Comprehensive single institute experience with melanoma TIL: Long term clinical results, toxicity profile, and prognostic factors of response.

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) mediates objective responses in 30% to 50% of patients with metastatic melanoma according to multiple, small phase 2 trials. Here we report the long-term clinical results, intent-to-treat analysis, predictors of response and toxicity profile in a large patient cohort. A total of 179 refractory melanoma patients were enrolled in the ACT trial. TIL were administered in combination with high-dose bolus interleukin-2 following preconditioning with cyclophosphamide and fludarabine. Patients were followed-up for a median of 7.2 years. A total of 107 (60%) of 179 enrolled patients were treated. The main reason for the drop out of the study was clinical deterioration. Of 103 evaluated patients, 29 patients (28%) achieved an objective response (OR), including complete remission (8%) or partial response (20%). Sixteen pateints exhibited stable disease. Predictors of response were performance status, time of TIL in culture and CD8 frequency in the infusion product. The absolute lymphocyte count 1 and 2 weeks after TIL infusion was the most predictive parameter of response. With a medium follow-up time of 7.2 years, OR patients reached a median overall survival (OS) of 58.45 months and a median progression-free survival (PFS) of 15.43 months, as compared with nonresponders, with 6.73 months OS and 2.60 months PFS. By 6 years, 50% of OR patients were alive and 43% had no documented progression. TIL ACT can yield durable objective responses, even as salvage therapy in highly advanced metastatic melanoma patients.

Locally produced CD19 CAR T cells leading to clinical remissions in medullary and extramedullary relapsed acute lymphoblastic leukemia.

Autologous CD19 chimeric-antigen receptor (CAR) T cells demonstrated remarkable remission rates in relapsed and refractory acute lymphoblastic leukemia (R/R ALL). Here, we report results from a phase 1b/2 study of in-house produced CD19 CAR with a CD28 costimulatory domain. Twenty-one patients with R/R ALL were enrolled, and 20 infused. The median age was 11 years (range, 5-48). Patients had a median of 4 prior regimens, including blinatumomab in 6 and prior stem-cell transplantation in 10. In total 8 patients had extramedullary (EM) leukemic involvement, and prior to lymphodepletion and CAR 7 had active lesions, a group underrepresented in previous trials. In vivo expansion of CAR T cells was observed in 18 patients. In total 16 patients developed cytokine release syndrome, and 11 patients developed neurotoxicity, with no toxic deaths. All responding patients were referred to an allogeneic hematopoietic stem-cell transplantation. The remission rate was 90%, including resolution of all refractory EM sites. Four responding patients relapsed, 3 who had a PCR-MRD positive remission at 28 days following CAR-T cells and 1 patient 21 months after an MRD-negative response. The estimated 1-year event-free survival and overall survival are 73% and 90%, respectively. Patients with R/R EM ALL may also benefit from CAR-T cell therapy.

An improved design of optical sensor for long-term measurement of arterial blood flow waveform.

We present here the improved design and development of optical sensor for non-invasive measurements of arterial blood flow waveform. The sensor is based on a physical principle of reflective photoplethysmography (PPG). As the light source we used serially connected infrared diodes whereas NPN silicon phototransistors were used as light detectors. The electronic components were molded into square package and poured with silicone. Such preparation produced an elastic superficies that allowed excellent attachment of the sensor on the skin's surface. Moreover, a serial connection of infrared diodes and phototransistors completely eliminated signal artifacts caused by minor muscle contractions. The sensor recording performances were examined at the photoplethysmographic sites on three different arteries; the commune carotid, femoral and radial and, on each site the sensor demonstrated remarkable capability to make a consistent, reproducible measurements. Because of the advantageous physical and electrical properties, the new sensor is suitable for various cardiovascular diagnostics procedures, especially when long-term measurements of arterial blood flow waveform are required, for monitoring of different parameters in cardiovascular units and for research.

A comparative analysis of total serum miRNA profiles identifies novel signature that is highly indicative of metastatic melanoma: a pilot study.

CONTEXT: Quantification of circulating microRNAs (miRNAs) has recently become feasible and reliable, with most efforts focusing on miRNAs overexpressed by cancer cells. OBJECTIVE: Identification of a characteristic circulating miRNAs profile in melanoma patients. METHODS: We conducted a pilot study comprised of unbiased qPCR comparison of serum miRNA profiles between metastatic melanoma patients and healthy donors. RESULTS: Loss of two normal serum-miRNAs, miR-29c and miR-324-3p, is highly indicative of metastatic melanoma. Hierarchical clustering analysis supported the results and clearly distinguished melanoma patients from healthy donors, metastatic colon and renal cancer patients. DISCUSSION AND CONCLUSIONS: This approach is independent of tumor heterogeneity and is expected to have superior biomarker performances.

