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Supplementary Figure from Cell-Free HPV DNA Provides an Accurate and Rapid Diagnosis of HPV-Associated Head and Neck Cancer

Up to 50% of patients treated with curative esophagectomy for esophageal cancer will develop recurrence, contributing to the dismal survival associated with this disease. Regional recurrence may represent disease that is not yet widely metastatic and may therefore be amenable to more-aggressive treatment. We sought to assess all patients treated with curative esophagectomy for esophageal cancer who developed regional recurrence. We retrospectively identified all patients who underwent esophagectomy for esophageal adenocarcinoma and esophageal squamous cell carcinoma at a single institution from January 2000 to August 2019. In total, 1626 patients were included in the study cohort. As of June 2022, 595 patients had disease recurrence, which was distant or systemic in 435 patients (27%), regional in 125 (7.7%), and local in 35 (2.2%). On multivariable analysis, neoadjuvant chemoradiation with a total radiation dose45 Gy (hazard ratio [HR], 3.5 [95% CI, 1.7-7.3]; P=0.001), pathologic node-positive disease (HR, 1.9 [95% CI, 1.3-3.0]; P=0.003), and lymphovascular invasion (HR, 1.6 [95% CI, 1.0-2.5]; P=0.049) were predictors of isolated nodal recurrence, whereas increasing age (HR, 0.97 [95% CI, 0.96-0.99]; P=0.001) and increasing number of excised lymph nodes (HR, 0.98 [95% CI, 0.95-1.00]; P=0.021) were independently associated with decreased risk of regional recurrence. Patients treated with a combination of local and systemic therapies had better survival outcomes than patients treated with systemic therapy alone (P0.001). In patients with recurrence of esophageal cancer limited to regional lymph nodes, salvage treatment may be possible. Higher radiation doses and more-extensive lymphadenectomy may reduce the risk of regional recurrence. This article is protected by copyright. All rights reserved.

PARPi-FL is a molecularly specific fluorescent agent that targets poly ADP-ribose polymerase 1, a DNA repair enzyme overexpressed in the nuclei of tumor cells. This imaging agent is being investigated in a clinical trial (NCT03085147) for the detection of oral cancer. The PARPi-FL mouthwash formulation currently being used in the phase I/II clinical trial comprises 1,000 nM of PARPi-FL dissolved first in 4.5 ml of polyethylene glycol (PEG) 300 and then in 9.5 ml of water. This formulation requires a 2-step process that can be cumbersome for routine clinical use. To minimize errors and simplify the formulation process, we have developed a new one-step formulation, which requires only the direct addition of water into a vial containing a mixture of the PARPi-FL and PEG 3350, which is also a powder. In a series of analytical and preclinical studies, we demonstrate that the new formulation of PARPi-FL is stable over 365 days, sustains its characteristics, and performs similar to the previous formulation. Moving forward, the new formulation of the PARPi-FL will be used for patients accrued in the phase II clinical trial.

New ultrasensitive methods for detecting residual disease after surgery are needed in human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OPSCC).To determine whether the clearance kinetics of circulating tumor human papillomavirus DNA (ctHPVDNA) is associated with postoperative disease status, a prospective observational study was conducted in 33 patients with HPV+OPSCC undergoing surgery. Blood was collected before surgery, postoperative days 1 (POD 1), 7, and 30 and with follow-up. A subcohort of 12 patients underwent frequent blood collections in the first 24 hours after surgery to define early clearance kinetics. Plasma was run on custom droplet digital polymerase chain reaction (ddPCR) assays for HPV genotypes 16, 18, 33, 35, and 45.In patients without pathologic risk factors for recurrence who were observed after surgery, ctHPVDNA rapidly decreased to1 copy/mL by POD 1 (n = 8/8). In patients with risk factors for macroscopic residual disease, ctHPVDNA was markedly elevated on POD 1 (350 copies/mL) and remained elevated until adjuvant treatment (n = 3/3). Patients with intermediate POD 1 ctHPVDNA levels (1.2-58.4 copies/mL) all possessed pathologic risk factors for microscopic residual disease (n = 9/9). POD 1 ctHPVDNA levels were higher in patients with known adverse pathologic risk factors such as extranodal extension1 mm (P = .0481) and with increasing lymph nodes involved (P = .0453) and were further associated with adjuvant treatment received (P = .0076). One of 33 patients had a recurrence that was detected by ctHPVDNA 2 months earlier than clinical detection.POD 1 ctHPVDNA levels are associated with the risk of residual disease in patients with HPV+OPSCC undergoing curative intent surgery and thus could be used as a personalized biomarker for selecting adjuvant treatment in the future.Human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OPSCC) is increasing at epidemic proportions and is commonly treated with surgery. This report describes results from a study examining the clearance kinetics of circulating tumor HPV DNA (circulating tumor human papillomavirus DNA [ctHPVDNA]) following surgical treatment of HPV+OPSCC. We found that ctHPVDNA levels 1 day after surgery are associated with the risk of residual disease in patients with HPV+OPSCC and thus could be used as a personalized biomarker for selecting adjuvant treatment in the future. These findings are the first to demonstrate the potential utility of ctHPVDNA in patients with HPV+OPSCC undergoing surgery.

