T-cell-based immunotherapy revolutionized the treatment of immune suppressive cancers. Blocking T cell inhibitory receptors such as programmed death 1 (PD1) or their ligands (like PD-L1) enhances antitumor T cell responses. However, many cancers employ multiple methods of immune suppression, including the production of potent regulatory cytokines like Interleukin-10 (IL-10). B cell Chronic Lymphocytic Leukemia (CLL) is one such B cell malignancy that elicits widespread immune dysfunction including numerous functional deficits in T cells. Human and mouse CLL cells (Eμ-Tcl1) produce IL-10 and PD-L1, which dampen antitumor immunity. Since immune checkpoint blockade experienced limited success in CLL, we investigated IL-10 suppression as a novel combination therapy. Exogenous and CLL-derived IL-10 decreased human and mouse T cell functionality in vitro. Further, CLL-derived IL-10 can be blocked with antibodies or can be reduced with small molecule inhibitors to improve CD8+ T cell functionality in vivo. CLL IL-10 production depends on the transcription factor Sp1. Hence, we utilized a novel analogue of the Sp1 inhibitor mithramycin to reduce CLL IL-10 secretion. IL-10 inhibition improved responses to anti-PD-L1 therapy, with increased survival and decreased CLL burden compared to anti-PD-L1 alone. This method improved CD8+ T cell proliferation, effector-memory cell frequency, and interferon-γ production. CD8+ T cells were also more abundant, with fewer exhausted T cells even at very advanced stages of disease. Current cancer therapies do not target IL-10 and our studies provide evidence for targeting IL-10 to increase the efficacy of T cell immunotherapies in human CLL, and possibly other cancers with T cell suppression.

T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1. However, most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated analogue of the Sp1 inhibitor mithramycin (MTMox32E) to suppress CLL IL-10. MTMox32E treatment inhibited mouse and human CLL IL-10 production and maintained T-cell effector function in vitro. In the Eμ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden and increased CD8+ T-cell proliferation, effector and memory cell prevalence, and interferon-γ production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared to anti-PD-L1 alone. Combination therapy also produced more interferon-γ+, cytotoxic effector KLRG1+, and memory CD8+ T-cells, and fewer exhausted T-cells. Since current therapies for CLL do not target IL-10, this provides a novel strategy to improve immunotherapies.

Purpose: Acute myeloid leukemia (AML) is characterized by multiple somatically acquired mutations that affect genes of different functional categories. It has been well established in myelodysplastic syndrome (MDS) that the cumulative number of somatic mutations has an impact on overall survival. However, no such data exist for AML. In this study, we sought to determine the number of clinically significant somatic mutations for each cytogenetically defined risk group of AML and to determine whether this had an impact on overall survival (OS). Methods: In this retrospective, single-center study, all adult patients diagnosed with AML from August 2016–December 2017 were reviewed. Baseline characteristics, somatic mutations in the diagnostic bone marrow as detected by Next Generation Sequencing (NGS), and survival outcomes were analyzed. NGS panel was done in-house and could identify 94 genes. Patients were divided into favorable, intermediate, and poor risk groups based on cytogenetics, and molecular abnormalities using NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for AML, version 1.2018. Kaplan-Meier plots and Cox regression analyses were utilized. Results: A total of 105 AML patients were included; baseline characteristics and frequency of identified clinically significant (CS) mutations are described in the presentation. The FLT3 mutation occurred in the highest frequency (22%) followed by DNMT3A & ASXL1 (15%). 17 (16%) patients were favorable risk, 33 (31%) intermediate risk, and 55 (52%) were poor risk. 67.6% of patients were male, and the median age was 64 (20–79) years. There was a difference in the number of CS mutations between the intermediate risk group and favorable risk group (P=.007), but not between the favorable risk and poor risk groups (P=.221) or between the intermediate risk group and poor risk group (P=.093). Increased number of CS mutations (≥ 5) was seen with equal frequency across risk groups and predicted for shorter overall survival in both univariate (HR=2.80; P=.039) and by multivariate Cox regression analysis (P=.001) independently from assigned risk group. There were no differences in age, gender, smoke, geographic, and different risk groups by multivariate analyses. Conclusion: Our study shows that ≥ 5clinically significant somatic mutations were associated with adverse outcomes and decreased survival, independent of risk groups and induction regimen. Thus, it may be a useful prognostic factor. This finding needs to be validated using a larger sample size.

