Tumor mutational burden for the prediction of PD-(L)1 blockade efficacy in cancer: challenges and opportunities.

Tumor mutational burden (TMB) is a biomarker that measures the number of somatic mutations in a tumor's genome. TMB has emerged as a predictor of response to immune checkpoint inhibitors (ICIs) in various cancer types, and several studies have shown that patients with high TMB have better outcomes when treated with programmed death-ligand 1-based therapies. Recently, the Food and Drug Administration has approved TMB as a companion diagnostic for the use of pembrolizumab in solid tumors. However, despite its potential, the use of TMB as a biomarker for immunotherapy efficacy is limited by several factors. Here we review the limitations of TMB in predicting immunotherapy outcomes in patients with cancer and discuss potential strategies to optimize its use in the clinic.

Clonal KEAP1 mutations with loss of heterozygosity share reduced immunotherapy efficacy and low immune cell infiltration in lung adenocarcinoma.

BACKGROUND: KEAP1 mutations have been associated with reduced survival in lung adenocarcinoma (LUAD) patients treated with immune checkpoint inhibitors (ICIs), particularly in the presence of STK11/KRAS alterations. We hypothesized that, beyond co-occurring genomic events, clonality prediction may help identify deleterious KEAP1 mutations and their counterparts with retained sensitivity to ICIs. PATIENTS AND METHODS: Beta-binomial modelling of sequencing read counts was used to infer KEAP1 clonal inactivation by combined somatic mutation and loss of heterozygosity (KEAP1 C-LOH) versus partial inactivation [KEAP1 clonal diploid-subclonal (KEAP1 CD-SC)] in the Memorial Sloan Kettering Cancer Center (MSK) MetTropism cohort (N = 2550). Clonality/LOH prediction was compared to a streamlined clinical classifier that relies on variant allele frequencies (VAFs) and tumor purity (TP) (VAF/TP ratio). The impact of this classification on survival outcomes was tested in two independent cohorts of LUAD patients treated with immunotherapy (MSK/Rome N = 237; DFCI N = 461). Immune-related features were studied by exploiting RNA-sequencing data (TCGA) and multiplexed immunofluorescence (DFCI mIF cohort). RESULTS: Clonality/LOH inference in the MSK MetTropism cohort overlapped with a clinical classification model defined by the VAF/TP ratio. In the ICI-treated MSK/Rome discovery cohort, predicted KEAP1 C-LOH mutations were associated with shorter progression-free survival (PFS) and overall survival (OS) compared to KEAP1 wild-type cases (PFS log-rank P = 0.001; OS log-rank P < 0.001). Similar results were obtained in the DFCI validation cohort (PFS log-rank P = 0.006; OS log-rank P = 0.014). In both cohorts, we did not observe any significant difference in survival outcomes when comparing KEAP1 CD-SC and wild-type tumors. Immune deconvolution and multiplexed immunofluorescence revealed that KEAP1 C-LOH and KEAP1 CD-SC differed for immune-related features. CONCLUSIONS: KEAP1 C-LOH mutations are associated with an immune-excluded phenotype and worse clinical outcomes among advanced LUAD patients treated with ICIs. By contrast, survival outcomes of patients whose tumors harbored KEAP1 CD-SC mutations were similar to those with KEAP1 wild-type LUADs.

Dissecting the clinicopathologic, genomic, and immunophenotypic correlates of KRASG12D-mutated non-small-cell lung cancer.

