Surface Proteomics Reveals CD72 as a Target for In Vitro-Evolved Nanobody-Based CAR-T Cells in KMT2A/MLL1-Rearranged B-ALL.

Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully in vitro system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL. SIGNIFICANCE: Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully in vitro nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development.This article is highlighted in the In This Issue feature, p. 1861.

How I investigate bone marrow necrosis.

Hematopathologists encounter bone marrow biopsy specimens with marrow necrosis relatively infrequently; when necrosis is seen, determining the clinical significance can be challenging. While bone marrow necrosis is not uncommon in site-directed biopsy specimens or autopsy material, substantial necrosis is much less common in nondirected bone marrow biopsy specimens. Retrospective review showed the prevalence of bone marrow necrosis to vary between 0.3% and 2% antemortem, depending on the patient population. Numerous causes of bone marrow necrosis have been identified, including malignancy, radiation/chemotherapy, medication, infection, autoimmune disease, disseminated intravascular coagulation, antiphospholipid syndrome and other thrombotic disorders, granulocyte-colony stimulating factor (G-CSF) exposure, and hemoglobinopathies. Clinical findings associated with bone marrow necrosis include bone pain and fever, cytopenias, elevated LDH and ferritin, and leukoerythroblastosis. Rarely, such as in fat embolization syndrome (FES), bone marrow necrosis can be associated with thrombotic microangiopathy, neurologic dysfunction, and multiorgan failure. A thorough review of the patient's clinical record (including medical history, clinical presentation, and other laboratory findings), a thorough morphologic review of the bone marrow with appropriate ancillary stains, and an appreciation of the causes of bone marrow necrosis in different patient populations are required to determine the underlying cause of bone marrow necrosis. The purpose of this review is to present a strategy for evaluation of bone marrow necrosis found in an antemortem biopsy specimen.

BCL6 expression correlates with the t(1;19) translocation in B-lymphoblastic leukemia.

OBJECTIVES: Study to date suggests that BCL6 protein expression in B-cell neoplasia predominates in germinal center-derived tumors, but less is known regarding its expression in B-lymphoblastic leukemia. Therefore, we designed a comprehensive study of BCL6 expression in B-lymphoblastic leukemia. METHODS: BCL6, LMO, and HGAL protein expression in B-lymphoblastic leukemia was investigated using immunohistochemical staining of paraffin-embedded bone marrow specimens. Cryptic TCF3(E2A)-PBX1 rearrangements were investigated using interphase fluorescence in situ hybridization. RESULTS: Six (12%) of 52 B-lymphoblastic leukemias demonstrated BCL6 protein expression, with B-cell lymphoblastic leukemias containing a t(1;19) translocation demonstrating the strongest staining (three of three). Additional t(1;19) cases beyond the screening study showed similar results. Public microarray expression database mining showed that BCL6 messenger RNA expression levels in B-lymphoblastic leukemia correlated with the protein expression findings. Finally, other markers of B-cell development correlated with BCL6 expression in t(1;19) B-lymphoblastic leukemia cases, with LMO2 and HGAL proteins expressed in six (67%) of nine and eight (89%) of nine cases, respectively. CONCLUSIONS: BCL6 expression is present in a subset of B-lymphoblastic leukemias, especially in cases containing the 1;19 translocation. Investigation for TCF3(E2A)-PBX1 rearrangements may be useful in BCL6-positive B-lymphoblastic leukemia.

Self-enforcing feedback activation between BCL6 and pre-B cell receptor signaling defines a distinct subtype of acute lymphoblastic leukemia.

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.

Bone marrow necrosis: ten-year retrospective review of bone marrow biopsy specimens.

OBJECTIVES: Bone marrow can undergo necrosis for many different causes; malignant causes are reported to be more frequent. METHODS: We undertook a 10-year retrospective review of all bone marrow biopsy specimens with bone marrow necrosis at our institution. RESULTS: Identified cases represented approximately 0.3% of our bone marrow cases. Most identified bone marrow cases with necrosis were involved by metastatic tumor or hematolymphoid malignancy (90% of total) in relatively equal proportions. In those cases of bone marrow necrosis with hematolymphoid malignancy, lymphoid disease predominated and the necrosis was often seen in the setting of chemotherapy. In metastatic tumor cases, necrosis seemed to enrich in prostate adenocarcinoma and Ewing sarcoma/primitive neuroectodermal tumor; neuroblastoma showed much less necrosis. Ten percent of patients with bone marrow necrosis had no underlying malignancy, and the associated causes varied. CONCLUSIONS: The causes of bone marrow necrosis are diverse but should always prompt careful assessment for malignancy and infectious etiology.

A novel tumor suppressor protein promotes keratinocyte terminal differentiation via activation of type I transglutaminase.

Tazarotene-induced protein 3 (TIG3) is a recently discovered regulatory protein that is expressed in the suprabasal epidermis. In the present study, we show that TIG3 regulates keratinocyte viability and proliferation. TIG3-dependent reduction in keratinocyte viability is accompanied by a substantial increase in the number of sub-G1 cells, nuclear shrinkage, and increased formation of cornified envelope-like structures. TIG3 localizes to the membrane fraction, and TIG3-dependent differentiation is associated with increased type I transglutaminase activity. Microscopic localization and isopeptide cross-linking studies suggest that TIG3 and type I transglutaminase co-localize in membranes. Markers of apoptosis, including caspases and poly(ADP-ribose) polymerase, are not activated by TIG3, and caspase inhibitors do not stop the TIG3-dependent reduction in cell viability. Truncation of the carboxyl-terminal membrane-anchoring domain results in a complete loss of TIG3 activity. The morphology of the TIG3-positive cells and the effects on cornified envelope formation suggest that TIG3 is an activator of terminal keratinocyte differentiation. Our studies suggest that TIG3 facilitates the terminal stages in keratinocyte differentiation via activation of type I transglutaminase.