Chronic lymphocytic leukemia (CLL) patients progressively develop an immunosuppressive state. CLL patients have more plasma IL-10, an anti-inflammatory cytokine, than healthy controls. In vitro human CLL cells produce IL-10 in response to BCR cross-linking. We used the transgenic Eμ–T cell leukemia oncogene-1 (TCL1) mouse CLL model to study the role of IL-10 in CLL associated immunosuppression. Eμ-TCL mice spontaneously develop CLL because of a B cell–specific expression of the oncogene, TCL1. Eμ-TCL1 mouse CLL cells constitutively produce IL-10, which is further enhanced by BCR cross-linking, CLL-derived IL-10 did not directly affect survival of murine or human CLL cells in vitro. We tested the hypothesis that the CLL-derived IL-10 has a critical role in CLL disease in part by suppressing the host immune response to the CLL cells. In IL-10R−/− mice, wherein the host immune cells are unresponsive to IL-10–mediated suppressive effects, there was a significant reduction in CLL cell growth compared with wild type mice. IL-10 reduced the generation of effector CD4 and CD8 T cells. We also found that activation of BCR signaling regulated the production of IL-10 by both murine and human CLL cells. We identified the transcription factor, Sp1, as a novel regulator of IL-10 production by CLL cells and that it is regulated by BCR signaling via the Syk/MAPK pathway. Our results suggest that incorporation of IL-10 blocking agents may enhance current therapeutic regimens for CLL by potentiating host antitumor immune response.

Abstract Introduction: Low socioeconomic status (SES) has been shown to shorten survival in classical Hodgkin lymphoma (cHL). We sought to determine if differences in outcome of cHL varied according to SES and geographic setting in a population-based analysis. Methods: All adult patients diagnosed in 2000-2014 with cHL were collected through Kentucky Cancer Registry (KCR). Baseline SES and clinical variables were collected. Pearson Chi-square, log-rank, and cox regression tests were utilized. To minimize selection bias, cases with less than 6 months survival after cancer diagnosis were excluded. Results: A total of 1075 cHL patients were included, of which 605 (56.3%) were early stage vs. 405 (37.7%) were advanced stage. Most patients were under age 50 (63.2%) with a male predominance (55.4%). Nodular sclerosis was the most common histology (57.6%). Therapy consisted of chemotherapy alone in 729 (67.8%) patients, while 222 (20.7%) had received both chemotherapy and radiation. 633 (58.8%) lived in metro areas. Metro-area residents were more likely to come from non-Appalachian counties (93.7%), have more advanced education (76.8%) and have private insurance (60.1%) than non-metro-area residents (41.2%) (p<0.0001). There were no differences in age, gender, stage and histology at presentation according to geographic setting. Patients living in metro-area had a better OS by bivariate analysis (p=0.01) (Figure 1). A multivariate analysis identified age <50, early stage, privately insured, non-lymphocyte-depleted histology, and treatment with combined chemo-radiation as independent factors favoring an improved OS. Conclusion: Our study shows that despite the absence of differences in presenting clinical variables based on SES or geographic setting, significant discrepancies in outcome are observed. This disparity in outcomes may have been affected by differences in access to or quality of care delivery and may widen with the introduction of more costly novel interventions. Disclosures No relevant conflicts of interest to declare.

Prostate apoptosis response-4 (Par-4), a proapoptotic tumor suppressor protein, is downregulated in many cancers including renal cell carcinoma, glioblastoma, endometrial, and breast cancer. Par-4 induces apoptosis selectively in various types of cancer cells but not normal cells. We found that chronic lymphocytic leukemia (CLL) cells from human patients and from Eµ-Tcl1 mice constitutively express Par-4 in greater amounts than normal B-1 or B-2 cells. Interestingly, knockdown of Par-4 in human CLL-derived Mec-1 cells results in a robust increase in p21/WAF1 expression and decreased growth due to delayed G1-to-S cell-cycle transition. Lack of Par-4 also increased the expression of p21 and delayed CLL growth in Eμ-Tcl1 mice. Par-4 expression in CLL cells required constitutively active B-cell receptor (BCR) signaling, as inhibition of BCR signaling with US Food and Drug Administration (FDA)-approved drugs caused a decrease in Par-4 messenger RNA and protein, and an increase in apoptosis. In particular, activities of Lyn, a Src family kinase, spleen tyrosine kinase, and Bruton tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling. Together, these results suggest that Par-4 may play a novel progrowth rather than proapoptotic role in CLL and could be targeted to enhance the therapeutic effects of BCR-signaling inhibitors.