BACKGROUND: Allele-specific KRAS inhibitors are an emerging class of cancer therapies. KRAS-mutant (KRASMUT) non-small-cell lung cancers (NSCLCs) exhibit heterogeneous outcomes, driven by differences in underlying biology shaped by co-mutations. In contrast to KRASG12C NSCLC, KRASG12D NSCLC is associated with low/never-smoking status and is largely uncharacterized. PATIENTS AND METHODS: Clinicopathologic and genomic information were collected from patients with NSCLCs harboring a KRAS mutation at the Dana-Farber Cancer Institute (DFCI), Memorial Sloan Kettering Cancer Center, MD Anderson Cancer Center, and Imperial College of London. Multiplexed immunofluorescence for CK7, programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), Foxp3, and CD8 was carried out on a subset of samples with available tissue at the DFCI. Clinical outcomes to PD-(L)1 inhibition ± chemotherapy were analyzed according to KRAS mutation subtype. RESULTS: Of 2327 patients with KRAS-mutated (KRASMUT) NSCLC, 15% (n = 354) harbored KRASG12D. Compared to KRASnon-G12D NSCLC, KRASG12D NSCLC had a lower pack-year (py) smoking history (median 22.5 py versus 30.0 py, P < 0.0001) and was enriched in never smokers (22% versus 5%, P < 0.0001). KRASG12D had lower PD-L1 tumor proportion score (TPS) (median 1% versus 5%, P < 0.01) and lower tumor mutation burden (TMB) compared to KRASnon-G12D (median 8.4 versus 9.9 mt/Mb, P < 0.0001). Of the samples which underwent multiplexed immunofluorescence, KRASG12D had lower intratumoral and total CD8+PD1+ T cells (P < 0.05). Among 850 patients with advanced KRASMUT NSCLC who received PD-(L)1-based therapies, KRASG12D was associated with a worse objective response rate (ORR) (15.8% versus 28.4%, P = 0.03), progression-free survival (PFS) [hazard ratio (HR) 1.51, 95% confidence interval (CI) 1.45-2.00, P = 0.003], and overall survival (OS; HR 1.45, 1.05-1.99, P = 0.02) to PD-(L)1 inhibition alone but not to chemo-immunotherapy combinations [ORR 30.6% versus 35.7%, P = 0.51; PFS HR 1.28 (95%CI 0.92-1.77), P = 0.13; OS HR 1.36 (95%CI 0.95-1.96), P = 0.09] compared to KRASnon-G12D. CONCLUSIONS: KRASG12D lung cancers harbor distinct clinical, genomic, and immunologic features compared to other KRAS-mutated lung cancers and worse outcomes to PD-(L)1 blockade. Drug development for KRASG12D lung cancers will have to take these differences into account.

Antibiotic-exposed patients with non-small-cell lung cancer preserve efficacy outcomes following first-line chemo-immunotherapy.

BACKGROUND: Prior antibiotic therapy (pATB) is known to impair efficacy of single-agent immune checkpoint inhibitors (ICIs), potentially through the induction of gut dysbiosis. Whether ATB also affects outcomes to chemo-immunotherapy combinations is still unknown. PATIENTS AND METHODS: In this international multicentre study, we evaluated the association between pATB, concurrent ATB (cATB) and overall survival (OS), progression-free survival (PFS) and objective response rate (ORR) in patients with non-small-cell lung cancer (NSCLC) treated with first-line chemo-immunotherapy at eight referral institutions. RESULTS: Among 302 patients with stage IV NSCLC, 216 (71.5%) and 61 (20.2%) patients were former and current smokers, respectively. Programmed death-ligand 1 tumour expression in assessable patients (274, 90.7%) was ≥50% in 76 (25.2%), 1%-49% in 84 (27.9%) and <1% in 113 (37.5%). Multivariable analysis showed pATB-exposed patients to have similar OS {hazard ratio (HR) = 1.42 [95% confidence interval (CI): 0.91-2.22]; P = 0.1207} and PFS [HR = 1.12 (95% CI: 0.76-1.63); P = 0.5552], compared to unexposed patients, regardless of performance status. Similarly, no difference with respect to ORR was found across pATB exposure groups (42.6% versus 57.4%, P = 0.1794). No differential effect was found depending on pATB exposure duration (≥7 versus <7 days) and route of administration (intravenous versus oral). Similarly, cATB was not associated with OS [HR = 1.29 (95% CI: 0.91-1.84); P = 0.149] and PFS [HR = 1.20 (95% CI: 0.89-1.63); P = 0.222] when evaluated as time-varying covariate in multivariable analysis. CONCLUSIONS: In contrast to what has been reported in patients receiving single-agent ICIs, pATB does not impair clinical outcomes to first-line chemo-immunotherapy of patients with NSCLC. pATB status should integrate currently available clinico-pathologic factors for guiding first-line treatment decisions, whilst there should be no concern in offering cATB during chemo-immunotherapy when needed.

