Critical evaluation of the utility of pre- and post-therapy immunophenotypes in assessment of measurable residual disease in B-ALL.

Measurable residual disease (MRD) is an important parameter to predict outcome in B-cell acute lymphoblastic leukemia (B-ALL). Two different approaches have been used for the assessment of MRD by multiparametric flow cytometry that include the "Leukemia Associated Aberrant Immunophenotype (LAIP)" and "Difference from Normal (DFN)" approach. In this retrospective study, we analyzed 539 samples obtained from 281 patients of which 258 were paired samples and the remaining 23 samples were from post-induction time point only, to explore the utility of baseline immunophenotype (IPT) for MRD assessment. Single-tube 10-color panel was used both at diagnosis and MRD time points. Out of 281 patients, 31.67% (n = 89) were positive and 68.32% (n = 192) were negative for MRD. Among 258 paired diagnostic and follow-up samples, baseline IPT was required in only 9.31% (24/258) cases which included cases with hematogone pattern and isolated dim to negative CD10 expression patterns. Comparison of baseline IPT with post-induction MRD positive samples showed a change in expression of at least one antigen in 94.04% cases. Although the immunophenotypic change in expression of various antigens is frequent in post-induction samples of B-ALL, it does not adversely impact the MRD assessment. In conclusion, the baseline IPT is required in less than 10% of B-ALL, specifically those with hematogone pattern and/or dim to negative expression of CD10. Hence, a combination of DFN and LAIP approach is recommended for reliable MRD assessment.

Cytologic, flow cytometry, and molecular assessment of lymphoid infiltrate in fine-needle cytology samples of Hashimoto thyroiditis.

BACKGROUND: The thyroidal lymphoid infiltrate (TLI) in Hashimoto thyroiditis (HT) represents the substrate from which thyroid lymphoma may arise. The objective of the current study was to classify the TLI in HT by comparing the cytologic features with flow cytometry (FC) data and evaluating the kappa/lambda light chain ratio and its molecular assessment. METHODS: Fine-needle aspiration cytology (FNAC) was performed in 34 patients with HT with nodular or diffuse palpable enlargement of the gland. Two or 3 passes were performed to prepare traditional smears, FC, and immunophenotyping, and RNAlater suspensions for molecular assessment. FC was performed using the following antibodies: CD3, CD5, CD4, CD8, CD10, CD19, and kappa and lambda light chains. In 4 cases, high molecular weight DNA was extracted and used for polymerase chain reaction (PCR) to amplify the variable diversity joining region of the heavy chain immunoglobulin (Ig) genes (IgH). Statistical analysis was performed to evaluate possible associations between clinical ultrasound presentation, cytologic pattern, and TLI phenotype. Light chain expression was evaluated as the percentage of the expressing cells (20%) and as the kappa/lambda ratio. RESULTS: Smears were classified as "lymphocytic," "lymph node-like," or "mixed." FC demonstrated T cells (CD3 positive [+], CD5+) in all cases, and T cells and B cell (CD19+, CD10+/-) lymphocytes in 22 cases. Light chains were expressed in 30 cases (in <20% of the gated cells in 13 cases and in >20% of the gated cells in 17 cases). Five cases demonstrated small kappa/lambda ratio imbalances and PCR analysis demonstrated diffuse bands in the gel and Gaussian curves at the heteroduplex. Statistical analysis indicated significant associations between the "lymphocytic" pattern and T-cell phenotype and between the "lymph node-like" pattern and B-cell phenotype. A significant association also was observed between light chain restriction and low light chain expression (P < .005). CONCLUSIONS: The cytologic pattern of TLI in HT is quite representative of the clinical presentation and phenotypic cell type. Small light chain imbalances are not sustained by heavy chain Ig gene (IgH) rearrangements. FNA coupled with FC may contribute to making the distinction between florid TLI and non-Hodgkin lymphoma.

Primary cutaneous follicular lymphoma: an assessment of clinical, histopathologic, immunophenotypic, and molecular features.