Although mutations in RNA splicing genes occur frequently in patients with clonal cytopenias of unknown significance (CCUS) and myelodysplastic syndromes (MDS), very often additional common myeloid gene driver mutations are present at diagnosis. Thus, the clinical significance of isolated mutations in the most commonly mutated RNA splicing genes remains unknown. Here we report five unusual patients with an isolated mutation causing a loss of function of ZRSR2, a protein required for recognition of a functional 3' splice site. Two of the patients had a diagnosis of CCUS and three patients had an MDS disorder characterized by low risk features and absence of complex cytogenetic abnor-malities. Notably, all five cases were characterized predominantly by macrocytic anemia. In addition, one CCUS patient followed for more than 15 years with a transfusion dependent macrocytic anemia was found to have an inactivating ZRSR2 mutation with an allele frequency of >60%. We conclude that the common clinical features of patients with an isolated mutation of ZRSR2 are a macrocytic anemia without leukopenia, thrombocytopenia or an increase in marrow blast percentage. At least in some cases, the presence of an isolated ZRSR2 mutation can accompany a dominant hematopoietic clone with a low risk for transformation to frank dysplasia or acute leukemia.

Introduction: Early stage classical Hodgkin lymphoma (cHL) carries good prognosis. Patients with B symptoms have been found to have unfavorable risk. Due to overall good outcome, studies are focusing on minimizing toxicity by omission of consolidative radiation. The aim of our study is to review outcome of this cohort in a population based analysis. Methods: All early stage (stage I and II) cHL adult patients diagnosed 2005-2014 were collected through Kentucky Cancer Registry (KCR). Patients reported to have had B symptoms (Unexplained fevers >38°C; drenching night sweats; or weight loss >10% of body weight within 6 months prior to diagnosis) were included in the current study. Baseline characteristics as well as survival outcome were compared between chemotherapy alone and combined chemo-radiation treatment. Pearson Chi-square, log-rank, and cox regression tests were used in the analysis. To minimize selection bias, events during the first 6 months of therapy were censored. 10-year survival data were then compared to Surveillance, Epidemiology and End Results (SEER) registry. Results: A total of 130 adult patients were included in the study; 76 patients got chemotherapy alone and 54 patients got chemo-radiation therapy. Median age was 35 (ranged 18-88). Most patients were younger than 50 year-old (76.9%). Most patients had nodular sclerosis (N=91) while 29 patients had unknown histology. There was no statistical difference in pretreatment features between the group receiving chemotherapy and those who received chemo-radiation (See table). There was no difference in 10-years overall survival between the chemotherapy group (73%) and chemo-radiation (80%) (p=0.830) (See figure). When adjusting for multivariate analysis, age younger than 50 was the only statistically significant variable affecting survival with a HR 0.17 (95% CI: 0.058-0.505). Only 4 (5.3%) patients developed second primary cancer in chemotherapy alone versus 1 (1.9%) in chemo-radiation therapy (p =0.4). Compared to overall survival in SEER database, younger patients ( Conclusion: Our study shows lack of benefit of the combined approach for the management of unfavorable prognosis classical Hodgkin lymphoma in a population based analysis. Younger patients had worse outcome when compared to SEER registry while older age group had worse prognosis with the available therapies and might benefit from alternative interventions. Download : Download high-res image (95KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

