Spontaneous Corneal Clearing after Descemet Membrane Rupture and Near-Total Detachment in Keratoglobus: A Case Report.

Introduction: We present a case of a patient with osteogenesis imperfecta (OI) and keratoglobus (KG) who had a near-total rupture of Descemet's membrane followed by spontaneous corneal clearing. This case is unique in that it demonstrates the potentially excellent outcome of conservative treatment for Descemet's rupture in patients with KG and illustrates the impressive migratory potential of healthy endothelial cells. Case Presentation: An 11-year-old girl with OI and KG who had rupture and near-total detachment of Descemet's membrane presented for evaluation. This was managed conservatively and resulted in the eventual spontaneous clearing of the cornea. A similar process happened in the fellow eye some years later. Given the result of conservative management originally, the patient was once again treated conservatively, with significant improvement in corneal edema and visual acuity. Conclusion: Given the size of the ruptures, this case highlights the dynamic nature of the corneal endothelium and provides an extreme example of the migratory potential of corneal endothelial cells.

Intratumoral delivery of TransCon™ TLR7/8 Agonist promotes sustained anti-tumor activity and local immune cell activation while minimizing systemic cytokine induction.

BACKGROUND: Intratumoral (IT) delivery of toll-like receptor (TLR) agonists has shown encouraging anti-tumor benefit in preclinical and early clinical studies. However, IT delivery of TLR agonists may lead to rapid effusion from the tumor microenvironment (TME), potentially limiting the duration of local inflammation and increasing the risk of systemic adverse events. METHODS: To address these limitations, TransCon™ TLR7/8 Agonist-an investigational sustained-release prodrug of resiquimod that uses a TransCon linker and hydrogel technology to achieve sustained and predictable IT release of resiquimod-was developed. TransCon TLR7/8 Agonist was characterized for resiquimod release in vitro and in vivo, in mice and rats, and was assessed for anti-tumor efficacy and pharmacodynamic activity in mice. RESULTS: Following a single IT dose, TransCon TLR7/8 Agonist mediated potent tumor growth inhibition which was associated with sustained resiquimod release over several weeks with minimal induction of systemic cytokines. TransCon TLR7/8 Agonist monotherapy promoted activation of antigen-presenting cells in the TME and tumor-draining lymph nodes, with evidence of activation and expansion of CD8+ T cells in the tumor-draining lymph node and TME. Combination of TransCon TLR7/8 Agonist with systemic immunotherapy further promoted anti-tumor activity in TransCon TLR7/8 Agonist-treated tumors. In a bilateral tumor setting, combination of TransCon TLR7/8 Agonist with systemic IL-2 potentiated tumor growth inhibition in both injected and non-injected tumors and conferred protection against tumor rechallenge following complete regressions. CONCLUSIONS: Our findings show that a single dose of TransCon TLR7/8 Agonist can mediate sustained local release of resiquimod in the TME and promote potent anti-tumor effects as monotherapy and in combination with systemic immunotherapy, supporting TransCon TLR7/8 Agonist as a novel intratumoral TLR agonist for cancer therapy. A clinical trial to evaluate the safety and efficacy of TransCon TLR7/8 Agonist, as monotherapy and in combination with pembrolizumab, in cancer patients is currently ongoing (transcendIT-101; NCT04799054).

TransCon IL-2 ß/γ: a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2 variant with improved pharmacokinetics and potent activation of cytotoxic immune cells for the treatment of cancer.

