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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

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

Supplementary Figure from 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

Analytical sensitivity for SARS-CoV-2 detection is a key performance metric for the evaluation of viral detection assays. We determined analytical limits of detection for seven SARS-CoV-2 assays using serial dilutions of pooled patient material quantified with droplet digital PCR. Limits of detection ranged from ≤10 to 74 copies/ml for commercial high-throughput laboratory analyzers (Roche Cobas, Abbott m2000, and Hologic Panther Fusion) and 167 to 511 copies/ml for sample-to-answer (DiaSorin Simplexa, GenMark ePlex) and point-of-care instruments (Abbott ID NOW)., Analytical sensitivity for SARS-CoV-2 detection is a key performance metric for the evaluation of viral detection assays. We determined analytical limits of detection for seven SARS-CoV-2 assays using serial dilutions of pooled patient material quantified with droplet digital PCR. Limits of detection ranged from ≤10 to 74 copies/ml for commercial high-throughput laboratory analyzers (Roche Cobas, Abbott m2000, and Hologic Panther Fusion) and 167 to 511 copies/ml for sample-to-answer (DiaSorin Simplexa, GenMark ePlex) and point-of-care instruments (Abbott ID NOW). The CDC assay yielded limits of detection ranging from 85 to 499 copies/ml, depending on the extraction method and thermocycler used. These results can help to inform the assay choice for testing approaches to manage the current COVID-19 outbreak.

Alternate strategies are needed for B-cell malignancy patients relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) 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 CARs, and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of CD72’s role in inhibiting B-cell receptor signaling, we found that pharmacologic SHIP1 inhibition increased CD72 surface density. We establish CD72-nanobody CAR T’s as a promising therapy for MLLr B-ALL.

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.

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.

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.

Purpose The 2015 American College of Medical Genetics and Genomics–Association for Molecular Pathology (ACMG–AMP) guidelines were a major step toward establishing a common framework for variant classification. In practice, however, several aspects of the guidelines lack specificity, are subject to varied interpretations, or fail to capture relevant aspects of clinical molecular genetics. A simple implementation of the guidelines in their current form is insufficient for consistent and comprehensive variant classification. Methods We undertook an iterative process of refining the ACMG–AMP guidelines. We used the guidelines to classify more than 40,000 clinically observed variants, assessed the outcome, and refined the classification criteria to capture exceptions and edge cases. During this process, the criteria evolved through eight major and minor revisions. Results Our implementation: (i) separated ambiguous ACMG–AMP criteria into a set of discrete but related rules with refined weights; (ii) grouped certain criteria to protect against the overcounting of conceptually related evidence; and (iii) replaced the “clinical criteria” style of the guidelines with additive, semiquantitative criteria. Conclusion Sherloc builds on the strong framework of 33 rules established by the ACMG–AMP guidelines and introduces 108 detailed refinements, which support a more consistent and transparent approach to variant classification.