Abstract Introduction We conducted a study to see the activity of hypomethylating agent (HMA) in relapsed refractory lymphoma. In parallel, we studied KIR expression and promoter methylation before and after treatment with HMA. Allele-specific stochastic KIR expression is maintained in mature natural killer (NK) cells by a combination of DNA methylation and histone modification, with DNA methylation being dominant. The aim of this part of the study is to understand how KIR gene expression changes in response to epigenetic therapy, so that we then can manipulate NK cells to optimize the effect of chemo-immunotherapy of neoplastic diseases. Methods Blood samples were collected from patients enrolled in an open-labeled phase I trial of the combination of azacitidine with cyclophosphamide, vincristine and rituximab in relapsed/refractory lymphoma. Regimen: Azacitidine on days one through five (starting at 25mg/m2), followed by oral cyclophosphamide at 300 mg/m2 on days six through nine, vincristine 1.4 mg/m2 day eight, and rituximab 375 mg/m2 day eight. Each cycle was to be repeated every 21 days, up to eight cycles if participants continued to benefit from therapy. Sampling: Between 10-20uL whole blood was collected from eligible patients before commencement of therapy and on Cycle 1 Day 5, Cycle 2 Day 1 and Cycle 3 Day 1. Peripheral blood mononuclear cells (PBMC) were isolated immediately ex vivo using Lymphocyte Separation Media (Lonza), and were divided into two aliquots: one for flowcytometry and the other for pyrosequencing (Epigendx, Hopkinton, MA). Samples from each time point were stained with fluorescently-labelled antibodies to CD3, CD16, CD56, CD14, CD20 and four different KIRs. For calculating the number of KIRs per NK cell, we used a gating strategy in which CD56dim NK cells are identified, and then the number of KIR expression is counted on those by sequential gating. The percentages of monocytes and lymphocytes among the PBMC population were also determined using CD14 and CD20 antibodies. Monocytes were further characterized according to their CD16 expression into Classical (CD14highCD16negative), Intermediate (CD14highCD16low), and Non-classical (CD14lowCD16high). Results PBMC from 10 patients were available for flow cytometric analysis and pyrosequencing. There were statistically significant differences (p=0.0089) in percent of DNA methylation determined by PBMC pyrosequencing, with significant decrease from Cycle 1 Day 1 to Cycle 1 Day 5 and then an increase from Cycle 1 day 5 to Cycle 2 Day 1. The decline in the percent of DNA methylation in PBMC at day 5 of cycle 1 of the chemotherapy was also found to be statistically significant (Figure 1; p=0.0202). There was a large variation in the types and numbers of KIR molecules expressed at baseline (Table 1) on CD56dim NK cells among the patients in the cohort studied, with no significant differences between the various time points and response to treatment. Despite an apparent trend of increasing percentages of non-classical and intermediate monocytes (at the expense of classical monocytes), the data was not statistically significant (Figure 2; p=0.06). We also noticed a trend of decreasing percentage of monocytes and increasing percentage of lymphocytes in the PBMC population with increasing treatment cycles. However, this was not statistically significant either. Conclusions KIR expression may play role in HMA induced tumor cell killing. In our study, no significant changes were seen in the number of KIR receptors on NK cells post-treatment with hypomethylating agent despite observing a reduction in global DNA methylation in PBMC. This could be due to a number of reasons, including the small size of the study, the rapid turnover of NK cells, or that the reduced methylation was not sufficient to induce changes in KIR expression. The significance of the increase in the number of non-classical and intermediate monocytes following treatment with HMA is not well understood and needs further investigation. (The study was supported by Celgene) Disclosures No relevant conflicts of interest to declare.

In contrast to traditional chemotherapy, patients responding to biological or targeted therapies often are treated indefinitely until progression or toxicity. This therapeutic model, however, increases treatment costs, may induce greater toxicity and theoretically could select for earlier emergence of drug resistance. Moreover, little data is available regarding the outcomes of patients who discontinue targeted therapies after achieving remission. In this regard, we report two patients with relapsed/refractory CLL who chose to stop therapy unrelated to toxicity or disease status after the induction of remission by the BTK inhibitor ibrutinib. Patient A started ibrutinib for progressive CLL at an absolute lymphocyte count (ALC) of 137,000mm3 and recurrent hemolytic anemia. After 5 months, the hemolysis had resolved (Hgb 15.6 g/dl) while the ALC had declined to 9200mm3. Treatment was then interrupted due to patient preference. One month after drug discontinuation, the ALC was in the normal range at 1400mm3 and remained within or near the normal range for a total of 12 months. Two months later, the ALC was again markedly elevated at 68,000mm3and anemia recurred. The patient then agreed to restart ibrutinib. Thus far, after 5 months of re-treatment, he has had prompt resolution of the anemia and achieved a partial remission with a slowly decreasing lymphocytosis. Patient B was started on ibrutinib for a rising ALC (26,000mm3) and severe hemolytic anemia. After 9 months of treatment, the hemoglobin was 13 g/dl and the ALC was in the normal range at 3300mm3. Due to unrelated medical problems, ibrutinib therapy was stopped. Currently, 6 months since drug discontinuation, the ALC remains in the normal range and no other signs of CLL are present. These clinical observations suggest that interruption of ibrutinib may be feasible in at least some CLL patients who achieve remission. Even if flow cytometry were performed at monthly intervals to detect early recurrence and ensure prompt re-institution of therapy, the cost savings would still be considerable. Of course, clinical trials will be necessary to confirm equivalent long-term efficacy and overall survival for intermittent versus continuous ibrutinib therapy in CLL.