BACKGROUND: Recombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe toxicities including cytokine storm and vascular leak syndrome (VLS). IL-2 promotes antitumor function of IL-2Rß/γ+ natural killer (NK) cells and CD8+, CD4+ and gamma delta (γδ) T cells. However, IL-2 also potently activates immunosuppressive IL-2Rα+ regulatory T cells (Tregs) and IL-2Rα+ eosinophils and endothelial cells, which may promote VLS. Aldesleukin is rapidly cleared requiring frequent dosing, resulting in high Cmax likely potentiating toxicity. Thus, IL-2 cancer immunotherapy has two critical drawbacks: potent activation of undesired IL-2Rα+ cells and suboptimal pharmacokinetics with high Cmax and short half-life. METHODS: TransCon IL-2 ß/γ was designed to optimally address these drawbacks. To abolish IL-2Rα binding yet retain strong IL-2Rß/γ activity, IL-2 ß/γ was created by permanently attaching a small methoxy polyethylene glycol (mPEG) moiety in the IL-2Rα binding site. To improve pharmacokinetics, IL-2 ß/γ was transiently attached to a 40 kDa mPEG carrier via a TransCon (transient conjugation) linker creating a prodrug, TransCon IL-2 ß/γ, with sustained release of IL-2 ß/γ. IL-2 ß/γ was characterized in binding and primary cell assays while TransCon IL-2 ß/γ was studied in tumor-bearing mice and cynomolgus monkeys. RESULTS: IL-2 ß/γ demonstrated selective and potent human IL-2Rß/γ binding and activation without IL-2Rα interactions. TransCon IL-2 ß/γ showed slow-release pharmacokinetics with a low Cmax and a long (>30 hours) effective half-life for IL-2 ß/γ in monkeys. In mouse tumor models, TransCon IL-2 ß/γ promoted CD8+ T cell and NK cell activation and antitumor activity. In monkeys, TransCon IL-2 ß/γ induced robust activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells, Tregs and eosinophils, with no evidence of cytokine storm or VLS. Similarly, IL-2 ß/γ enhanced proliferation and cytotoxicity of primary human CD8+ T cells, NK cells and γδ T cells. SUMMARY: TransCon IL-2 ß/γ is a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2. It has remarkable and durable pharmacodynamic effects in monkeys and potential for improved clinical efficacy and tolerability compared with aldesleukin. TransCon IL-2 ß/γ is currently being evaluated in a Phase 1/2 clinical trial (NCT05081609).

Intraocular Lens Calculation after Refractive Surgery: A Long-Term Retrospective Comparison of Eight Formulas.

The aim of this study was to compare the accuracy of 8 IOL power calculation formulas for eyes post-refractive surgery. In this Retrospective study, a chart review and data analysis of post-corneal refractive surgery patients who subsequently underwent cataract surgery with IOL implantation in Tertiary surgical center, Draper, UT, USA. The surgery was done in a single surgical center in Draper, UT by one surgeon. The study was approved by the organization's ethics board. The IOL power formulas used were Barrett True K (BTK), Average Pupil Power (APP), Shammas, Haigis, Galilei, Potvin-Hill Pentacam (PVP), OCT and Barrett True K No History (BTKNH). The percent of time each formula was within ±0.5 D and ±0.75 D of refractive prediction error was calculated. Statistical analysis was performed comparing these 8 methodologies at four post-operative follow-up time points and on the summative time points. Mean follow-up time periods were: 4 weeks, 3 months, 6 months, and 12 months. A total of 64 eyes were included in the study. All IOL formulas showed a myopic trend except APP and Shammas, which showed a hyperopic trend. All tests showed a statistically significant mean absolute value difference from zero. OCT, BTKNH, and BTK had consistently high percentages within ±0.5D and ±0.75 D of refractive error. Linear mixed model analysis showed a statistically significant change in predictive value over time for all formulas. Linear mixed model analysis suggests that it is inadequate to evaluate the performance of IOL power formulae in the short term. Longer-term follow-up is needed to determine accuracy as several factors can result in refractive changes greater than 3 months postoperatively. Our analysis did not demonstrate any formula that was clearly superior to the other methods for predicting IOL power at any time point.

Visual Prognosis after Explantation of Small-Aperture Corneal Inlays in Presbyopic Eyes: A Case Series.

