RHOA mutated with wild-type case for clinicopathologic differences. Targeted exome and Sanger sequencing was performed on 27 AITLs and 10 PTCL-NOS. The RHOA G17V mutation was identified in 63% of the AITL cases and in none of the PTCL-NOS cases. The median variant allelic frequency was 14%, with a range of 0.4 to 50% in positive cases. RHOA G17V-mutated cases had a significantly higher incidence of splenomegaly and B symptoms at diagnosis, but there was no difference in overall survival between mutated and wild-type subgroups. Cases with the RHOA G17V mutation had a significantly higher mean microvessel density (P<0.01) and expressed a greater number of follicular helper T-cell markers (P<0.05) than wild-type cases. RHOA G17V is present in a significant proportion of angioimmunoblastic lymphomas and is associated with classic pathologic features of AITL. Additional studies are needed to provide a biological or functional link between altered RHOA function and these pathologic features.

Distinction between lymphocyte-rich thymoma and T-lymphoblastic lymphoma/leukemia (T-LBL) can be problematic because of a predominance of precursor T cells in both, particularly if the epithelial component in a thymoma is undersampled. Because of very different clinical implications, accurate diagnosis is critical. The NOTCH1 signaling pathway is frequently activated in T-LBL and plays a central role in the pathogenesis of this disease. Antibodies to NOTCH1 intracellular domain (N1ICD), recognizing the active form of NOTCH1, have been developed. We hypothesized that detection of N1ICD would be useful in distinguishing T-LBL from thymoma and investigated a series of formalin-fixed, paraffin-embedded tissues for immunoreactivity with an N1ICD antibody using automated immunohistochemistry. Slides were scored using a 25% nuclear reactivity threshold for positivity. Hyperplastic tonsil showed positivity in few scattered interfollicular lymphoid cells, suprabasilar epithelial cells, and endothelial cells. Thymocytes from non-neoplastic thymus were largely negative for N1ICD. All thymomas tested (n=23) were negative for N1ICD, although epithelial cells and a small minority of thymocytes may be positive, requiring careful interpretation. All T-LBL cases (n=16) were scored positive for N1ICD: 8 (50%) of these showed diffuse and mostly strong immunoreactivity, whereas the remaining 8 (50%) had less extensive positivity, but with consistently >25% nuclear staining. In conclusion, normal thymocytes do not express significant levels of N1ICD. In keeping with this pattern, thymomas are negative for N1ICD, whereas a high percentage of T-LBL expresses N1ICD. Thus, N1ICD immunohistochemistry appears to be a useful method in distinguishing T-LBL from thymoma.

BRAF V600E mutations are present in virtually all cases of hairy cell leukemia (HCL). We hypothesized that detection of phospho-ERK (pERK) in tissue sections may be a useful marker for diagnosis of HCL. pERK/CD20 double immunostaining was performed on 90 formalin-fixed bone marrow trephine samples affected with small B-cell lymphoproliferative disorders, including 28 cases of HCL. pERK staining was observed in all 28 cases of HCL and in 1 of 62 cases of non-HCL B-cell lymphoproliferative disorders. By allele-specific polymerase chain reaction, all 11 cases of HCL with available DNA were positive for BRAF V600E, as was the 1 pERK non-HCL case. The remaining 31 non-HCL cases tested were negative for BRAF V600E. The sensitivity and specificity of pERK for diagnosis of HCL was 100% and 98%, respectively. We conclude that the presence of pERK as detected by immunohistochemical staining is a useful surrogate marker for BRAF V600E in the diagnosis of HCL.

Peripheral benzodiazepine receptor (PBR), a mitochondrial protein involved in cell proliferation and differentiation, and carbonic anhydrase IX (CA IX), an intrinsic marker of hypoxia, have been studied in the panel of human breast (MCF-7, BT- 20, MDA-MB-453, MDA-MB-231) and ovarian (A2780, A2780/CP, A2780/ADR, CH1, SKOV-3) carcinoma cell lines that differ by malignant progression. The expression of both antigens was detected by staining with the PBR-specific 8D7 and CA IX-specific M75 monoclonal antibodies and quantitated by flow cytometry. PBR was related to mitochondrial mass and CA IX to the cell density. Breast carcinoma cell lines showed higher relative fluorescence intensity of PBR expression than ovarian cell lines, with the exception of A2780/CP cisplatin-resistant subline that was comparable to highly invasive MDA-MB-231 breast line. Among the breast cell lines, PBR expression increased with their invasive potential. The ovarian cell lines showed greater variability in fluorescence intensities and the expression of PBR did not correlate with the amount of mitochondria. Mitochondrial PBR density disclosed significant difference between cisplatin-sensitive (low PBR density) and -resistant (high PBR density) ovarian cell lines. MTT test showed higher sensitivity of 2 breast cell lines MCF-7 and MDA-MB-231 (IC50 < 75 microM) to PBR ligand PK 11195 than all examined ovarian cell lines (IC50 > 90 microM, in chemo- and radio- resistant lines IC50 > 110 microM). Growth inhibitory effect of PK 11195 did not correlate with the amount of PBR and was mediated probably by another, PBRindependent mechanisms. The expression of CA IX was only marginal in majority of tested cell lines in subconfluent conditions and was inducible by high cell density. More than 5% of positive cells in sparse culture have been found in MDA-MB-231 and MDA-MB-453 breast cell lines while more than 15% of A2780/ADR adriamycin-resistant ovarian cells were positive for CA IX expression under the same conditions. Our data indicate that PBR expression in breast and ovarian carcinoma cell lines is not proportional to the amount of mitochondria and should be expressed relatively to the cell mitochondrial mass. This assessment allows establishing high PBR density as a measure of aggressiveness (invasion in breast and resistance in ovarian cancer). Observation of relatively high CA IX expression in A2780/ADR cells evokes the assumption that multidrug resistance might be connected with selection advantage towards CA IX expressing cells.