Key message Exceedance of critical limits in soil solution samples was more frequent in intensively monitored forest plots across Europe with critical loads for acidity and eutrophication exceeded compared to other plots from the same network. Elevated inorganic nitrogen concentrations in soil solution tended to be related to less favourable nutritional status. Context Forests have been exposed to elevated atmospheric deposition of acidifying and eutrophying sulphur and nitrogen compounds for decades. Critical loads have been identified, below which damage due to acidification and eutrophication are not expected to occur. Aims We explored the relationship between the exceedance of critical loads and inorganic nitrogen concentration, the base cation to aluminium ratio in soil solutions, as well as the nutritional status of trees. Methods We used recent data describing deposition, elemental concentrations in soil solution and foliage, as well as the level of damage to foliage recorded at forest plots of the ICP Forests intensive monitoring network across Europe. Results Critical loads for inorganic nitrogen deposition were exceeded on about a third to half of the forest plots. Elevated inorganic nitrogen concentrations in soil solution occurred more frequently among these plots. Indications of nutrient imbalances, such as low magnesium concentration in foliage or discolouration of needles and leaves, were seldom but appeared more frequently on plots where the critical limits for soil solution were exceeded. Conclusion The findings support the hypothesis that elevated nitrogen and sulphur deposition can lead to imbalances in tree nutrition.

The present study was aimed at testing the prediction of the Pavlovian excitatory conditioning explanation of context-specific sensitization that the sensitized effect (SE) should persist as long as the post-sensitization conditioned activity (CR). C57BL/6J mice were tested for the expression of cocaine-induced conditioned and sensitized locomotion on several intervals after the establishment of a sensitization in an unchanging context. A group of mice received 10 once-daily injections of 10 mg/kg cocaine (s.c.) in a short time prior to being tested in activity-meters for 60 min sessions (cocaine-pretreated group), mice from a control group receiving saline in the same manner (saline-pretreated group). On the test sessions, taking place 1, 8 and 28 days after cocaine pretreatment, half of the animals of each pretreatment group ( n = 8) received a challenge test with saline and the other half with 10 mg/kg cocaine in the pretreatment context room (for CR and SE tests, respectively). The CR was significantly expressed on the three successive saline-challenge tests, albeit the activity levels were markedly decreased on the 28-day retention test. In contrast, the SE was significantly expressed only during the first half of the 1-day test session and the first 10 min of the 8-day test session, no SE effect being expressed on the 28-day retention test. The results, suggesting a functional uncoupling of the CR from the SE, disprove the theories of context-specificity of sensitization based completely or partially on Pavlovian excitatory conditioning mechanisms.