The purpose of this study was to report visual prognosis after explantation of a small-aperture corneal inlay used for the treatment of presbyopia. This is a retrospective case series conducted at a single site in Draper, Utah, USA (Hoopes Vision). Medical records of 176 patients who had received a small-aperture corneal inlay (KAMRA™, AcuFocus Inc., Irvine, CA, USA) were reviewed. Patients who had undergone explantation of the device were identified. Uncorrected distance visual acuity (UDVA), uncorrected near visual acuity (UNVA), corrected distance visual acuity (CDVA), and manifest refraction spherical equivalent (MRSE) were measured pre-implantation, post-implantation, pre-explantation, and post-explantation of the inlay. Ten eyes from ten patients were included in this study. The explantation rate was 5.7% over 31 months, with blurry vision as the most common complaint. After explantation, six patients achieved pre-implantation UDVA, and six achieved pre-implantation UNVA. Eight of nine patients who underwent final manifest refraction achieved pre-operative CDVA. All patients had residual donut-shaped corneal haze in the stroma at the previous position of the inlay. All patients experienced improvement in haze with 20% experiencing complete resolution. The degree of stromal haze was not related to the duration of implantation. Of the subset of patients who underwent explantation of their small-aperture corneal inlay, there was persistent loss of CDVA in 10%. The majority of patients experienced some level of residual stromal haze, which may contribute to deficits in UNVA and CDVA in few patients. A hyperopic shift induced by the corneal inlay may contribute to the blurry vision these patients experienced; there was a reduction of this shift post-explantation. While this device is removable, patients should expect some post-explantation changes such as residual haze with a small subset experiencing persistent deficits in CDVA.

Special Care in Lichen Planus Patients Undergoing LASIK: A Review Article.

Laser-Assisted in Situ Keratomileusis (LASIK) is a common surgery for the correction of refractive errors. The majority of patients who undergo this procedure often have excellent results. However, uncontrolled autoimmune disorders and dry eye have both been listed as contraindications to this surgery. Lichen planus (LP) is an autoimmune, inflammatory disorder that characteristically affects mucocutaneous membranes. The etiology is unknown, but it most commonly affects middle-aged adults and presents with bilateral, purple papules. Clinical presentation is used to diagnose the condition, and a punch biopsy is confirmatory. LP may present with multiple different symptoms depending on the type, with ocular manifestations being rare. Multiple viruses and autoimmune conditions have been associated with the disorder, and physicians should take care when gathering a full history of the patient. Exacerbation of symptoms may happen if mood disorders such as depression and anxiety are not well controlled. There are several additional factors physicians must carefully consider before recommending LASIK to patients with LP. These include lichenoid reactions, current medications, and past or present ocular lesions. LASIK may be carefully considered in patients with well-controlled LP in the absence of ocular manifestations. Patients with ocular LP are not candidates for LASIK.

Visual Prognosis after Explantation of a Corneal Shape-Changing Hydrogel Inlay in Presbyopic Eyes.

The purpose of this case series is to report visual outcomes in patients who underwent explantation of the Raindrop® hydrogel corneal inlay. Retrospective chart review comprising four cases of explantation of the Raindrop® corneal shape-changing hydrogel inlay: pre-implantation, pre-explantation, and post-explantation values for uncorrected distance visual acuity, uncorrected near visual acuity, and corrected distance visual acuity (CDVA) were measured; keratometric and tomographic data were collected using the Pentacam system (Oculus, Inc). Three eyes were explanted for progressive haze after implantation that persisted even after removal; one eye was explanted due to poor visual acuity with no haze formation. All patients experienced decreased unaided and corrected distance visual acuity. Persistent increase in corneal thickness and mean keratometry was noted post-explantation. All four patients regained their original near visual acuities, but one patient had persistent one-line loss in CDVA. There are long lasting tomographic corneal changes following Raindrop inlay explantation. In addition, persistent increased corneal thickness could be related to semi-permanent changes in corneal structure and may account for residual haze experienced by patients. After explantation, patients may not return to baseline CDVA.

Comparative Analysis of LASIK Flap Diameter and its Centration Using Two Different Femtosecond Lasers.

