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Purpose:Infectious complications constitute a leading cause of morbidity and mortality in chronic lymphocytic leukemia (CLL). Patients respond poorly to vaccines, particularly pneumococcal polysaccharide and influenza vaccines. In addition, patients with genetically high-risk disease are at increased risk for early disease progression and death. Lenalidomide, an oral immunomodulatory agent with demonstrated clinical activity in CLL, can potentially restore immune system dysfunction associated with CLL while improving disease outcomes.Patients and Methods:Phase II study randomized 49 patients with genetically high-risk CLL or small lymphocytic lymphoma [SLL; defined as unmutated Ig heavy chain variable region, deletion(17p) or (11q), and/or complex abnormal karyotype], to receive lenalidomide either concurrent (arm A) or sequential to (arm B) two doses of 13-valent protein-conjugated pneumococcal vaccine (PCV13) administered 2 months apart, in patients not meeting International Workshop on Chronic Lymphocytic Leukemia treatment criteria.Results:Four serotypes (3, 4, 5, 6B) achieved the additional seroprotection definition of a fourfold increase in arm A, and six serotypes (3, 4, 5, 6B, 19A, 19F) in arm B. All patients achieved the defined concentration of 0.35 μg/mL for at least one serotype tested. No significant difference was observed with the addition of lenalidomide. At median time on treatment of 3.6 years, median progression-free survival (PFS) was 5.8 years [95% confidence interval (CI), 3.1—not reached]. PFS at 1, 2, and 3 years was 85% (95% CI, 72–93), 79% (95% CI, 64–88), and 72% (95% CI, 57–83), respectively.Conclusions:Lenalidomide is efficacious with manageable toxicities as an early intervention strategy in patients with high-risk CLL, but did not enhance humoral response to PCV13 vaccine.

Targeting BCR and BCL-2 signaling is a widely used therapeutic strategy for chronic lymphocytic leukemia. C481S mutation decreases the covalent binding affinity of ibrutinib to BTK, resulting in reversible rather than irreversible inhibition. In addition to BTK, mutations in PLCG2 have been demonstrated to mediate acquired ibrutinib resistance. Venetoclax, a highly selective BCL2 inhibitor, has high affinity to the BH3-binding grove of BCL2. Mutation in BCL2 (Gly101Val) decreases the affinity of BCL2 for venetoclax and confers acquired resistance in cell lines and primary patient cells. This review discusses the common mechanisms of resistance to targeted therapies in chronic lymphocytic leukemia.

PURPOSE: To determine how photorefractive keratectomy (PRK) and mitomycin C (MMC) affect corneal nerves and their regeneration over time after surgery. METHODS: Twenty-eight New Zealand rabbits had corneal epithelial scraping with (n = 3) and without (n = 3) MMC 0.02% or −9.00 diopter PRK with (n = 6) and without (n = 16) MMC 0.02%. Corneas were removed after death and corneal nerve morphology was evaluated using acetylcholinesterase immunohistochemistry and beta-III tubulin staining after 1 day for all groups, after 1 month for PRK with and without MMC, and 2, 3, and 6 months after PRK without MMC. Image-Pro software (Media Cybernetics, Rockville, MD) was used to quantitate the area of nerve loss after the procedures and, consequently, regeneration of the nerves over time. Opposite eyes were used as controls. RESULTS: Epithelial scraping with MMC treatment did not show a statistically significant difference in nerve loss compared to epithelial scraping without MMC ( P = .40). PRK with MMC was significantly different from PRK without MMC at 1 day after surgery ( P = .0009) but not different at 1 month after surgery ( P = .90). In the PRK without MMC group, nerves regenerated at 2 months ( P < .0001) but did not return to the normal preoperative level of innervation until 3 months after surgery ( P = .05). However, the morphology of the regenerating nerves was abnormal—with more tortuosity and aberrant innervation compared to the preoperative controls—even at 6 months after surgery. CONCLUSIONS: PRK negatively impacts the corneal nerves, but they are partially regenerated by 3 months after surgery in rabbits. Nerve loss after PRK extended peripherally to the excimer laser ablated zone, indicating that there was retrograde degeneration of nerves after PRK. MMC had a small additive toxic effect on the corneal nerves when combined with PRK that was only significant prior to 1 month after surgery. [ J Refract Surg . 2018;34(12):790–798.]

The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin (αSMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP+ αSMA+ myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45+ fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present in the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP+ CD45+ cells were also αSMA+ myofibroblasts. At 21 days after PTK, few GFP+ αSMA+ cells persisted in the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP+ CD45+ SMA+ cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6—C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature αSMA+ myofibroblasts. Most GFP+ CD45+ αSMA+ myofibroblasts that did develop subsequently underwent apoptosis—likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGFβ requisite for myofibroblast survival.