Introduction: B-cell acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr; also known as KMT2Ar) have particularly dismal clinical outcomes. Although CAR T immunotherapies targeting CD19 have shown impressive responses treating MLLr B-ALL and other B cell malignancies, relapse, often with loss of relevant CD19 epitope, remains a major clinical concern. The mixed results of CD19 CAR T as a monotherapy underscores the need to pursue additional immunotherapy targets and novel therapeutic modalities for high-risk patients. Results and Methods: Data with existing CAR-T's suggest that increased target antigen density frequently correlates with increased tumor elimination. Therefore, we aimed to define the cell surface proteomic landscape of B-ALL to identify novel, MLLr-enriched candidates for targeted immunotherapy of this poor-prognosis subtype. As an initial screen, using N-glycoprotein capture and mass spectrometry, we quantified differentially abundant cell surface proteins in MLLr (n= 4) versus non-MLLr (n= 5) B-ALL cell lines (Figure 1). Label-free proteomics (n= 3 replicates) quantified >900 high-confidence membrane proteins (FDR=0.05). Principal component analysis identified unique cell surfaceome signatures between B-ALL subtypes, implying different surface landscapes associated with specific genetic alterations. The MLLr B-ALL "surfaceome" is notably characterized by increased expression of adhesion molecules not identified by RNA-sequencing alone. We focused on CD72 as a novel immunotherapy target given significant enrichment on MLLr B-ALL vs. other B-ALL subtypes, near equivalent antigen density to CD19, undetectable expression on HSPCs, T-cells, and other normal tissues, and reported widespread expression on other mature B-cell malignancies. Analysis of transcriptome and ChIP-seq data suggested increased CD72 expression in MLLr B-ALL is not regulated directly by the MLL-AF4 oncoprotein but instead a function of increased CD72 expression at pro-B-cell stage. Flow cytometry and immunohistochemistry on primary samples confirmed high expression of CD72 both in MLLr B-ALL as well as DLBCL. Recombinant CD72 ECD was panned against a fully in vitro nanobody yeast display library (McMahon et al., Nat Struct Mol Biol(2018)) resulting in isolation of multiple unique, highly-specific CD72 nanobody binders with KD's < 5nM. Nanobodies were incorporated into 2nd generation CAR constructs and transduced into normal donor CD8+ T-cells and assessed in vitro for tumor cell lysis, cytokine release, and exhaustion marker expression. Nanobody clone Nb.D4 outperformed others in lysis of B-ALL and DLBCL cells lines displaying a broad range of CD72 expression, had no activity versus CD72 negative cells, and showed similar efficacy to that found with a clinically-used CD19 CAR. To assess in vivo activity, CD72(Nb.D4) CAR-T's at 1:1 CD4:CD8 ratio were injected at an effector:tumor ratio of 5:1 into tumor-bearing NSG mice (luciferase-labeled SEM or MLLr PDX). In vivo results confirmed strong anti-tumor effect of CD72 nanobody CAR-T's, equivalent to clinical CD19 CAR, and significantly increased survival in mice (Figure 2). A CRISPR interference-generated antigen escape model of CD19 was also effectively eliminated by CD72 CAR-T's. We also introduce "antigen escape profiling", where cell surface proteomics of a CRISPRi CD72-knockdown model demonstrated extensive surfaceome rewiring with potential implications for leukemia cell trafficking and adhesion in the setting of acquired resistance. Given CD72's role as a BCR signaling inhibitory receptor, we are currently examining its influence on proximal B-cell receptor signaling and relationship to combination therapies affecting this pathway. Conclusions:By characterizing the surface proteomic landscape of B-ALL, we develop a resource for the research community and identify CD72 as a promising therapeutic target. We demonstrate that a novel, fully recombinant nanobody library can generate potent cellular therapies, which may be extended to other targets in the future. We anticipate that antigen escape profiling will prove broadly useful for anticipating mechanisms of resistance to novel immunotherapies. CD72 CAR-T's are a promising strategy across a range of B-cell malignancies, particularly those refractory to CD19 therapy. Disclosures Nix: UCSF: Patents & Royalties. Wiita:UCSF: Patents & Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Pathogenic variants in the mitofusin 2 gene (MFN2) are the most common cause of autosomal dominant Charcot-Marie-Tooth (CMT2) disease, which is typically characterized by axonal sensorimotor neuropathy. We report on a 7-month-old white female with hypotonia, motor delay, distal weakness, and motor/sensory axonal neuropathy in which next-generation sequencing analysis identified compound heterozygous pathogenic variants (c.2054_2069_1170del and c.392A>G) in MFN2. A review of the literature reveals that sporadic and familial cases of compound heterozygous or homozygous pathogenic MFN2 variants have been infrequently described, which indicates that MFN2 can also be inherited in a recessive manner. This case highlights several clinical findings not typically associated with MFN2 pathogenic variants, including young age of onset and rapidly progressing diaphragmatic paresis that necessitated tracheostomy and mechanical ventilation, and adds to the growing list of features identified in autosomal recessive MFN2-related CMT2. Our patient with MFN2-related CMT2 expands the clinical and mutational spectrum of individuals with autosomal recessive CMT2 and identifies a new clinical feature that warrants further observation. © 2016 Wiley Periodicals, Inc.

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.

Bone marrow can undergo necrosis for many different causes; malignant causes are reported to be more frequent.We undertook a 10-year retrospective review of all bone marrow biopsy specimens with bone marrow necrosis at our institution.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.The causes of bone marrow necrosis are diverse but should always prompt careful assessment for malignancy and infectious etiology.