The aim of this study was to compare the diameter, accuracy, variability, and centration with respect to the limbus of corneal flaps created by two femtosecond lasers, the VisuMax, and Wavelight FS200, for laser in situ keratomileusis (LASIK) and how these flaps affect visual outcomes. This is a retrospective chart review of flap morphology created during LASIK Surgery. Overall, 168 eyes underwent flap creation using the WaveLight FS200 laser, and on 189 eyes, the VisuMax laser was used. Of these total number, flap morphology was analyzed in a random sample of 158 eyes; 80 with the Visumax laser and 78 with the WaveLight FS200 laser. Intraoperative photos of the flaps taken by the Wavelight Allegretto EX500 were analyzed. Flap diameters and centration were measured using Adobe Acrobat Pro. All patients had visual acuity measurements including uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), spherical equivalent refraction (SE) and refractive astigmatism recorded three months postoperatively. Greater than 90% of patients in both groups achieved a UDVA of 20/20 postoperatively. The mean difference between targeted and achieved flap diameter was 0.50 +/- 0.15 mm in the VisuMax group and 0.35 +/- 0.15 millimeters (mm) in the FS200 group (P<0.01). The flap diameters of the VisuMax group were more precise with a variance of 0.024 mm compared to a variance of 0.038 mm in the FS200 group (P<0.05). VisuMax flaps were more nasally displaced (log(NA/TA) = -0.21 +/- 0.10 mm) compared to the FS200 flaps (log(NA/TA) = 0.03 +/- 0.10 mm), (P< 0.01). We concluded that both the VisuMax and FS200 created flaps larger than the preoperative targeted diameter. VisuMax created corneal flaps that had a greater degree of deviation from the targeted diameter when compared to flaps from the FS200. However, there was less variance in the VisuMax flap diameter. In addition, VisuMax flaps were more nasally displaced. There were no statistically significant differences in visual outcomes when comparing the two femtosecond lasers.

Should Patients with Hidradenitis Suppurativa Undergo LASIK?

Hidradenitis suppurativa (HS) is a relatively common chronic inflammatory disease with immune dysregulation. While eye manifestations of HS are rare, a dilemma arises when these patients seek treatment for refractive errors. Although excimer laser surgery can be safely performed in patients with autoimmune and immune-mediated inflammatory disease, there are caveats. Aside from the routine laser-assisted in situ keratomileusis (LASIK) screening tests, in some instances, we recommend additional screening tests in patients with HS, such as dry eye tests, consultation with specialists regarding HS diagnosis and treatment, careful assessment of the eyelids and periorbital structures, and thorough history of past and current lesions and treatments. After these patients undergo LASIK, careful, frequent, and long-term follow-up is necessary. Any adverse event or complication should be managed immediately. FUNDING: Research to Prevent Blindness funded the study. Hoopes Vision funded the Rapid Service Fees.

Dyskeratosis Congenita and Corneal Refractive Surgery.

Dyskeratosis congenita is a syndrome of bone marrow failure secondary to unstable telomeres. It is characterized by a range of mucocutaneous diseases. Due to premature telomere shortening, these patients have limbal stem cell deficiency leading to poor regeneration and maintenance of the cornea. Many of these patients will require hematopoietic stem cell transplant in their lifetime, which poses a significant risk for acute and chronic graft-versus-host disease with and without ocular manifestations. We advise against elective corneal refractive surgery in patients with dyskeratosis congenita due to the compounded and long-term risks of delayed healing secondary to limbal stem cell deficiency and ocular complications of graft-versus-host disease post-allogeneic hematopoietic stem cell transplant.

Keratoconus Screening in Elementary School Children.

Keratoconus is a degenerative structural disease of the cornea. Progression leads to poor acuity that is not easily correctable by standard means. New treatments, such as collagen cross-linking, lead to better long-term outcomes if performed early in the disease course. Currently, children in the USA are screened for acuity in school, but not for keratoconus. Due to the severity of the disease in children, we recommend topographic screening in elementary schools as a way to provide early detection and treatment.Funding Research to Prevent Blindness provided funding for the Rapid Service Fees.