Autoimmune hemolytic anemia (AIHA) occurs in CLL at some time during the course of the disease in up to 7-10% of patients. The acute onset of AIHA may occur unrelated to therapy but has also been linked to treatment with chemo­therapeutic agents including chlor­ambucil, benda­mustine and particu­larly purine nucleosides such as fludarabine. Although the mechanism is still not well understood, chemo­therapy-induced changes in regulatory T-cells have been proposed as a trigger for autoimmunity and clinical hemolysis. In contrast to these cytotoxic therapies, ibrutinib, an inhibitor of Bruton’s tyrosine kinase recently approved for the treatment of CLL, appears to have a different mechanism of action and thus far has not been associated with AIHA in published reports. However, we report here a patient with CLL and a history of prior AIHA, who developed a recurrence of acute hemolysis after the initiation of ibrutinib. The patient is a 67-year-old man diagnosed with CLL in 2002 and treated for progressive disease with a single cycle of bendamustine in 2009. Although the lymphocytosis resolved rapidly, the hemoglobin also decreased from 14 g/dL to 5.2 g/dL by 3 weeks after the start of therapy. Due to the onset of Coombs-positive AIHA, chemotherapy was dis­con­tinued. Hemolysis resolved with prednisone therapy and did not recur after a slow taper. The CLL then remained asymptomatic until 2012 when night sweats developed at a white blood cell (wbc) count of 95,000/µL. Benda­mustine was re-started and despite a negative Coombs test prior to treatment, Coombs-positive AIHA developed again with the hemoglobin falling from normal to 7.0 g/dL within 4 weeks. After stabilization with transfusions and steroids, an additional cycle of bendamustine plus rituximab was administered without further complications and the patient’s symptoms and lympho­cytosis resolved. After the discontinuation of prednisone, hemolysis did not recur clinically although the Coombs test remained 1+ positive through early 2014. By May 2014 the wbc count had increased to 144,000/µL with the onset of a mild anemia (Hgb 12.3 g/dL) and symptomatic night sweats. Due to the history of repeated chem­otherapy-associated AIHA, alter­native therapy with ibrutinib, which had not been associated with AIHA, was instituted at 420 mg daily. However, within 2 weeks the hemoglobin decreased to 7.0 g/dL while the wbc count increased to 300,000/µL. A reticulocyte count was 16%, total bilirubin 3.2 mg/dL, haptoglobin <10 mg/dL, and the Coombs test was reported 3+ positive for IgG. Prednisone was started at 1 mg/Kg daily and ibrutinib was held. After 10 days of steroid therapy, the hemoglobin improved to 10 g/dL and ibrutinib was restarted. Over the next month, the hemoglobin continued to increase to 12.7 g/dL and the wbc count gradually decreased to 44,000/µL with 24% neutrophils, a marked increase in the absolute neutrophil count as compared to pre-treatment. The patient currently continues on ibrutinib with a slow prednisone taper and is now asymptomatic. In summary, this case suggests that some CLL patients may experience the acute onset of AIHA after starting ibrutinib with a clinical course similar to cases reported after fludarabine and other agents. In addition, a response to steroid therapy may allow the successful resumption of ibrutinib treatment. The actual likelihood of acute hemolysis after ibrutinib therapy in CLL is unclear, but may have been much greater in this case than in CLL patients without prior episodes of AIHA or a positive Coombs test. However, a potential mechanism for the precipitation or accel­eration of auto­immunity is suggested by the recent report by Byrd and colleagues (Blood 2013; 122:2539-49) showing that ibrutinib inhibits interleukin-2-inducible kinase (ITK). This inhibition skewed the CD4 T-cell popul­a­tions isolated from treated CLL patients toward a Th1 profile, a regulatory T-cell subset previously reported to drive the early auto­antibody response in an animal model of AIHA (Blood 2009; 113:389-95). Disclosures No relevant conflicts of interest to declare.