Summary: In this issue, Blombery and colleagues show that the chronic lymphocytic leukemia (CLL) cells bearing Gly101Val mutation confer resistance to venetoclax by reducing the affinity of BCL2 for venetoclax by 180-fold in cell lines and in patient cells. Detection of this mutation provides a potential biomarker for impending disease progression and an opportunity for targeted and combinational therapy to treat CLL. See related article by Blombery et al., p. 342.

Introduction: The development of Bruton tyrosine kinase inhibitors (BTKi) and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL). Ibrutinib and other second generation BTKi as monotherapies generally do not produce minimal residual disease negative (MRD-) complete remissions even with extended therapy. The reason for lack of continued elimination of CLL to a MRD- status over time is unknown, and we hypothesized that biological differences in the CLL cells or immune microenvironment might make them resistant to elimination. Methods: Samples were obtained from patients on continuous ibrutinib who hadn't relapsed at time points of 3 years on treatment and 5 years on treatment; and pre-ibrutinib. Isolated CLL cells were subject to B-cell receptor (BCR) sequencing using NEBNext Immune Sequencing Kit by New England Biolabs (NEB, Inc., USA). In a separate cohort, 10X VDJ+5'-sequencing was performed on peripheral blood mononuclear cells. Flow cytometry and ELISA were used to identify alterations in immune cell subtype and identify immune profiles associated with MRD positive (MRD+) status. Results: To identify the clonal pattern in MRD+, we performed deep sequencing of the BCR repertoire on samples from 13 patients with 3 time points each. We found that dominant clones tended to remain constant, but new clones appeared in later time points (Figure 1). MiXCR (v3.0.5) was used with default parameters to identify preprocessed reads containing CDR3 regions from B-cell heavy, kappa, and lambda chains, generating a list of unique productive and nonproductive CDR3 sequences associated with their relative abundances and specific V(D)J gene usage. Two out of three patients (patients 1 and 3) showed significant change in the clone over time. In patients 1 and 2, we saw that heavy chain clones emerge at later time points. In patient 3 alone, we observed that at 5 years there are two dominant clones. Our findings suggest that each patient shows a diverse repertoire of CLL clones and that the dominant clone does not change significantly across time points. To identify cell populations based on gene expression patterns, we performed 10X VDJ+5'-seq. Based on the expression of known markers, we identified CLL cells and other immune cell subtypes. We identified differentially expressed genes (DEGs) for CLL cells in each time points. Over time, we observed upregulation of CD79a, LTB, TAGLN2, and LGALS, genes typically associated with leukemic cell survival. Suggesting differential expression of pro-survival genes contribute to continued presence of MRD over time. T cells are known to be dysfunctional in CLL and have not previously been extensively studied in the setting of long term BTKi. We performed flow cytometry to determine the repertoire and function of T cells at 3 and 5 years of ibrutinib therapy. We found that the percentage of CD3+ T cells increases at later time points in all the 8 patients (p Conclusion: Extended ibrutinib treatment yields a subset of patients who become MRD- whereas a large majority remain MRD+. Our findings suggest that BCR repertoire in CLL MRD might change in long term ibrutinib therapy and induce necessary genes for its survival in the microenvironment. Although T cells and NK cells are non-functional at later time points, better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells. Differentiating the biology of why certain patients attain MRD- status on BTK inhibitor is of high interest as it could provide rationale for therapy discontinuation or add on approaches. Figure 1 Figure 1. Disclosures Rogers: AbbVie Inc.: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy, Research Funding; Innate Pharma: Consultancy; Pharmacyclics LLC: Consultancy; Janssen Pharmaceuticals, Inc: Research Funding; ovartis Pharmaceuticals Corporation: Research Funding. Bhat: Beigene: Consultancy; AstraZeneca: Consultancy; Aptitude Health: Honoraria; Onclive: Honoraria. Kittai: Bristol-Meyers Squibb: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees. Woyach: AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; Gilead Sciences Inc: Other: Data & Safety.

Basement membranes are thin connective tissue structures composed of organ-specific assemblages of collagens, laminins, proteoglycan-like perlecan, nidogens, and other components. Traditionally, basement membranes are thought of as structures which primarily function to anchor epithelial, endothelial, or parenchymal cells to underlying connective tissues. While this role is important, other functions such as the modulation of growth factors and cytokines that regulate cell proliferation, migration, differentiation, and fibrosis are equally important. An example of this is the critical role of both the epithelial basement membrane and Descemet’s basement membrane in the cornea in modulating myofibroblast development and fibrosis, as well as myofibroblast apoptosis and the resolution of fibrosis. This article compares the ultrastructure and functions of key basement membranes in several organs to illustrate the variability and importance of these structures in organs that commonly develop fibrosis.