I-motif nanospheres: unusual self-assembly of long cytosine strands.

The synthesis and characterization of novel DNA structures based on tetraplex cytosine (C) arrangements, known as i-motifs or i-tetraplexes, is reported. Atomic force microscopy (AFM) investigation shows that long C-strands in mild acidic conditions form compact spherically shaped nanostructures. The DNA nanospheres are characterized by a typical uniform shape and narrow height distribution. Electrostatic force microscopy (EFM) measurements performed on the i-motif spheres clearly show their electrical polarizability. Further investigations by scanning tunneling microscopy (STM) at ultrahigh vacuum reveals that the structures exhibit an average voltage gap of 1.9 eV, which is narrower than the voltage gap previously measured for poly(dG)-poly(dC) molecules in similar conditions.

Specific high-affinity binding of thiazole orange to triplex and G-quadruplex DNA.

Interaction of Thiazole Orange (TO) with double-, triple-, and quadruple-stranded forms of DNA was studied. We have demonstrated by UV-vis absorption, circular dichroism (CD), and fluorescence spectroscopy that TO binds with much higher affinity to triplex and G-quadruplex DNA structures compared to double-stranded (ds) DNA. Complexes of the dye with DNA triplexes and G-quadruplexes are very stable and do not dissociate during chromatography and gel electrophoresis. TO binding to either triple- or quadruple-stranded DNA structures results in a >1000-fold increase in dye fluorescence. The fluorescence titration data showed that TO to triad and tetrad ratios, in tight complexes with the triplex and the G-quadruplex, are equal to 0.5 and 1, respectively. Preferential binding of TO to triplexes and G-quadruplexes enables selective detection of only these DNA forms in gels in the absence of free TO in electrophoresis running buffer. We have also demonstrated that incubation of U2OS cells with submicromolar concentrations of TO results in preferential staining of certain areas in the nucleus in contrast to DAPI which binds to dsDNA and efficiently stains regions that are unstained with TO. We suggest that TO staining may be useful for the detection of noncanonical structural motifs in genomic DNA.

Synthesis and AFM characterization of poly(dG)-poly(dC)-gold nanoparticle conjugates.

In the present work, we have synthesized conjugates between the 5 nm gold nanoparticles (Au-NP) and 5' thiol-functionalized, 700 bp poly(dG)-poly(dC). We have completely separated and purified to homogeneity conjugates bearing different number of poly(dG)-poly(dC) molecules per Au-NP by electrophoresis and HPLC. The conjugates were directly visualized by atomic force microscopy. We have demonstrated that Au NP-bound poly(dG)-poly(dC) can be considerably extended by Klenow exo(-) polymerase in the presence of dCTP and dGTP.

Long-lived fluorescence of homopolymeric guanine-cytosine DNA duplexes.

The fluorescence spectrum of the homopolymeric double helix poly(dG) x poly(dC) is dominated by emission decaying on the nanosecond time-scale, as previously reported for the alternating homologue poly(dGdC) x poly(dGdC). Thus, energy trapping over long periods of time is a common feature of GC duplexes which contrast with AT duplexes. The impact of such behaviour on DNA photodamage needs to be evaluated.

Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin-biotin recognition.

We describe a method for the preparation of novel long (hundreds of nanometers), uniform, inter-molecular G4-DNA molecules composed of four parallel G-strands. The only long continuous G4-DNA reported so far are intra-molecular structures made of a single G-strand. To enable a tetra-molecular assembly of the G-strands we developed a novel approach based on avidin-biotin biological recognition. The steps of the G4-DNA production include: (i) Enzymatic synthesis of long poly(dG)-poly(dC) molecules with biotinylated poly(dG)-strand; (ii) Formation of a complex between avidin-tetramer and four biotinylated poly(dG)-poly(dC) molecules; (iii) Separation of the poly(dC) strands from the poly(dG)-strands, which are connected to the avidin; (iv) Assembly of the four G-strands attached to the avidin into tetra-molecular G4-DNA. The average contour length of the formed structures, as measured by AFM, is equal to that of the initial poly(dG)-poly(dC) molecules, suggesting a tetra-molecular mechanism of the G-strands assembly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA molecules having similar contour length. The CD spectra of the tetra- and mono-molecular G4-DNA are markedly different, suggesting different structural organization of these two types of molecules. The tetra-molecular G4-DNA nanostructures showed clear electrical polarizability. This suggests that they may be useful for molecular electronics.