The identification of Jak2(V617F) mutations in more than 90% of patients with polycythemia vera (PV) has greatly improved the diagnostic accuracy for this uncommon myeloproliferative disorder. Although previous cases of presumptive PV in patients with hereditary spherocytosis (HS) have been described, these earlier reports either preceded the establishment of widely accepted criteria for the diagnosis of PV or lacked definitive studies to rule out secondary causes of polycythemia. In contrast, the author describes here a novel case of PV confirmed at the molecular level in a patient with hereditary spherocytosis by the finding of a Jak2(V617F) mutation. Based on recent advances in understanding the role of Jak2 signaling in the pathogenesis of PV, the author proposes 2 independent biological mechanisms that could account for more than a chance association of these 2 disorders.

HoxA11 and HoxD11 are homeobox genes critical for normal development of the forearm and thus are potential candidate genes for involvement in the pathogenesis of the thrombocytopenia/absent radius (TAR) syndrome. However, we previously reported an absence of coding sequence mutations in either HoxA11 or HoxD11 in a series of 10 unrelated TAR syndrome patients (Fleischman RA et al., Br J Haematol., 116:367-75, 2002). Despite this negative finding, interest in the potential role of homeobox genes in the TAR syndrome has been supported by a report of a HoxA11 mutation occurring in two kindreds with amegakaryocytic thrombocytopenia and radio-ulnar synostosis, a less pronounced more proximal pattern of radial malformation (Thompson AA and Nguyen LT. Nat Genet., 26:397-8, 2000). Unlike HoxA11, however, no mutations in the human HoxD11 gene have been described thus far that would help elucidate the potential role of this paralogous gene in megakaryopoiesis or the TAR syndrome. We now describe a novel mutation in human HoxD11 that results in a polyalanine sequence expansion, (GCG)6→ (GCG)8, and report that this mutation is associated with a unilateral absent radius in the affected propositus. A familial syndrome is suggested in this kindred, moreover, by the prior observation of a bilateral absent radius in a deceased maternal aunt. This mutation was not present in more than 100 unrelated normal subjects or 8 other unrelated individuals with sporadic absence of the radius. Two other living maternal relatives also carried the mutation but did not exhibit any radial defects, a finding consistent with autosomal dominance with incomplete penetrance, an inheritance pattern reported for short polyalanine expansion mutations in the related homeobox gene HoxD13 which cause synpolydactyly. In contrast to the reported HoxA11 mutation, however, neither the propositus nor the mutation carriers of this HoxD11 mutation exhibited thrombocytopenia or any other cytopenias or congenital defect. The results suggest that at least one class of mutation in human HoxD11 may be sufficient to cause an absent radius syndrome but unlike the reported HoxA11 mutation, does not adversely affect megakaryopoiesis. The findings further suggest that additional studies of the TAR syndrome may be necessary to exclude as yet undetected non-coding mutations in promoter or enhancer sequences that alter the expression of HoxA11, HoxD11 or other homeobox genes critical for radial development and/or megakaryopoiesis. This work was supported by a VA Merit Award. Disclosures: No relevant conflicts of interest to declare.