The potential differential effects of isolated and collective housing through the testing phase on sensitisation to cocaine-induced locomotion, the subsequent conditioned locomotion and the context-dependent expression of sensitisation were examined in C57Bl/6J male mice. Sensitisation was first generated in mice receiving seven once-daily subcutaneous injections of either saline or 10 mg/kg cocaine, before being placed in a testing chamber (singly) or in their home cage. On Day 8, mice were tested for conditioned locomotion (under saline). On Day 12, after three daily sessions of reinstatement of sensitisation, they were tested for contextual sensitisation (under cocaine). Whereas little or no effect of housing on the development of sensitisation was found, postsensitisation conditioned activity was significantly greater in isolation-kept mice (in comparison with the group-kept animals) and the likelihood of inducing a context-dependent expression of sensitisation was greater in grouped-housed mice. The results indicate that some of the aspects of contextual sensitisation can be influenced by the social conditions of lodging, not only when these conditions start several weeks before or during previous developmental periods of animals life (as previously published), but also when social isolation or social grouping are initiated after sexual maturity and applied through the period of treatments and testing. Moreover, the differential effect of social conditions of lodging on conditioned activity and contextual expression of sensitisation disagrees with the excitatory conditioning account of contextual sensitisation.

The study tested the possibility of a positive relationship between the dose of cocaine and the size of the placebo effect generated after contextual sensitisation to the behavioural effects of cocaine. Male C57BL/6J mice were first injected (subcutaneous, s.c.) over seven successive daily sessions with saline or one of three doses of cocaine (2.5, 5 or 7.5 mg/kg), either in the test room or in the colony room (before being placed in a novel cage tub). On the test day, 24 h after chronic pre-treatment, mice from the four conditions were challenged under saline in the test room. Mice were video-recorded and their behaviours were scored using a time-sampling technique. A dose-dependent development of sensitisation was first generated. On the saline challenge test day, significant levels of placebo hyperlocomotion were obtained for mice previously given 5 and 7.5 mg/kg, but not 2.5 mg/kg cocaine, the effect being significantly greater in the mice pretreated with the highest dose than in those receiving the intermediate one, which exhibited a placebo effect that was greater than that of the mice pretreated with 2.5 mg/kg cocaine. Therefore, the magnitude of the placebo effect was a function of the intensity of the unconditioned stimulus (the dose used to generate sensitisation). Such results directly support the Pavlovian conditioning account of post-sensitisation placebo effects.

The aims of the study were to test whether drug dose is positively related to the magnitude of the conditioned response following sensitization to the behavioural effects of cocaine and to investigate the relationship between the conditioned response and cocaine-induced sensitization. Male mice (C57BL/6J) were first injected over seven successive days with either saline or cocaine at 2.5, 5, 10 or 20 mg/kg s.c., in the testing room. On the test day, 24 h after the last injection, mice from all conditions were challenged with saline in the testing room to test for conditioned cocaine effects. Mice were video-recorded and various behaviours were later scored using a time-sampling technique. Cocaine-elicited orofacial stereotypy was significantly sensitized at the two highest doses and dose-dependently conditioned at the three highest doses. Cocaine-increased locomotion was sensitized at the three highest doses and significantly conditioned at 10 and 20mg/kg. Cocaine-increased sniffing did not change over pretreatment at any dose, and was conditioned only at 10 mg/kg. Cocaine-decreased immobility also did not change over pretreatment at any dose, but was conditioned at 10 and 20mg/kg. Concomitantly, rearing was reduced by cocaine at 10 and 20mg/kg, without sensitization being induced, and it was reduced under saline challenge after 5 mg/kg cocaine, while cocaine-decreased grooming was sensitized at the three highest doses and conditioned at 10 and 20 mg/kg cocaine. There was a positive relation between the size of the conditioned response for orofacial stereotypy and the magnitude of the unconditioned stimulus (the doses), a result conforming to the Pavlovian account of the placebo effect. This could also be concluded from considering the behaviour patterns as components of a unique placebo effect (hyperkinetic syndrome), since orofacial stereotypy, very apparent at 20 mg/kg cocaine, interfered at that dose with the full-blown expression of locomotion and sniffing, both yielding (approximately) inverted U-shaped dose-effect curves. However, no correlation was found between the magnitude of the conditioned response and the amplitude of sensitization (the difference between the initial unconditioned non-sensitized response and the last unconditioned sensitized response), a finding which indicates that conditioned responding does not participate in the generation of the sensitized effects, contrary to the 'excitatory conditioning model of contextual sensitization'.