Bone Marrow Histopathological Changes in Neoplastic and Non-Neoplastic Diseases

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Hairy cell leukemia (HCL) is a B-cell lymphoproliferative disorder characterized by distinct immunophenotype (positive for CD19, CD20, PAX5, CD22, CD11c, CD25, CD103, CD123, and CD200). Immunophenotypic analysis by flow cytometry (FC) is considered the gold standard for diagnosis of HCL. However, both FC and immunohistochemistry (IHC) can be used to determine these markers. Although both trephine bone marrow biopsy and aspirate are vital for assessment of the extent of bone marrow infiltration, in some cases a cellular aspirate cannot be obtained because of extensive fibrosis (ie, dry tap). In such cases, IHC stains are crucial for assessment of bone marrow leukemic involvement. To date, IHC detection of HCL in bone marrow sections has been limited to overt disease and could not be used reliably in patients with very low-level HCL involvement. Monoclonal anti-CD103 antibody has been previously established as a specific HCL marker that reliably distinguishes HCL from most other B-cell lymphomas. However, CD103 is also expressed in dendritic cells and in background T cells in the bone marrow, which makes CD103 difficult to interpret by light microscopy. Tartrate-resistant alkaline phosphatase (TRAP) is another IHC marker for HCL that lacks adequate specificity. In trephine biopsies, TRAP stains nonneoplastic cells such as macrophages and osteoclasts. Annexin A1, BRAF V600E, and DBA.44 are other IHC stains used in the evaluation of HCL. However, Annexin A1 is strongly positive in myeloid cells; this makes morphological interpretation challenging, especially in the setting of low disease burden. BRAF V600E stain sometimes has low reliability in decalcified bone marrow core biopsy specimens. Although neoplastic cells can be differentiated using all of these single IHC stains based on morphologic features, interpretation can be challenging in the setting of low disease burden. We have developed dual IHC staining with a specific B-cell marker (PAX5; nuclear stain) along with either CD103 or TRAP (membranous stain) to help overcome these limitations, especially in patients with low-level HCL involvement. We evaluated the efficacy of these dual IHC staining assays (PAX5/CD103 and PAX5/TRAP) in a large cohort (148 patients) evaluated for HCL involvement in marrow core biopsies and compared the results with concurrently collected FC data. We evaluated efficacy of two dual immunohistochemistry (IHC) staining assays in assessing hairy cell leukemia (HCL) involvement in core biopsies and compared the results with concurrently collected flow cytometric data. Overall, 148 patients with HCL (123 male, 25 female; mean age: 59.8 years; range: 25-81 years) had multiparameter flow cytometry performed using CD19, CD20, CD22, CD11c, CD25, CD103, CD123, surface light chains, CD5, and CD23. In parallel, bone marrow IHC was done using PAX5/CD103 and PAX5/tartrate-resistant alkaline phosphatase (TRAP) dual IHC stains. Overall sensitivity of dual IHC stains was 81.4%, positive predictive value was 100%, and negative predictive value was 81.7%. All IHC-positive cases concurred with flow cytometry data, even when HCL burden was extremely low in the flow cytometry specimens (as low as 0.02% of all lymphoid cells). The results revealed that dual IHC staining is a sensitive tool in detecting HCL, even in cases with minimal disease involvement. Therefore, dual IHC staining could be used as a reliable surrogate in the clinical practice in the event of non-availability of aspirate specimens for FC analysis. In a separate study, we searched for molecular underpinnings of clinical disparity patterns in African American vs. Caucasian American multiple myeloma patients. Caucasian Americans (CA) compared with African Americans (AA) have a twofold increased incidence of multiple myeloma (MM) and have an earlier age of diagnosis. However, there is sparse information regarding underlying biological differences across racial/ethnic groups. We characterized genetic alterations using a targeted next-generation sequencing assay called myTYPE, developed at MSKCC, allowing capture of somatic mutations, IgH translocations, gains/losses, and hyperdiploidy. Samples were obtained from the NIH Plasma Cell Dyscrasia Racial Disparity Cohort. In total, 68 patient samples were successfully sequenced and manually curated based on well-established databases. Of the 68 patient samples (47 CA, 21 AA), 84% had at least one type of genomic alteration. Importantly, the IgH translocation, t(11;14), was observed more frequently in the AA group (0 vs. 29%, p = 0.001). Known oncogenic somatic non-synonymous mutations were found in 18 genes and indels in 2 genes. KRAS mutations were the most common mutation found in 16% of patients followed by NRAS and BRAF mutations. TP53 somatic mutations appeared to be more common in CA but lacked significance. This proof-of-principle study indicated the presence of varying underlying tumor biology between racial groups and supported the need of future prospective trials to capture these molecular characteristics. A separate study investigated clinical and biological implications of target occupancy in chronic lymphocytic leukemia (CLL) treated with the BTK inhibitor acalabrutinib. Inhibition of the B-cell receptor pathway, and specifically of Bruton tyrosine kinase (BTK), is a leading therapeutic strategy in B-cell malignancies, including CLL. Target occupancy is a measure of covalent binding to BTK and has been applied as a pharmacodynamic parameter in clinical studies of BTK inhibitors. However, the kinetics of de novo BTK synthesis, which determines occupancy, and the relationship between occupancy, pathway inhibition and clinical outcomes remain undefined. The randomized phase 2 study led by Dr. Wiestners group investigated the safety, efficacy, and pharmacodynamics of a selective BTK inhibitor acalabrutinib at 100 mg twice daily (BID) or 200 mg once daily (QD) in 48 patients with relapsed/refractory or high-risk treatment-nave CLL. Acalabrutinib was well tolerated and yielded an overall response rate (ORR) of partial response or better of 95.8% (95% confidence interval CI, 78.9-99.9) and an estimated progression-free survival (PFS) rate at 24 months of 91.5% (95% CI, 70.0-97.8) with BID dosing and an ORR of 79.2% (95% CI, 57.9-92.9) and an estimated PFS rate at 24 months of 87.2% (95% CI, 57.2-96.7) with QD dosing. BTK resynthesis was faster in patients with CLL than in healthy volunteers. BID dosing maintained higher BTK occupancy and achieved more potent pathway inhibition compared with QD dosing. Small increments in occupancy attained by BID dosing relative to QD dosing compounded over time to augment downstream biological effects. The impact of BTK occupancy on long-term clinical outcomes remains to be determined.