Summary. Recent studies have suggested the HoxA10 , HoxA11 and HoxD11 homeobox genes as candidate loci for the thrombocytopenia with absent radius (TAR) syndrome. For example, targeted disruptions of these Hox genes result in abnormal development of the mouse radius, while overexpression of HoxA10 stimulates mouse megakaryocyte (MK) development in vitro . To examine the expression of Hox genes in human MK cells, we utilized reverse transcription polymerase chain reaction with degenerate oligonucleotides to study megakaryocytic cell lines (MEG‐01, DAMI), and primary human MK purified from adult and cord blood. Using this approach, 13 out of 40 clones isolated from cell lines, 10 out of 21 from cord MK, and 11 out of 21 from adult MK were identified as HoxA10 , while HoxA11 and HoxD11 sequences were not detected. The normal genomic sequences for the human HoxA10 , ‐A11 , and ‐D11 genes were then determined and sequenced in 10 unrelated individuals with TAR syndrome. In all patients the derived amino acid sequence for the three Hox genes was identical to normal controls. Southern blotting did not reveal genomic rearrangements or deletions at these loci, and in two patients intact HoxA10 transcripts were detected by amplification in myeloid cells. Although these studies cannot completely exclude the possibility that the TAR syndrome results from non‐coding mutations that affect the level of Hox gene expression in megakaryocytes, mutations in the coding sequence of the Hox genes known to affect radial development are not a common cause of TAR syndrome.

Heterozygous mutations in the gene for the Kit transmembrane receptor have been identified recently in human piebaldism and mouse "dominant spotting." Interestingly, all of the 14 known missense mutations that cause depigmentation in these species map to the tyrosine kinase domain of the receptor, whereas none have involved the extracellular ligand-binding domain. In an attempt to detect these uncommon mutations, we screened the nine exons encoding the extracellular portion of Kit for single-strand conformation polymorphisms (SSCP) in eight piebald subjects previously reported to be negative for kinase mutations. Four of these eight kindreds proved to carry novel mutations. The first mutation, found in two apparently unrelated probands with mild piebaldism and English ancestry, substitutes an arginine for a highly conserved cysteine at codon 136. This substitution disrupts a putative disulfide bond required for formation of the second Ig-like (D2) loop of the Kit ligand-binding domain. The second mutation, detected in a piebald kindred characterized by unusually limited depigmentation, substitutes a threonine for an alanine at codon 178, a site just proximal to conserved cysteines at codons 183 and 186. The third mutation, occurring in a kindred with more extensive depigmentation, is a novel four-base insertion in exon 2 that results in a proximal frameshift and premature termination. The data strongly suggest that piebaldism can result from missense mutations in the Kit ligand-binding domain, although the resulting phenotype may be milder than that observed for null or kinase mutations. The apparent clustering of these uncommon mutations at or near the conserved cysteines for the D2 Ig-like loop further suggests a critical role for this region in Kit receptor function.

American Journal of HematologyVolume 48, Issue 4 p. 293-293 Letters and CorrespondenceFree Access Acute Onset of Severe Autoimmune Hemolytic Anemia After Treatment With 2-Chlorodeoxyadenosine for Chronic Lymphocytic Leukemia Roger A. Fleischman, Roger A. Fleischman Division of Hematology/Oncology, University of Kentucky Medical Center and Veterans Administration Hospital, Lexington, KentuckySearch for more papers by this authorDavid Croy, David Croy Division of Hematology/Oncology, University of Kentucky Medical Center and Veterans Administration Hospital, Lexington, KentuckySearch for more papers by this author Roger A. Fleischman, Roger A. Fleischman Division of Hematology/Oncology, University of Kentucky Medical Center and Veterans Administration Hospital, Lexington, KentuckySearch for more papers by this authorDavid Croy, David Croy Division of Hematology/Oncology, University of Kentucky Medical Center and Veterans Administration Hospital, Lexington, KentuckySearch for more papers by this author First published: April 1995 https://doi.org/10.1002/ajh.2830480423Citations: 21AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Carrera CJ, Saven A, Piro LD: Purine metabolism of lymphocytes. Targets for chemotherapy drug development. Hematol Oncol Clin North Am 8: 357–381, 1994. 2 Bastion Y, Coiffier B, Dumontet C, Espinouse D, Bryon PA: Severe autoimmune hemolytic anemia in two patients treated with fludarabine for chronic lymphocytic leukemia. Ann Oncol 3: 171–172, 1992. 3 Tosti S, Caruso R, D'Adamo F, Picardi A, Ali Ege M, Girelli G, Mauro FR, Maurillo L, Amadori S: Severe autoimmune hemolytic anemia in a patient with chronic lymphocytic leukemia responsive to fludarabine-based treatment. Ann Hematol 65: 238–239, 1992. 4 Di Raimondo F, Giustolisi R, Cacciola E, O'Brien S, Kantarjian H, Robertson LB, Keating MJ: Autoimmune hemolytic anemia in chronic lymphocytic leukemia patients treated with fludarabine. Leuk Lymphoma 11: 63–68, 1993. 5 Juliusson G, Elmhorn-Rosenberg A, Liliemark J: Response to 2-chlorodeoxyadenosine in patients with B-cell chronic lymphocytic leukemia resistant to fludarabine. N Engl J Med 327: 1056, 1992. Citing Literature Volume48, Issue4April 1995Pages 293-293 ReferencesRelatedInformation