PURPOSE: Unlike nodal follicular lymphoma (NFL), Primary cutaneous follicular lymphomas (PCFLs) rarely express Bcl-2 protein or t(14;18)(q32;q21) (Bcl-2/IgH). The aim of this study was to further characterize PCFL in a large series from North America. PATIENTS AND METHODS: Clinical data and archival formalin-fixed, paraffin-embedded tissue were obtained from 32 patients. PCFL was defined as follicular lymphoma limited to the skin at the time of diagnosis and within the first 6 months after diagnosis. Specimens were analyzed for the expression of CD3, CD10, CD20, Bcl-2, and Bcl-6 proteins by immunohistochemistry as well as for the presence of t(14;18)(q32;q21) by polymerase chain reaction. RESULTS: The male-to-female ratio was 1.5:1, with a median age of 60 years. Twenty-four patients had lesions on the head and neck, five had lesions on the trunk, and three had lesions on both head and trunk. Follow-up data were available in all cases, with a mean length of 35.8 months. The majority of the patients were treated with radiation therapy. All patients were alive at last follow-up except one. Recurrence was noted in seven patients (22%), after a mean disease-free survival time of 17.7 months. CD10 and Bcl-6 expression were seen in 29 (91%) of 32 and 31 (97%) of 32 cases, respectively. Bcl-2 expression was noted in 13 (41%) of 32 cases. PCR results for t(14;18)(q32;q21) were positive in 11 (34%) of 32 patients and showed correlation with Bcl-2 protein expression. The sequencing of the t(14;18)(q32;q21) amplicons confirmed unique breakpoints in each of the seven tested cases. Comparison between the Bcl-2 and/or t(14;18)(q32;q21)-positive and t(14;18)(q32;q21)-negative cases revealed no significant difference in age, site, clinical course, or outcome. CONCLUSION: We demonstrated Bcl-2 protein expression and t(14;18)(q32;q21) in a significant minority of cases, suggesting a relationship with NFL. It remains to be seen whether, on longer follow-up, there is any clinical difference in cases with and without t(14;18)(q32;q21).

Assessment of CD10 in the diagnosis of small B-cell lymphomas: a multiparameter flow cytometric study.

We evaluated the usefulness of multiparameter flow cytometry with cluster analysis in the diagnosis of a series of 100 well-characterized small B-cell lymphomas (SBCLs). The histologic diagnoses in the 100 cases were follicular lymphoma (FL) in 58, marginal zone lymphoma (MZL) in 17, small lymphocytic lymphoma in 15, and mantle cell lymphoma (MCL) in 10. Of the 58 FLs, 57 were CD10 positive (98% sensitivity). The 1 negative case was unusual in that it occurred in the small intestine. However; architectural, cytologic, and immunohistochemicalfeatures were diagnostic of FL. Of 42 other SBCLs, 2 were CD10+ (95% specificity); 1 was a CD5+/cyclin D1 + MCL, and the other was an extranodal MZL. We found that assessment of CD10 expression using multiparameter flow cytometry with cluster analysis is highly sensitive and specific for the diagnosis of FL, validating its usefulness in situations in which adequate tissue is not available for definitive histologic diagnosis.

Monoclonal antibodies recognizing CD5, CD10 and CD23 in formalin-fixed, paraffin-embedded tissue: production and assessment of their value in the diagnosis of small B-cell lymphoma.

AIMS: Assessment of the expression of antigens CD5, CD10 and CD23 can be of value in the differential diagnosis of small B-cell lymphoma. Correct subclassification is important since optimal treatment regimes differ between the subtypes. The aim of this study was to generate monoclonal antibodies recognizing these antigens in paraffin-embedded tissue and to assess their efficacy using a panel of cases of small B-cell lymphoma of various subtypes. METHODS AND RESULTS: For each antibody synthetic recombinant protein and conventional murine hybridoma technology was employed. Monoclonal antibodies effective in formalin-fixed, paraffin-embedded tissue were successfully generated, designated NCL-CD5-4C7, NCL-CD10-270 and NCL-CD23-1B12, respectively. A series of 58 cases of small B-cell lymphoma including examples of each subtype (lymphocytic, follicle centre cell, mantle cell, marginal zone and lymphoplasmacytoid) was assembled and immunostaining for the respective antigens carried out using the monoclonal antibodies produced. Our results indicate that the antibodies are specific for their respective antigens and give the predicted phenotypic profile in the small B-cell lymphoma subtypes. CONCLUSIONS: These novel monoclonal antibodies may be of value in routine diagnostic practice.