Linac4 H⁻ ion sources.

CERN's 160 MeV H(-) linear accelerator (Linac4) is a key constituent of the injector chain upgrade of the Large Hadron Collider that is being installed and commissioned. A cesiated surface ion source prototype is being tested and has delivered a beam intensity of 45 mA within an emittance of 0.3 π ⋅ mm ⋅ mrad. The optimum ratio of the co-extracted electron- to ion-current is below 1 and the best production efficiency, defined as the ratio of the beam current to the 2 MHz RF-power transmitted to the plasma, reached 1.1 mA/kW. The H(-) source prototype and the first tests of the new ion source optics, electron-dump, and front end developed to minimize the beam emittance are presented. A temperature regulated magnetron H(-) source developed by the Brookhaven National Laboratory was built at CERN. The first tests of the magnetron operated at 0.8 Hz repetition rate are described.

Charge neutralized low energy beam transport at Brookhaven 200 MeV linac.

The H(-) magnetron source provides about 100 mA H(-) beam to be match into the radio-frequency quadrupole accelerator. As H(-) beam traverses through low energy transport, it ionizes the residual gas and electrons are repelled and positive ions are trapped in the beam, due to negative potential of the beam, providing charge neutralization for the H(-) beam. The neutralization time for the critical density depends upon the background gas and its pressure. Critical density for xenon gas at 35 keV is about 43 times smaller than that of hydrogen and stripping cross section is only 5 times than that of hydrogen gas. We are using xenon gas to reduce neutralization time and to improve transmission through the 200 MeV linac. We are also using pulse nitrogen gas to improve transmission and stability of polarized H(-) beam from optically pumped polarized ion source.

Ion optics of RHIC electron beam ion source.

RHIC electron beam ion source has been commissioned to operate as a versatile ion source on RHIC injection facility supplying ion species from He to Au for Booster. Except for light gaseous elements RHIC EBIS employs ion injection from several external primary ion sources. With electrostatic optics fast switching from one ion species to another can be done on a pulse to pulse mode. The design of an ion optical structure and the results of simulations for different ion species are presented. In the choice of optical elements special attention was paid to spherical aberrations for high-current space charge dominated ion beams. The combination of a gridded lens and a magnet lens in LEBT provides flexibility of optical control for a wide range of ion species to satisfy acceptance parameters of RFQ. The results of ion transmission measurements are presented.

Investigation of ISIS and Brookhaven National Laboratory ion source electrodes after extended operation.

Linac4 accelerator of Centre Européen de Recherches Nucléaires is under construction and a RF-driven H(-) ion source is being developed. The beam current requirement for Linac4 is very challenging: 80 mA must be provided. Cesiated plasma discharge ion sources such as Penning or magnetron sources are also potential candidates. Accelerator ion sources must achieve typical reliability figures of 95% and above. Investigating and understanding the underlying mechanisms involved with source failure or ageing is critical when selecting the ion source technology. Plasma discharge driven surface ion sources rely on molybdenum cathodes. Deformation of the cathode surfaces is visible after extended operation periods. A metallurgical investigation of an ISIS ion source is presented. The origin of the deformation is twofold: Molybdenum sputtering by cesium ions digs few tenths of mm cavities while a growth of molybdenum is observed in the immediate vicinity. The molybdenum growth under hydrogen atmosphere is hard and loosely bound to the bulk. It is, therefore, likely to peel off and be transported within the plasma volume. The observation of the cathode, anode, and extraction electrodes of the magnetron source operated at BNL for two years are presented. A beam simulation of H(-), electrons, and Cs(-) ions was performed with the IBSimu code package to qualitatively explain the observations. This paper describes the operation conditions of the ion sources and discusses the metallurgical analysis and beam simulation results.

Development of electron beam ion source charge breeder for rare isotopes at Californium Rare Isotope Breeder Upgrade.