Vesicle priming and recruitment by ubMunc13-2 are differentially regulated by calcium and calmodulin.

Ca2+ regulates multiple processes in nerve terminals, including synaptic vesicle recruitment, priming, and fusion. Munc13s, the mammalian homologs of Caenorhabditis elegans Unc13, are essential vesicle-priming proteins and contain multiple regulatory domains that bind second messengers such as diacylglycerol and Ca2+/calmodulin (Ca2+/CaM). Binding of Ca2+/CaM is necessary for the regulatory effect that allows Munc13-1 and ubMunc13-2 to promote short-term synaptic plasticity. However, the relative contributions of Ca2+ and Ca2+/CaM to vesicle priming and recruitment by Munc13 are not known. Here, we investigated the effect of Ca2+/CaM binding on ubMunc13-2 activity in chromaffin cells via membrane-capacitance measurements and a detailed simulation of the exocytotic machinery. Stimulating secretion under various basal Ca2+ concentrations from cells overexpressing either ubMunc13-2 or a ubMunc13-2 mutant deficient in CaM binding enabled a distinction between the effects of Ca2+ and Ca2+/CaM. We show that vesicle priming by ubMunc13-2 is Ca2+ dependent but independent of CaM binding to ubMunc13-2. However, Ca2+/CaM binding to ubMunc13-2 specifically promotes vesicle recruitment during ongoing stimulation. Based on the experimental data and our simulation, we propose that ubMunc13-2 is activated by two Ca2+-dependent processes: a slow activation mode operating at low Ca2+ concentrations, in which ubMunc13-2 acts as a priming switch, and a fast mode at high Ca2+ concentrations, in which ubMunc13-2 is activated in a Ca2+/CaM-dependent manner and accelerates vesicle recruitment and maturation during stimulation. These different Ca2+ activation steps determine the kinetic properties of exocytosis and vesicle recruitment and can thus alter plasticity and efficacy of transmitter release.

Predictors of tumor-infiltrating lymphocyte efficacy in melanoma.

In the past decades, the increasing knowledge in cellular immunology and tumor-host immune interactions, led to the development of immunotherapy approaches. Immunotherapy, based on adoptive cell transfer of ex vivo activated and expanded tumor-infiltrating T lymphocytes (TILs), has shown promising clinical results in patients with metastatic melanoma. TIL therapy yields response rates of around 50% and significant survival benefit in refractory melanoma patients, even after failing other immunotherapies, such as checkpoint inhibitors or cytokine-based therapy. Identifying predictors of TIL efficacy and detection of TIL subsets with specific reactivity against the patient's tumor might be an important milestone toward further improvement of clinical responses and prolonged survival.

Effect of viscosity on the wave propagation: Experimental determination of compression and expansion pulse wave velocity in fluid-fill elastic tube.

The velocity by which the disturbance travels through the medium is the wave velocity. Pulse wave velocity is one of the main parameters in hemodynamics. The study of wave propagation through the fluid-fill elastic tube is of great importance for the proper biophysical understanding of the nature of blood flow through of cardiovascular system. The effect of viscosity on the pulse wave velocity is generally ignored. In this paper we present the results of experimental measurements of pulse wave velocity (PWV) of compression and expansion waves in elastic tube. The solutions with different density and viscosity were used in the experiment. Biophysical model of the circulatory flow is designed to perform measurements. Experimental results show that the PWV of the expansion waves is higher than the compression waves during the same experimental conditions. It was found that the change in viscosity causes a change of PWV for both waves. We found a relationship between PWV, fluid density and viscosity.

Immunotherapy for the management of advanced melanoma: the next steps.

Melanoma is an immunogenic tumor that can induce a natural immune response. A number of immunotherapy-based approaches have been developed over the past decades, and certain degrees of effectiveness were achieved by the use of cytokines, adoptive cell transfer and T-cell immune modulators. Currently, interleukin-2 and the immune stimulatory antibody, ipilimumab, are the only two approved immunotherapies for metastatic melanoma, but various new therapies are in promising developmental stages. This comprehensive review will discuss the latest achievements of immunotherapy and emerging directions for the management of advanced melanoma.