The epidermis is a dynamic and continually renewing surface that provides and maintains a life-sustaining interface with the environment. The epidermal keratinocyte, the major cell type of the epidermis, undergoes a complex and carefully choreographed program of differentiation. This process requires a balance between keratinocyte proliferation, differentiation, and apoptosis. This overview will concentrate on cascades that regulate the balance between keratinocyte cell proliferation and survival, and apoptosis and cell differentiation, with a particular emphasis on the role of the mitogen-activated protein kinase cascades. A summary of the literature suggests that extracellular regulated kinases function to promote keratinocyte proliferation and survival, whereas p38 mitogen-activated protein kinase functions to promote differentiation and apoptosis.

Calcium is an important physiologic regulator of keratinocyte function that may regulate keratinocyte differentiation via modulation of protein kinase C (PKC) activity. PKCalpha and PKCdelta are two PKC isoforms that are expressed at high levels in keratinocytes. In the present study, we examine the effect of PKCdelta and PKCalpha on calcium-dependent keratinocyte differentiation as measured by effects on involucrin (hINV) gene expression. Our studies indicate that calcium increases hINV promoter activity and endogenous hINV gene expression. This response requires PKCdelta, as evidenced by the observation that treatment with dominant-negative PKCdelta inhibits calcium-dependent hINV promoter activity, whereas wild type PKCdelta increases activity. PKCalpha, in contrast, inhibits calcium-dependent hINV promoter activation, a finding that is consistent with the ability of dominant-negative PKCalpha and the PKCalpha inhibitor, Go6976, to increase hINV gene expression. The calcium-dependent regulatory response is mediated by an AP1 transcription factor-binding site located within the hINV promoter distal regulatory region that is also required for PKCdelta-dependent regulation; moreover, both calcium and PKCdelta produce similar, but not identical, changes in AP1 factor expression. A key question is whether calcium directly influences PKC isoform function. Our studies show that calcium does not regulate PKCalpha or delta levels or cause a marked redistribution to membranes. However, tyrosine phosphorylation of PKCdelta is markedly increased following calcium treatment. These findings suggest that PKCalpha and PKCdelta are required for, and modulate, calcium-dependent keratinocyte differentiation in opposing directions.

Typing of STR (short tandem repeat) alleles is used in a variety of applications in clinical molecular pathology, including evaluations for maternal cell contamination. Using a commercially available STR typing assay for maternal cell contamination performed in conjunction with prenatal diagnostic testing, we were posed with apparent nonmaternity when the two fetal samples did not demonstrate the expected maternal allele at one locus. By designing primers external to the region amplified by the primers from the commercial assay and by performing direct sequencing of the resulting amplicon, we were able to determine that a guanine to adenine sequence variation led to primer mismatch and allele dropout. This explained the apparent null allele shared between the maternal and fetal samples. Therefore, although rare, allele dropout must be considered whenever unexplained homozygosity at an STR locus is observed.

The epidermis is a dynamic renewing structure that provides life-sustaining protection from the environment. The major cell type of the epidermis, the epidermal keratinocyte, undergoes a carefully choreographed program of differentiation. Alteration of these events results in a variety of debilitating and life-threatening diseases. Understanding how this process is regulated is an important current goal in biology. In this review, we summarize the literature regarding regulation of involucrin, an important marker gene that serves as a model for understanding the mechanisms that regulate the differentiation process. Current knowledge describing the role of transcription factors and signaling cascades in regulating involucrin gene expression are presented. These studies describe a signaling cascade that includes the novel protein kinase C isoforms, Ras, MEKK1, MEK3, and a p38delta-extracellular signal regulated kinase 1/2 complex. This cascade regulates activator protein one, Sp1, and CCATT/enhancer-binding protein transcription factor DNA binding to two discrete involucrin promoter regions, the distal- and proximal-regulatory regions, to regulate involucrin gene expression.