Recently, the Californium Rare Isotope Breeder Upgrade (CARIBU) to the Argonne Tandem Linac Accelerator System (ATLAS) was commissioned and became available for production of rare isotopes. Currently, an electron cyclotron resonance ion source is used as a charge breeder for CARIBU beams. To further increase the intensity and improve the purity of neutron-rich ion beams accelerated by ATLAS, we are developing a high-efficiency charge breeder for CARIBU based on an electron beam ion source (EBIS). The CARIBU EBIS charge breeder will utilize the state-of-the-art EBIS technology recently developed at Brookhaven National Laboratory (BNL). The electron beam current density in the CARIBU EBIS trap will be significantly higher than that in existing operational charge-state breeders based on the EBIS concept. The design of the CARIBU EBIS charge breeder is nearly complete. Long-lead components of the EBIS such as a 6-T superconducting solenoid and an electron gun have been ordered with the delivery schedule in the fall of 2011. Measurements of expected breeding efficiency using the BNL Test EBIS have been performed using a Cs(+) surface ionization ion source for external injection in pulsed mode. In these experiments we have achieved ∼70% injection∕extraction efficiency and breeding efficiency into the most abundant charge state of ∼17%.

The Brookhaven National Laboratory electron beam ion source for RHIC.

As part of a new heavy ion preinjector that will supply beams for the Relativistic Heavy Ion Collider and the National Aeronautics and Space Administration Space Radiation Laboratory, construction of a new electron beam ion source (EBIS) is now being completed. This source, based on the successful prototype Brookhaven National Laboratory Test EBIS, is designed to produce milliampere level currents of all ion species, with q/m=(1/6)-(1/2). Among the major components of this source are a 5 T, 2-m-long, 204 mm diameter warm bore superconducting solenoid, an electron gun designed to operate at a nominal current of 10 A, and an electron collector designed to dissipate approximately 300 kW of peak power. Careful attention has been paid to the design of the vacuum system, since a pressure of 10(-10) Torr is required in the trap region. The source includes several differential pumping stages, the trap can be baked to 400 C, and there are non-evaporable getter strips in the trap region. Power supplies include a 15 A, 15 kV electron collector power supply, and fast switchable power supplies for most of the 16 electrodes used for varying the trap potential distribution for ion injection, confinement, and extraction. The EBIS source and all EBIS power supplies sit on an isolated platform, which is pulsed up to a maximum of 100 kV during ion extraction. The EBIS is now fully assembled, and operation will be beginning following final vacuum and power supply tests. Details of the EBIS components are presented.

Model simulations of continuous ion injection into electron-beam ion source trap with slanted electrostatic mirror.

The efficiency of trapping ions in an electron-beam ion source (EBIS) is of primary importance for many applications requiring operations with externally produced ions: RIA breeders, ion sources, and traps. At the present time, the most popular method of ion injection is pulsed injection, when short bunches of ions get trapped in a longitudinal trap while traversing the trap region. Continuous trapping is a challenge for EBIS devices because mechanisms which reduce the longitudinal ion energy per charge in a trap (cooling with residual gas, energy exchange with other ions, and ionization) are not very effective, and accumulation of ions is slow. A possible approach to increase trapping efficiency is to slant the mirror at the end of the trap which is opposite to the injection end. A slanted mirror will convert longitudinal motion of ions into transverse motion, and, by reducing their longitudinal velocity, prevent these ions from escaping the trap on their way out. The trade-off for the increased trapping efficiency this way is an increase in the initial transverse energy of the accumulated ions. The slanted mirror can be realized if the ends of two adjacent electrodes, drift tubes, which act as an electrostatic mirror, are machined to produce a slanted gap, rather than an upright one. Applying different voltages to these electrodes will produce a slanted mirror. The results of two-dimensional (2D) and three-dimensional (3D) computer simulations of the ion injection into an EBIS are presented using simplified models of an EBIS with conical (2D simulations) and slanted (3D simulations) mirror electrodes.

Polarized proton collisions at 205 GeV at RHIC.