Should I Get LASIK If I'm Breastfeeding?

Changes in keratometric values and refraction can occur during pregnancy. For this reason, changing a patient's refractive prescription or undergoing corneal refractive surgery is not recommended during pregnancy. However, the extent to which these corneal changes persist during lactation is not as well reported. Pregnancy and lactation lead to hormonal changes that affect the corneal structure. LASIK, or other types of refractive surgery, is not recommended until all of the following conditions are met: cessation of lactation, the return of regular menses, and a return to pre-pregnancy refraction. Additionally, patients should be cautioned that refractive regression may occur if they become pregnant within 1 year of LASIK. FUNDING: Research to Prevent Blindness, NY, USA.

GITR Agonism Enhances Cellular Metabolism to Support CD8+ T-cell Proliferation and Effector Cytokine Production in a Mouse Tumor Model.

GITR is a costimulatory receptor currently undergoing phase I clinical trials. Efficacy of anti-GITR therapy in syngeneic mouse models requires regulatory T-cell depletion and CD8+ T-cell costimulation. It is increasingly appreciated that immune cell proliferation and function are dependent on cellular metabolism. Enhancement of diverse metabolic pathways leads to different immune cell fates. Little is known about the metabolic effects of GITR agonism; thus, we investigated whether costimulation via GITR altered CD8+ T-cell metabolism. We found activated, GITR-treated CD8+ T cells upregulated nutrient uptake, lipid stores, glycolysis, and oxygen consumption rate (OCR) in vitro Using MEK, PI3Kδ, and metabolic inhibitors, we show increased metabolism is required, but not sufficient, for GITR antibody (DTA-1)-induced cellular proliferation and IFNγ production. In an in vitro model of PD-L1-induced CD8+ T-cell suppression, GITR agonism alone rescued cellular metabolism and proliferation, but not IFNγ production; however, DTA-1 in combination with anti-PD-1 treatment increased IFNγ production. In the MC38 mouse tumor model, GITR agonism significantly increased OCR and IFNγ and granzyme gene expression in both tumor and draining lymph node (DLN) CD8+ T cells ex vivo, as well as basal glycolysis in DLN and spare glycolytic capacity in tumor CD8+ T cells. DLN in GITR-treated mice showed significant upregulation of proliferative gene expression compared with controls. These data show that GITR agonism increases metabolism to support CD8+ T-cell proliferation and effector function in vivo, and that understanding the mechanism of action of agonistic GITR antibodies is crucial to devising effective combination therapies. Cancer Immunol Res; 6(10); 1199-211. ©2018 AACR.

Provider variability in intensity modulated radiation therapy utilization among Medicare beneficiaries in the United States.

BACKGROUND: In this study, we sought to examine the variation in intensity modulated radiation therapy (IMRT) use among radiation oncology providers. METHODS AND MATERIALS: The Medicare Physician and Other Supplier Public Use File was queried for radiation oncologists practicing during 2014. Healthcare Common Procedural Coding System code 77301 was designated as IMRT planning with metrics including number of total IMRT plans, rate of IMRT utilization, and number of IMRT plans per distinct beneficiary. RESULTS: Of 2759 radiation oncologists, the median number of total IMRT plans was 26 (mean, 33.4; standard deviation, 26.2; range, 11-321) with a median IMRT utilization rate of 36% (mean, 43%; standard deviation, 25%; range, 4% to 100%) and a median number of IMRT plans per beneficiary of 1.02 (mean, 1.07; range, 1.00-3.73). On multivariable analysis, increased IMRT utilization was associated with male sex, academic practice, technical fee billing, freestanding practice, practice in a county with 21 or more radiation oncologists, and practice in the southern United States (P < .05). The top 1% of users (28 providers) billed a mean 181 IMRT plans with an IMRT utilization rate of 66% and 1.52 IMRT plans per beneficiary. Of these 28 providers, 24 had billed technical fees, 25 practiced in freestanding clinics, and 20 practiced in the South. CONCLUSIONS: Technical fee billing, freestanding practice, male sex, and location in the South were associated with increased IMRT use. A small group of outliers shared several common demographic and practice-based characteristics.

Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP).

BACKGROUND: Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity. METHODS: Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates. RESULTS: Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples. CONCLUSIONS: SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.

Development and validation of a single-cell network profiling assay-based classifier to predict response to induction therapy in paediatric patients with de novo acute myeloid leukaemia: a report from the Children's Oncology Group.

Single cell network profiling (SCNP) is a multi-parameter flow cytometry technique for simultaneous interrogation of intracellular signalling pathways. Diagnostic paediatric acute myeloid leukaemia (AML) bone marrow samples were used to develop a classifier for response to induction therapy in 53 samples and validated in an independent set of 68 samples. The area under the curve of a receiver operating characteristic curve (AUC(ROC)) was calculated to be 0·85 in the training set and after exclusion of induction deaths, the AUC(ROC) of the classifier was 0·70 (P = 0·02) and 0·67 (P = 0·04) in the validation set when induction deaths (intent to treat) were included. The highest predictive accuracy was noted in the cytogenetic intermediate risk patients (AUC(ROC) 0·88, P = 0·002), a subgroup that lacks prognostic/predictive biomarkers for induction response. Only white blood cell count and cytogenetic risk were associated with response to induction therapy in the validation set. After controlling for these variables, the SCNP classifier score was associated with complete remission (P = 0·017), indicating that the classifier provides information independent of other clinical variables that were jointly associated with response. This is the first validation of an SCNP classifier to predict response to induction chemotherapy. Herein we demonstrate the usefulness of quantitative SCNP under modulated conditions to provide independent information on AML disease biology and induction response.

Maternal decidual macrophages inhibit NK cell killing of invasive cytotrophoblasts during human pregnancy.

Human pregnancy is an immunological paradox. Semiallogeneic (fetal) placental cells (extravillous cytotrophoblasts [CTBs]) invade the uterine lining (decidua), which contains a unique decidual natural killer (dNK) cell population, identified by the cell surface phenotype CD56(bright) CD16(-) CD3(-) and CD14(+) CD206(+) macrophages (dMac). Previous reports suggested that human dNK cells are not a threat to the fetoplacental unit because they are anergic. In contrast, here we showed that purified and exogenously stimulated dNK cells are capable killers of cellular targets, including semiallogeneic CTBs. However, dMacs in the decidual leukocyte (DL) population restrained dNK killing through a transforming growth factor beta1 (TGF-beta1)-dependent mechanism. Our findings support a new model whereby dNK cells, capable of killing CTBs, are prevented from doing so by neighboring macrophages, thus protecting the fetal cells from NK cell attack. We speculate that this mechanism would inhibit dNK cell-mediated killing, even under conditions where high levels of cytokines may stimulate dNK cells, which could pose a threat to the developing placenta.

Functional pathway analysis using SCNP of FLT3 receptor pathway deregulation in AML provides prognostic information independent from mutational status.

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.

AKT signaling as a novel factor associated with in vitro resistance of human AML to gemtuzumab ozogamicin.

Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1) derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ(1) resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1). Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.

Assessing signaling pathways associated with in vitro resistance to cytotoxic agents in AML.

This study uses single cell network profiling (SCNP) to characterize biological pathways associated with in vitro resistance or sensitivity to chemotherapeutics commonly used in acute myeloid leukemia (AML) (i.e. cytarabine/daunorubicin, gemtuzumab ozogamicin (GO), decitabine, azacitidine, clofarabine). Simultaneous measurements at the single cell level of changes in DNA damage, apoptosis and signaling pathway responses in AML blasts incubated in vitro with the above drugs showed distinct profiles for each sample and mechanistically different profiles between distinct classes of agents. Studies are ongoing to assess the clinical predictive value of these findings.