The novel protein kinase C (nPKC) isoforms are important regulators of human involucrin (hINV) gene expression during keratinocyte differentiation (Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601-1607). Although the regulatory mechanism involves mitogen-activated protein kinase (MAPK) activation, the role of individual MAPK isoforms has not been elucidated. We therefore examined the effects of individual nPKCs on MAPK activation. We observe unique changes whereby nPKC expression simultaneously increases p38 activity and decreases ERK1 and ERK2 activity. Although p38 alpha, p38 beta, and p38 delta are expressed in keratinocytes, only a single isoform, p38 delta, accounts for the increased p38 activity. Parallel studies indicate that this isoform is also activated by treatment with the keratinocyte regulatory agents, 12-O-tetradecanoylphorbol-13-acetate, calcium, and okadaic acid. These changes in MAPK activity are associated with increased C/EBP alpha transcription factor expression and DNA binding to the hINV promoter and increased hINV gene expression. Expression of PKC delta, PKC epsilon, or PKC eta causes a 10-fold increase in hINV promoter activity, whereas C/EBP alpha expression produces a 25-fold increase. However, simultaneous expression of both proteins causes a synergistic 100-fold increase in promoter activity. These responses are eliminated by the dominant-negative C/EBP isoform, GADD153, and are also inhibited by dominant-negative forms of Ras, MEKK1, MEK3, and p38. These results suggest that the nPKC isoforms produce a unique shift in MAPK activity via a Ras, MEKK1, MEK3 pathway, to increase p38 delta and inhibit ERK1/2 and ultimately increase C/EBP alpha binding to the hINV promoter and hINV gene expression.

Introduction Hereditary hemochromatosis (HH) is a genetic form of iron overload. In cases of excessive iron deposition, serious clinical manifestations may occur, such as liver damage, cardiomyopathy, diabetes, and arthritis. First described in 1996, the HFE gene leads to autosomal recessive HH with reduced penetrance. In other words, two mutations in the HFE gene need to be present in a patient in order to develop symptoms of HFE-related HH, but not all patients with two mutations are affected. In the last 15 years, 4 additional genes were discovered that cause HH: HAMP (hepcidin), HFE2 (hemojuvelin), SLC40A1 (ferroportin), and TFR2 (transferrin receptor 2). HAMP, HFE2 and TFR2 mutations are inherited in a recessive pattern, whereas SLC40A1 mutations are inherited in a dominant pattern. HAMP and HFE2 mutations cause a severe, early-onset form of HH. There is some evidence that sequence changes in HAMP, HFE2 and TFR2 may interact with homozygous HFE mutations, causing a more severe phenotype. Current HH testing guidelines only exist for the most common HFE mutations (C282Y and H63D), with no specific recommendations regarding full gene sequencing for any of the HH genes. Recent research suggests that sequential sequencing may be beneficial in patients who test negative for the most common HFE mutations, exhibit a more severe or early-onset phenotype compared to what is normally seen in HFE-related HH, and/or are of non-Caucasian ethnicity. Next Generation Sequencing (NGS) is a new high-throughput sequencing technology that allows testing of multiple genes concurrently and can detect rare and novel HH-causing mutations that are not typically assayed using targeted methods. However, sequencing can also identify sequence changes known as variants of uncertain significance (VUS) - changes that have not yet been characterized as disease-causing or benign. This abstract summarizes the results of clinical NGS for the five HH-related genes, and shows preliminary evidence as to its’ increased diagnostic yield for HH diagnosis. Methods Patients were referred for clinical full gene sequencing of HFE, HAMP, HFE2, SLC40A1, and/or TFR2 using Next Generation Sequencing (Illumina MiSeq). Results from patients with a clinical indication of iron overload or HH who were tested from 9/2013 - 7/2014 were reviewed. The diagnostic yield of sequencing for all five HH genes was determined. Patients who only had sequencing for a subset of the five genes were analyzed separately. Patients who had testing for a familial mutation were excluded from the review. Results In total, 56 patients underwent HH-related NGS. Thirty-five (62.5%) were males and 21 (37.5%) were females. Ages ranged from 3-77yrs (avg. 40.9yrs). Fifty-one percent were Caucasian, 9% Hispanic, 4% African American, 16% Asian, and 20% not specified. Forty-one patients were tested for all five genes. HH-causing mutations were found in 9 patients: 5 (12.2%) were c.187C>G (p.H63D) HFE homozygous, 1 (2.4%) was c.845G>A (p.C282Y) HFE homozygous, and 3 (7.3%) had mutations in non-HFE genes: SLC40A1 c.430A>T (p.N144Y) heterozygous, SLC40A1 c.533G>A (p.R178Q) heterozygous, and HFE2 c.959G>T (p.R178Q) homozygous. Nine patients (22%) were heterozygous carriers of an HFE mutation. One or more VUSes were found in 5 patients (12.2%). In 18 patients (43.9%), no pathogenic mutations or VUSes were found. There were 15 additional patients who only had sequencing of 1-3 of the available genes. Results for those patients consisted of 1 H63D HFE homozygote, 3 HFE heterozygotes (2 H63D and 1 C282Y) and 1 VUS. Conclusions The new sequencing technology of NGS makes it possible to test multiple genes at the same time. In this sample, sequencing of HFE, HAMP, HFE2, SLC40A1, and TFR2 genes resulted in an additional diagnostic yield compared to HFE C282Y and H63D testing alone. In patients who have a genetic explanation for their HH, management can be personalized based on genotype-phenotype correlation (e.g. N144Y SLC40A1 mutations may lead to reduced phlebotomy tolerance) and at-risk family members can be screened. In addition, all patients in this sample with non-HFE positive results were reportedly Caucasian, highlighting the benefit of sequencing regardless of ethnic background. This preliminary study is an important step toward gaining a better understanding of the genetics of HH. Ultimately, NGS data may make it possible to update current clinical guidelines for HH. Disclosures Rabideau: Invitae: Employment, Equity Ownership.

Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential display–PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA,tazarotene-induced gene 3(TIG3;Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene,H-rev107. Tazarotene treatment increasesTIG3expression in primary human keratinocytes andin vivoin psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3 may be a growth regulator that mediates some of the growth suppressive effects of retinoids.

Rat liver fatty acid binding protein (FABP) is believed relevant to understanding of homeostasis in lipid metabolism and lipid related diseases. Relatively little is known about endocrine control of FABP production. Thus, we examined endocrine effects on its hepatic content.Hypophysectomy of 300-325 g males caused statistically significant drops of FABP levels averaging 62.2% and 67.0%, expressed g/liver or 100 g/body weight, 30-52 days after surgery. Cortisol administration (3.8 mg/kg, daily, 32-36 days) did not significantly alter this effect of hypophysectomy. Recombinant human growth hormone (GH, 2.0 U/kg, b.i.d, 17-20 days) greatly decreased the effect of hypophysectomy on FABP but had no effect in intact males. Supporting the control of FABP content by GH, FABP levels decreased significantly in 12-13 and 16-22 month old males, but not in growing, 4-6 or 10-11 month old males. FABP levels in 12-13.5 month old females also dropped significantly compared to 4-6 month old females.The importance of the data to metabolism, growth, and aging is discussed.

SummaryStudying 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.

TIG3 is a recently discovered class II tumor suppressor protein, originally isolated from retinoid-treated cultured epidermal keratinocytes, that suppresses the proliferation of a variety of epithelial cell types. In the present study, we examine the ability of this protein to reduce CHO, T 47 D and HaCaT cell proliferation, and the role of the carboxy-terminal hydrophobic domain in this regulation. Vector-mediated expression of the full length TIG3 protein, TIG3 1-164 , results in a 50-70% reduction colony formation efficiency. Expression of a truncated mutant, TIG3 1-134 , that lacks the putative carboxy-terminal membrane-anchoring domain, results in a partial loss of ability to suppress colony formation. The fact that the truncated protein remains partially active suggests that both the amino- and carboxy-terminal regions of TIG3 are required for optimal growth suppression. The full-length protein is distributed in a perinuclear location, and is not present in the nucleus. TIG3 1-134 , in contrast, is distributed in the cytoplasm. Thus, a change in location is associated with the partial loss of activity. We also monitored the distribution of green fluorescent protein (GFP)-TIG3 fusion proteins. GFP-TIG3 1-164 was localized in a pattern similar to that observed for TIG3 1-164 , while GFP-TIG3 1-134 displayed a distribution pattern similar to GFP. This suggests that the C-terminal hydrophobic domain has an important role in determining the intracellular localization of TIG3. In addition, GFP-TIG3 1-164 retains the ability to inhibit cell function, while GFP-TIG3 1-134 is inactive.