The Brookhaven Relativistic Heavy Ion Collider (RHIC) has been providing collisions of polarized protons at a beam energy of 100 GeV since 2001. Equipped with two full Siberian snakes in each ring, polarization is preserved during acceleration from injection to 100 GeV. However, the intrinsic spin resonances beyond 100 GeV are about a factor of 2 stronger than those below 100 GeV making it important to examine the impact of these strong intrinsic spin resonances on polarization survival and the tolerance for vertical orbit distortions. Polarized protons were first accelerated to the record energy of 205 GeV in RHIC with a significant polarization measured at top energy in 2005. This Letter presents the results and discusses the sensitivity of the polarization survival to orbit distortions.

Observation of a hybrid spin resonance

A new type of spin depolarization resonance has been observed at the Brookhaven Alternating Gradient Synchrotron (AGS). This spin resonance is identified as a strong closed-orbit sideband around the dominant intrinsic spin resonance. The strength of the resonance was proportional to the 9th harmonic component of the horizontal closed orbit and proportional to the vertical betatron oscillation amplitude. This "hybrid" spin resonance cannot be overcome by the partial snake at the AGS, but it can be corrected by the harmonic orbit correctors.

In vivo testing of an infection-resistant vascular graft material.

Present prosthetic arterial conduits continue to suffer the clinically and economically catastrophic complication of infection. We recently described a novel technique for binding quinolone antibiotics to Dacron based on principles of textile chemistry. This thermofixation procedure ("pad/heat") utilizes the limited fibrophilic characteristics of the quinolone antibiotic ciprofloxacin (Cipro) to permit pad/heat application and allowed controlled, sustained release from Dacron in several in vitro assays. The objective of this study was to test this infection-resistant prosthetic vascular graft material in an in vivo model. Dacron segments (1 cm2, either plain, dipped into antibiotic immediately prior to implantation, or Cipro pad/heat treated) were implanted in the dorsal subcutaneous tissue of the rabbit and directly contaminated with 10(6) Staphylococcus aureus. After 1 week, the samples were sterily harvested. Wounds were blindly graded on a scale from 1 (no evidence of infection, good tissue incorporation) to 4 (suppurative infection extending outside of the graft pocket, no gross tissue incorporation). Plain Dacron was easily infected in this model (mean grade 3.1 +/- 0.6, 92% culture positive). Notably, however, a significant (P < 0.05) wound grade difference between the dipped (2.3 +/- 1.0) and pad/heat (1.4 +/- 0.6) samples was demonstrated. Determination of adherent bacteria present on the implanted Dacron pieces by sonication and culture studies again revealed a significant difference between the dipped (56% culture positive) and pad/heat (12% culture) groups (P < 0.025). Histologic studies confirmed good tissue incorporation of the pad/heat samples. This project opens new avenues in the development of infection-resistant biomaterials.

Application of the quinolone antibiotic ciprofloxacin to Dacron utilizing textile dyeing technology.

Prosthetic arterial graft infection continues to be a significant and often devastating complication of vascular surgery. The organisms Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) are the primary pathogens causing acute and late graft infections, respectively. The objective of this study was to develop an infection-resistant prosthetic arterial graft by applying the bacteriocidal quinolone antibiotic ciprofloxacin to polyethylene terepthalate (Dacron) via thermofixation (pad/heat), a new application method founded on established textile procedures. We hypothesize that the limited fibrophilic characteristics of ciprofloxacin will permit binding to Dacron and at the same time allow persistent controlled release over an extended period of time. Using pad/heat technology, 33 micrograms (+/- 2.97 micrograms, n = 12) of ciprofloxacin was successfully bound to a 1-cm2 piece of woven Dacron. A full complement of microbiologic assays demonstrated superior, sustained antistaphylococcal activity of the pad/heat Dacron when compared to Dacron dipped into an equivalent concentration of ciprofloxacin. The sustained antimicrobial efficacy of ciprofloxacin pad/heat-treated Dacron opens new avenues in the development of infection-resistant biomaterials based on an understanding of textile chemistry.