Adoptive T-cell transfer in melanoma.

Immunotherapy holds a highly promising treatment approach for metastatic melanoma patients. Adoptive cell transfer (ACT) involves the ex vivo expansion of autologous antitumor reactive lymphocytes and their reinfusion into lymphodepleted patients, accompanied by IL-2 administration. ACT with tumor-infiltrating T lymphocytes demonstrates objective clinical responses in 50-72% of the patients, including 10-40% complete responses and was shown to produce durable disease control with long progression-free survival. Tumor-infiltrating T-lymphocyte ACT might even have curative potential as the vast majority of the complete responders are without any evidence of disease many years after treatment. Other adoptive transfer studies employ the genetic modification of T lymphocytes with genes encoding tumor-specific T cell receptors or antibody-based chimeric antigen receptors. These approaches opened numerous possibilities to treat cancers other than melanoma. In this article we will summarize the ACT strategies in melanoma, the new developments in this field and combinations with other therapies.

Adoptive transfer of tumor-infiltrating lymphocytes in patients with metastatic melanoma: intent-to-treat analysis and efficacy after failure to prior immunotherapies.

PURPOSE: Adoptive cell transfer (ACT) using autologous tumor-infiltrating lymphocytes (TIL) was reported to yield objective responses in about 50% of metastatic patients with melanoma. Here, we present the intent-to-treat analysis of TIL ACT and analyze parameters predictive to response as well as the impact of other immunotherapies. EXPERIMENTAL DESIGN: Eighty patients with stage IV melanoma were enrolled, of which 57 were treated with unselected/young TIL and high-dose interleukin-2 (IL-2) following nonmyeloablative lymphodepleting conditioning. RESULTS: TIL cultures were established from 72 of 80 enrolled patients. Altogether 23 patients were withdrawn from the study mainly due to clinical deterioration during TIL preparation. The overall response rate and median survival was 29% and 9.8 months for enrolled patients and 40% and 15.2 months for treated patients. Five patients achieved complete and 18 partial remission. All complete responders are on unmaintained remission after a median follow-up of 28 months and the 3-year survival of responding patients was 78%. Multivariate analysis revealed blood lactate-dehydrogenase levels, gender, days of TIL in culture, and the total number of infused CD8+ cells as independent predictive markers for clinical outcome. Thirty-two patients received the CTLA-4-blocking antibody ipilimumab prior or post TIL infusion. Retrospective analysis revealed that nonresponders to ipilimumab or IL-2 based therapy had the same overall response rate to ACT as other patients receiving TIL. No additional toxicities to TIL therapy occurred following ipilimumab treatment. CONCLUSION: Adoptive transfer of TIL can yield durable and complete responses in patients with refractory melanoma, even when other immunotherapies have failed.

Establishment and large-scale expansion of minimally cultured "young" tumor infiltrating lymphocytes for adoptive transfer therapy.

Treatment of metastatic melanoma patients with adoptively transferred tumor infiltrating lymphocytes (TIL) has developed into an effective therapy. Various studies reported objective responses of 50% and more. The use of unselected, minimally cultured, bulk TIL (Young-TIL) has simplified the TIL production process and may therefore, allow the accessibility of this approach to cancer centers worldwide. This article describes the precise process leading to the large-scale production of Young-TIL for therapy. We have enrolled 55 melanoma patients and optimized their Young-TIL generation process. Young-TIL cultures were successfully established for 51 of 55 (93%) patients in 16.7 ± 5.5 days. In a large-scale expansion procedure Young-TIL of 32 patients were further expanded to treatment levels, resulting in a final number of 4.5 x 10¹° ± 2.0 x 10¹° TIL. Fifteen of 31 (48%) patients, who were evaluated, achieved a clinical response, including 4 complete and 11 partial responses. We confirmed the significant correlation between short culture duration, high number of infused cells, and tumor regression. A high percentage of CD8 T cells in the infusion product was beneficial to achieve an objective response. All responding patients were treated with Young-TIL cultures established in < 20 days. In summary, we describe here an efficient and reliable method to generate Young-TIL for adoptive transfer therapy, which may easily be adopted by other cancer centers and can lead to objective responses in 50% of refractory melanoma patients. In the future this approach may be used also in other types of malignancies.