The purpose of this study was to investigate whether myofibroblast-related fibrosis (scarring) after microbial keratitis was modulated by the epithelial basement membrane (EBM) injury and regeneration. Rabbits were infected with Pseudomonas aeruginosa after epithelial scrape injury and the resultant severe keratitis was treated with topical tobramycin. Corneas were analyzed from one to four months after keratitis with slit lamp photos, immunohistochemistry for alpha-smooth muscle actin (α-SMA) and monocyte lineage marker CD11b, and transmission electron microscopy. At one month after keratitis, corneas had no detectible EBM lamina lucida or lamina densa, and the central stroma was packed with myofibroblasts that in some eyes extended to the posterior corneal surface with damage to Descemet’s membrane and the endothelium. At one month, a nest of stromal cells in the midst of the SMA+ myofibroblasts in the stroma that were CD11b+ may be fibrocyte precursors to myofibroblasts. At two to four months after keratitis, the EBM fully-regenerated and myofibroblasts disappeared from the anterior 60 to 90% of the stroma of all corneas, except for one four-month post-keratitis cornea where anterior myofibroblasts were still present in one localized pocket in the cornea. The organization of the stromal extracellular matrix also became less disorganized from two to four months after keratitis but remained abnormal compared to controls at the last time point. Myofibroblasts persisted in the posterior 10% to 20% of posterior stroma even at four months after keratitis in the central cornea where Descemet’s membrane and the endothelium were damaged. This study suggests that the EBM has a critical role in modulating myofibroblast development and fibrosis after keratitis—similar to the role of EBM in fibrosis after photorefractive keratectomy. Damage to EBM likely allows epithelium-derived transforming growth factor beta (TGFβ) to penetrate the stroma and drive development and persistence of myofibroblasts. Eventual repair of EBM leads to myofibroblast apoptosis when the cells are deprived of requisite TGFβ to maintain viability. The endothelium and Descemet’s membrane may serve a similar function modulating TGFβ penetration into the posterior stroma—with the source of TGFβ likely being the aqueous humor.

Adipocytes and their products play essential roles in tumor establishment and progression. As the main cellular component of the bone marrow, adipocytes may contribute to the development of hematologic tumors.This review summarizes experimental data on adipocytes and their interaction with various cancer cells. Special focus is set on the interactions of bone marrow adipocytes and normal and transformed cells of the hematopoietic system such as myeloma and leukemia cells. Current in vitro and in vivo data are summarized and the potential of novel therapeutic targets is critically discussed.Targeting lipid metabolism of cancer cells and adipocytes in combination with standard therapeutics might open novel therapeutic avenues in these cancer entities. Adipocyte-derived products such as free fatty acids and specific adipokines such as adiponectin may be vital anti-cancer targets in hematologic malignancies. However, available data on lipid metabolism is currently mostly referring to peripheral fat cell/cancer cell interactions and results need to be evaluated specifically for the bone marrow niche.

