Bone Marrow Histopathological Changes in Neoplastic and Non-Neoplastic Diseases

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Abstract

In collaboration with Dr. Landgren, MOB, CCR, NCI group, we studied bone marrow changes in myeloma patients. In multiple myeloma (MM), increased angiogenesis of the bone marrow (BM) involves a complex interplay of proangiogenic and antiangiogenic molecules induced by plasma cells (PCs) within the BM microenvironment, with eventual balance tipped in favor of an angiogenic switch, as the disease transitions to MM from preceding monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). The dynamic nature of angiogenesis can be exploited by highly active anti-myeloma agents, such as thalidomide, its derivatives, the immunomodulatory drugs (IMiDs) and proteasome inhibitors, which not only interfere with tumor vessel growth, but also cause significant regression of the tumor vasculature both in vitro and in vivo. Therefore, there is great interest in studying the role of angiogenesis in MM to define therapeutic targets, prognostic and predictive biomarkers, as well as monitor disease activity longitudinally. In a prospective clinical trial, we evaluated 30 patients with MGUS, SMM and MM to comprehensively characterize the levels of angiogenesis from MGUS to frank malignancy. We compared angiogenesis using three parallel approaches: (i) BM microvessel density (MVD), (ii) dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) of lumbar vertebrae and (iii) serum panel of 17 angiogenic markers. Subjects (12 males and 18 females, with a median age 58.5 years) with Eastern Cooperative Oncology Group performance status of 02, diagnosed in accordance with standard criteria were included. Immunohistochemistry was performed on sections of B-Plus-fixed paraffin-embedded posterior iliac crest BM core biopsies using anti-CD34 (clone QBEnd 10, DAKO, Carpinteria, CA, USA) and anti-CD138 (clone B-A38, Cell Marque, Rocklin, CA, USA) antibodies on automated stainer according to manufacturer instructions. Stained slides were microscopically evaluated in a blinded manner and results expressed as mean vessel density based on CD34 endothelial immunostaining. From the dynamic imaging data obtained with DCE-MRI, curves were generated to mathematically fit a two compartment pharmacokinetic model. In this model, an injected contrast agent leaks into the extravascular, extracellular space at a rate predicted by the vessels permeability and surface area. Angiogenic vessels would be expected to be very leaky and thus have a rapid forward leakage rate and back flow. Based on this, the kinetic parameters Ktrans (transendothelial transport of gadolinium from vascular compartment to the tumor interstitium (wash in)) and kep (reverse transport of gadolinium back into the vascular space (wash out)) were calculated. Serum analysis included a panel of 17 vascular biomarkers (listed in Table 1B) using MILLIPLEX MAP human angiogenesis/growth factor magnetic bead panel (Millipore, Billerica, MA, USA). The results showed that while MVD was low in samples of BM obtained from patients with MGUS (median, 15.0), it increased in those with SMM (median, 19.4) and MM (median, 20.9). MVD linearly increased along the myeloma spectrum (MGUS<SMM<MM); P=0.008. Higher kep and MVD were seen in MM/SMM vs MGUS patients (kep median 7.1 vs 3.9; P=0.08; MVD median 20.30 vs 15.00; P=0.01). MVD and kep were moderately strongly correlated in all patients (r=0.59; P=0.001). Ktrans was weakly-to-moderately well correlated with MVD (r=0.43; P=0.03). Circulating levels of Ang2 (P=0.02), granulocyte-colony stimulating factor (G-CSF; P=0.06), follistatin (P=0.06), hepatocyte growth factor (HGF; P=0.01) and vascular endothelial growth factor-A (P=0.02) were elevated in MM/SMM patients in comparison to MGUS. The levels of HGF (r=0.45; P=0.02), Ang2 (r=0.37; P=0.06) and VEGFD (r=&#8722;0.36; P=0.07) were weakly-to-moderately well correlated with kep. However, there was no correlation between any of the tested angiogenic biomarkers and MVD. Looking forward, as more sophisticated tools become available to identify early myeloma, and as more refined therapeutic options with decreased toxicity are available, we may be seeing a trend for earlier intervention before symptoms develop. Establishing biomarkers associated with progression from MGUS and SMM to MM and that can be effectively targeted therapeutically remains an important challenge. It is possible that informative and accurate measurements of angiogenic biomarkers may prompt a clinician to initiate therapy sooner rather than later, or to decrease the surveillance interval of such patients with high-risk SMM or perhaps high-risk MGUS. Presence of a synchronous plasma cell myeloma and an additional leukemia or lymphoma in bone marrow specimens is a very rare finding. We described a patient with no previous significant history, who presented for baseline bone marrow evaluation as a potential donor for hematopoietic stem cell (HSC) transplant for a family member. Marrow aspirate smear revealed 7% myeloid precursors, 36% erythroid precursors, 52% lymphoid cells, and 5% plasma cells. The vast majority of lymphocytes exhibited a moderate amount of pale cytoplasm with fluffy projections, round-to-oval bland nuclei, and inconspicuous nucleoli, indicative of hairy cells. Plasma cells were predominantly larger than normal, some exhibiting prominent nucleoli. The normocellular marrow core biopsy showed extensive interstitial lymphoid infiltrate morphologically and immunophenotypically diagnostic of hairy cell leukemia (positive for pan B-cell markers, CD25, Annexin A1, and TRAP). There was also an increase in atypical plasma cells (10%-20%) with focal clustering/sheeting, most consistent with asymptomatic plasma cell myeloma because close monitoring detected no serum paraprotein or signs of end-organ damage. Dual immunohistochemical staining with cyclin D1 and CD138 demonstrated that both hairy cell leukemia and atypical plasma cells were positive for cyclin D1, with few normal plasma cells present.Cases of synchronous hairy cell leukemia and plasma cell myeloma have been only rarely reported. In this case, both the hairy cell leukemia cells and the atypical plasma cells were positive for cyclin D1. In addition, the patient has family history of a third type of hematopoietic malignancy with aberration in cyclin D1. A possible genetic component behind this observation warrants further investigation. In collaboration with Dr. Kochenderfer, NCI, we are participating in clinical trials using allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting B-cell/plasma cell antigens CD19 and BCMA. T cells for genetic modification with CD19 were obtained from each patient's alloHSCT donor. All patients had malignancy that persisted after alloHSCT and standard donor lymphocyte infusions (DLIs). Patients did not receive chemotherapy prior to the CAR T-cell infusions and were not lymphocyte depleted at the time of the infusions. The 10 treated patients received a single infusion of allogeneic anti-CD19-CAR T cells. Three patients had regressions of their malignancies. One patient with chronic lymphocytic leukemia (CLL) obtained an ongoing complete remission after treatment with allogeneic anti-CD19-CAR T cells, another CLL patient had tumor lysis syndrome as his leukemia dramatically regressed, and a patient with mantle cell lymphoma obtained an ongoing partial remission. None of the 10 patients developed graft-versus-host disease (GVHD). Toxicities included transient hypotension and fever. Cells containing the anti-CD19-CAR gene in the blood were detected in 8 of 10 patients. Bone marrows showed no presence of CD19 positive cells. These results showed for the first time that donor-derived allogeneic anti-CD19-CAR T cells can cause regression of B-cell malignancies. A study using anti-BCMA-CAR T-cells is ongoing.