Although macrophages account for 70-90% of the adherent cells in mouse long-term bone marrow cultures (LTBMC) and CFU-F colonies, the predominant nonhematopoietic stromal cell is endothelial-like (EL), expressing cytoplasmic collagen IV, laminin, and an antigen recognized by the monoclonal antibody MECA-10. We report the isolation of this stromal cell lineage from primary LTBMC by immunomagnetic cell selection using MECA-10. More than 95% of the cells in the MECA-10-positive fraction are EL cells as judged by morphology, surface staining for MECA-10, cytoplasmic staining for collagen IV, and electrophoretic analysis of MECA-10-positive cells isolated from radiation chimeras. When plated under LTBMC conditions, EL cell monolayers recharged with either wild-type or Sl/Sld marrow support an increased density and number of clonogenic and mature hematopoietic cells in short-term cultures. In accord with this finding, Northern blots of mRNA from unstimulated EL cells demonstrate constitutive expression of Kit ligand (KL). Moreover, in situ two-color immunofluorescence staining for cytoplasmic collagen IV and surface KL suggests that EL cells are the exclusive source of membrane-bound KL in mouse cultures. The ability to isolate EL cells from primary cultures without the need for repeated cell passage or immortalization provides a novel approach to dissecting the molecular basis of stem cell-stromal cell interactions.

We made use of a previously described in vivo model of chimeric mice created by embryo aggregation that allows the study of contributions to both lymphohematopoiesis and marrow stroma by two genotypically distinct cell populations. Day-2 embryos from C57BL/6 and DBA/2 strains were fused to produce allophenic chimeric mice that proved to have contributions from each strain in all the tissues of the body and that permitted study of competitive contributions to blood formation. Although the contribution of DBA/2 stem cells to hematopoiesis gradually ceased in an age-related manner so that all blood cells in aged chimeras were C57BL/6 in origin, here we show that, in contrast, the extent of stromal chimerism, determined by the fibroblast colony-forming unit (CFU-F) assay, was maintained and was remarkably uniform from one marrow site to another. This result is consistent with a polyclonal nonhematopoietic origin of the CFU-F and suggests that the decline in DBA/2 blood cells was not dependent on similar changes in genotype-matched stroma, but was instead an intrinsic property of this stem cell population. These intrinsic stem cell properties were further examined by serial bone marrow transplantation. When marrow from a chimera with no detectable DBA/2 blood cells was transplanted into irradiated recipients, cells of DBA/2 genotype significantly contributed to early hematopoietic engraftment, demonstrating that the DBA/2 stem cell population was not extinguished in the chimeric donor, but rather had entered a reversible state of quiescence. Reactivation of the DBA/2 stem cell population, however, was short-lived, and long-term engraftment of recipients was accomplished by donor cells of the partner strain (C57BL/6). However, transient reactivation of the quiescent (DBA/2) stem cell pool again occurred with a second round of transplantation. These surprising results demonstrate, for the first time, selective and reiterated inactivation and reactivation of a stem cell population depending on hematopoietic needs. Moreover, the results suggest that genetic differences in the stem cell populations of coexistent strains account for the selective responses described.

Recent advances in molecular genetics have made possible the use of retroviral "vectors" to transfer cloned human genes into somatic cells. With this new technology, the genetic defect underlying many recessive inherited disorders can probably be corrected by inserting a normal gene into the patient's hematopoietic stem cells. This article reviews the design and safety of the viral vectors and the results of in vivo studies in mice and large animals that have led to the first human trials. Other target cells for gene transfer, such as endothelial cells, fibroblasts, keratinocytes, and hepatocytes, are also discussed. The use of recombinant retroviruses for gene transfer in vivo is still a new area of research, but the feasibility of "gene therapy" for genetic disorders is rapidly gaining medical and scientific acceptance.