Introduction: The Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib (IB) is a standard therapy for previously untreated CLL patients. While therapy is currently indicated only for patients with progressive, symptomatic disease, the introduction of targeted therapies in CLL has re-opened the question of whether asymptomatic high risk patients would benefit from early intervention. As well, even in early stages CLL is associated with profound cellular, humoral and innate immune suppression and patients with CLL respond poorly to routine vaccinations. IB has been shown to reverse disease mediated immune dysfunction partly through Th1 skewing. We undertook a phase 2 study of IB in asymptomatic high risk CLL patients who did not otherwise require therapy to evaluate: 1) the safety and efficacy of 2 years of IB in this clinical setting and 2) ability of IB to improve the efficacy of routine vaccines. Methods: This is a single-stage phase II study of IB in previously untreated asymptomatic, genetically high-risk patients with CLL, who did not meet IWCLL treatment criteria. High risk genomics were defined as del(11)(q22.3), del(17)(p13.1), unmutated IGHV, and/or complex karyotype (≥3 cytogenetic abnormalities). Patients were randomized to receive IB 420mg PO daily either concurrent with the pneumococcal (PCV13), influenza and TdaP vaccines (Arm A) or following vaccination (Arm B) for a total of 27 cycles (2 years). The primary objective of the study was to determine the safety and 2-year progression-free survival (PFS) of asymptomatic, high-risk CLL patients treated with IB. Secondary objectives include determination of safety and immune responses to vaccines in relation to IB administration, development of resistance, and quality-of-life (QOL). QOL measures assessed general QOL (SF-12, EORTC), anxiety (GAD-7) and depression (PHQ-9). Results: Forty-four patients (pts; 21 in Arm A, 23 in Arm B) were enrolled from 1/2016 to 6/2017, with a median age of 58 (range 35-82). Sixty-six percent of pts were male and all were high-risk: 91% with unmutated IGHV, 14% with del(17)(p13.1), 34% with del(11)(q22.3), and 24% with complex karyotype (≥ 3 abnormalities). Median follow-up is 2.6 years (range: 0.2-3.2). Excluding 2 patients in Arm B who progressed prior to receipt of IB, 2-year PFS was 92%. From a landmark of 2-years and including 33 patients who reached this point and discontinued IB, 6-month PFS was 87% (95% CI: 69-95%; Figure 1). Of 9 pts who progressed after IB discontinuation, 3 have not required further treatment, 5 restarted IB or acalabrutinib, and 1 started venetoclax/rituximab, and all responded. For PCV13, in Arm A, 15/16 patients showed a significant increase in antibody titer 2 cycles following vaccination, but response disappeared by cycle 12. In Arm B, 3/13 patients had an increase in antibody titer, with no increase following second vaccination. For influenza, patients in Arm B showed a significant response to influenza A vaccination, while patients in both arms responded to influenza B vaccination. IB was generally well tolerated, and only one pt discontinued treatment due to toxicity (atrial fibrillation). Grade 3+ toxicities that occurred in >2% of patients included anemia (7%), atrial fibrillation (11%), dental caries (7%), hyperglycemia (7%), hypertension (43%), and neutropenia (7%). At baseline, patients' reports of physical health-related (M=48.52, SD=7.96) and mental health-related quality of life (M=52.20, SD=8.57) similar to U.S population norms, along with moderately high global health (M=78.49 of 100, SD = 20.51). Additionally, patients' anxiety (M=3.56, SD=4.71) and depressive symptom reports (M=3.65, SD=4.46) were in the "none/mild" range. There was no significant change in the latter measure from baseline to 12 months (p>0.25), excepting anxiety which slightly decreased (M=1.31, SD=2.33; F(3,44)=5.94, p Conclusions Early intervention with IB was well tolerated and significantly improved patient disease status. Vaccine response to PCV13 was improved by concurrent ibrutinib, but lost at 12 months, suggesting re-vaccination might be helpful. Quality of life remained good throughout treatment, with improvement in patient anxiety. Extended follow-up will be required to determine how long remissions can be maintained after drug discontinuation in this population. Disclosures Woyach: Verastem: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Karyopharm: Research Funding; AbbVie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding. Rogers:AbbVie: Research Funding; Acerta Pharma: Consultancy; Genentech: Research Funding; Janssen: Research Funding. Bhat:Pharmacyclics: Consultancy; Janssen: Consultancy. Grever:Acerta Pharma, LLC: Membership on an entity's Board of Directors or advisory committees. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding; Genentec: Research Funding. Byrd:TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau. Awan:Gilead: Consultancy; Sunesis: Consultancy; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy; Genentech: Consultancy. OffLabel Disclosure: Clinical trial of ibrutinib used in an off-label setting

Th17 cells infiltrate the kidneys of patients with lupus nephritis (LN) and are critical for the pathogenesis of this disease. In this study, we show that enhanced activity of Stat3 in CD4+CD45RA−Foxp3− and Foxp3low effector T cells from children with LN correlates with increased frequencies of IL-17–producing cells within these T cell populations. The levels of retinoic acid-related orphan receptor c and IL-17 mRNA are significantly higher in PBMCs from children with LN than in those from controls. Mammalian target of rapamycin inhibition by rapamycin reduces both Stat3 activation in effector T cells and the frequency of IL-17–producing T cells in lupus patients. Complement factor C5a slightly increases the expression of IL-17 and induces activation of Akt in anti-CD3–activated lupus effector T cells. Th17 cells from children with LN exhibit high Akt activity and enhanced migratory capacity. Inhibition of the Akt signaling pathway significantly decreases Th17 cell migration. These findings indicate that the Akt signaling pathway plays a significant role in the migratory activity of Th17 cells from children with LN and suggest that therapeutic modulation of the Akt activity may inhibit Th17 cell trafficking to sites of inflammation and thus suppress chronic inflammatory processes in children with LN.