Identification of proliferative and non-proliferative subpopulations of leukemic cells in CLL.

Pathogenesis in chronic lymphocytic leukemia (CLL) is strongly linked to the potential for leukemic cells to migrate to and proliferate within lymph-nodes. Previous in vivo studies suggest that all leukemic cells participate in cycles of migration and proliferation. In vitro studies, however, have shown heterogeneous migration patterns.To investigate tumor subpopulation kinetics, we performed in vivo isotope-labeling studies in ten patients with IgVH-mutated CLL (M-CLL). Using deuterium-labeled glucose, we investigated proliferation in sub-populations defined by CXCR4/CD5 and surface (sIgM) expression. Mathematical modeling was performed to test the likelihood that leukemic cells exist as distinct sub-populations or as a single population with the same proliferative capacity. Further labeling studies in two patients with M-CLL commencing idelalisib investigated the effect of B-cell receptor (BCR) antagonists on sub-population kinetics.Modeling revealed that data were more consistent with a model comprising distinct sub-populations (p = 0.008) with contrasting, characteristic kinetics. Following idelalisib therapy, similar labeling suppression across all sub-populations suggested that the most proliferative subset is the most sensitive to treatment. As the quiescent sub-population precedes treatment, selection likely explains the persistence of such residual non-proliferating populations during BCR-antagonist therapy. These findings have clinical implications for discontinuation of long-term BCR-antagonist treatment in selected patients.

Allogeneic Anti-BCMA CAR T Cells Are Superior to Multiple Myeloma-derived CAR T Cells in Preclinical Studies and May Be Combined with Gamma Secretase Inhibitors.

Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however, all patients ultimately still progress from their disease. Lack of CAR T-cell persistence, impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile, fitness, and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts, higher CD4/CD8 ratio, and expanded naïve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production, patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells, decreased central memory phenotype, and increased checkpoint inhibitory markers compared with HD-derived products, which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly, HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion, allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. Significance: Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately, patients still relapse. In this study, we propose to use T cells from HD volunteers, which have a stronger T-cell fitness, higher cancer killing capacity, and are ready to be administered when needed.

In vitro and in vivo evidence for uncoupling of B-cell receptor internalization and signaling in chronic lymphocytic leukemia.

B-cell receptor activation, occurring within lymph nodes, plays a key role in the pathogenesis of chronic lymphocytic leukemia and is linked to prognosis. As well as activation of downstream signaling, receptor ligation triggers internalization, transit to acidified endosomes and degradation of ligand-receptor complexes. Herein, we investigated the relationship between these two processes in normal and leukemic B cells. We found that leukemic B cells, particularly anergic cases lacking the capacity to initiate downstream signaling, internalize and accumulate ligand in acidified endosomes more efficiently than normal B cells. Furthermore, ligation of either surface CD79B, a B-cell receptor component required for downstream signaling, or surface Immunoglobulin M (IgM) by cognate agonistic antibody, showed that the two molecules internalize independently of each other in leukemic but not normal B cells. Since association with surface CD79B is required for surface retention of IgM, this suggests that uncoupling of B-cell receptor internalization from signaling may be due to the dissociation of these two molecules in leukemic cells. A comparison of lymph node with peripheral blood cells from chronic lymphocytic leukemia patients showed that, despite recent B-cell receptor activation, lymph node B cells expressed higher levels of surface IgM. This surprising finding suggests that the B-cell receptors of lymph node- and peripheral blood-derived leukemic cells might be functionally distinct. Finally, long-term therapy with the Bruton's tyrosine kinase inhibitors ibrutinib or acalabrutinib resulted in a switch to an anergic pattern of B-cell receptor function with reduced signaling capacity, surface IgM expression and more efficient internalization.

Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration.

Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4(dim)CD5(bright) phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of α4ß1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4(dim)CD5(bright) with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d(hi) CLL cells showed an enhanced capacity to activate T cells compared with CD49d(lo) subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4(+) T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells.

How I treat patients with relapsed chronic lymphocytic leukaemia.

As front line therapy has improved, the treatment of relapsed chronic lymphocytic leukaemia has become more difficult as the disease becomes resistant and the patient accumulates comorbidities. The outcome for those who relapse after immunochemotherapy with fludarabine, cyclophosphamide and rituximab is strongly influenced by the duration of initial response. Patients who relapse within the first year or with a TP53 abnormality have very high-risk disease and will not respond to chemotherapy. High dose glucocorticoid and alemtuzumab followed by an allogeneic stem cell transplant is probably the best approach for younger, fitter patients in this category. Those who relapse after 2-3 years without TP53 abnormality will probably respond to their initial therapy again. Relapse within 12-24 months carries an intermediate outlook. Additional options include bendamustine and rituximab, ofatumumab and lenalidomide. New therapies are on the horizon and patients should be discussed with a specialist centre and entered into a clinical trial whenever possible.

Pneumonitis post-haematopoeitic stem cell transplant - cytopathology clinches diagnosis.

BACKGROUND: Primary BK virus (BKV) infection is probably acquired by the respiratory route in childhood, and latent virus persists principally in the urinary tract. BKV reactivation is implicated in late onset haemorrhagic cystitis (HC) post Haematopoietic Stem Cell Transplant (HSCT). There is emerging evidence that BKV can cause life-threatening pneumonitis in immunocompromised individuals. OBJECTIVES: To describe the first known case of BKV pneumonitis in an adult HSCT recipient. STUDY DESIGN/RESULTS: A 19-year old male underwent an ABO-incompatible, volunteer unrelated donor allogeneic HSCT for high risk AML. The post-transplant period was complicated by moderate-severe cutaneous and gut acute graft-versus-host disease (aGVHD) and severe HC, attributable to BKV. Treatment encompassed intensification of immunosupression for aGVHD and weekly intravenous (IV) cidofovir (2.5mg/Kg) for BK viruria. He was readmitted with presumed septic shock and acute renal failure. After a transient improvement on broad spectrum antibacterials, he suffered significant respiratory deterioration. CT imaging revealed diffuse 'ground-glass' attenuation. Cytopathological assessment of a broncho-alveolar sample (BAL) was consistent with polyomavirus pneumonitis. No other cause was found to account for the respiratory deterioration. He did not respond to therapy and died of multi-organ failure. CONCLUSIONS: BKV is implicated in haemorrhagic cystitis in HSCT recipients but not routinely considered as a cause of pneumonitis. There are just 5 other cases in the literature, including 3 patients with AIDS. BKV should be considered as a possible cause of pneumonitis in HSCT recipients.

Life-threatening rectal bleeding due to cytomegalovirus colitis in a haemodialysis patient.

Cytomegalovirus (CMV) disease is a well-recognized complication in immunocompromised patients such as renal transplant recipients, occurring due to reactivation of latent infection or primary infection. It is, however, uncommon in immunocompetent patients. We report a haemodialysis patient who presented with pyrexia and life-threatening rectal bleeding due to CMV colitis, who had none of the classical risk factors for CMV disease, although was at risk. We review the literature surrounding CMV colitis in immunocompetent patients. This case highlights the importance of considering unusual causes for the common presentation of rectal bleeding in vulnerable patient populations.