Six-year experience of Australia's first dedicated cancer of unknown primary clinic.

BACKGROUND: Diagnosis and management of cancers of unknown primary (CUP) remain challenging. This study examines the referral patterns, management and outcomes of patients referred to Australia's first dedicated CUP clinic. METHODS: Retrospective medical record review was conducted for patients seen at the Peter MacCallum Cancer Centre CUP clinic between July 2014 and August 2020. Overall survival (OS) was analysed for patients with a CUP diagnosis where treatment information was available. RESULTS: Of 361 patients referred, fewer than half had completed diagnostic work-up at the time of referral. A diagnosis of CUP was established in 137 (38%), malignancy other than CUP in 177 (49%) and benign pathology in 36 (10%) patients. Genomic testing was successfully completed in 62% of patients with initial provisional CUP and impacted management in 32% by identifying a tissue of origin or actionable genomic alteration. The use of site-specific, targeted therapy or immunotherapy was independently associated with longer OS compared to empirical chemotherapy. CONCLUSION: Our specialised CUP clinic facilitated diagnostic work-up among patients with suspected malignancy and provided access to genomic testing and clinical trials for patients with a CUP diagnosis, all of which are important to improve outcomes in this patient population.

Survival difference according to mutation status in a prospective cohort study of Australian patients with metastatic non-small-cell lung carcinoma.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is a heterogeneous disease comprising not only different histological subtypes but also different molecular subtypes. AIM: To describe the frequency of oncogenic drivers in patients with metastatic NSCLC, the proportion of patients tested and survival difference according to mutation status in a single-institution study. METHODS: Metastatic NSCLC patients enrolled in a prospective Thoracic Malignancies Cohort Study between July 2012 and August 2016 were selected. Patients underwent molecular testing for epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK) gene rearrangements, Kirsten rat sarcoma (KRAS), B-Raf proto-oncogene (BRAF) mutations and ROS1 gene rearrangements. Survival was calculated using the Kaplan-Meier method for groups of interest, and comparisons were made using the log-rank test. RESULTS: A total of 392 patients were included, 43% of whom were female with median age of 64 years (28-92). Of 296 patients tested, 172 patients (58%) were positive for an oncogenic driver: 81 patients (27%) were EGFR positive, 25 patients (9%) were ALK positive, 57 patients (19%) had KRAS mutation and 9 patients (3%) were ROS1 or BRAF positive. Patients with an actionable mutation (EGFR/ALK) had a survival advantage when compared with patients who were mutation negative (hazard ratio (HR) 0.49; 95% confidence interval (CI) 0.33-0.71; P < 0.01). Survival difference between mutation negative and mutation status unknown was not statistically significant when adjusted for confounding factors in a multivariate analysis (HR 1.29; 95% CI 0.97-1.78, P = 0.08). CONCLUSION: In this prospective cohort, the presence of an actionable mutation was the strongest predictor of overall survival. These results confirm the importance of molecular testing and suggest likely survival benefit of identification and treatment of actionable oncogenes.

Direct access colonoscopy: impact of intervention on time to colorectal cancer diagnosis and treatment in North West Tasmania.

BACKGROUND: Direct access colonoscopy (DAC) allows general practitioners to refer directly for colonoscopy, without specialist review. Research suggests DAC reduces times to diagnosis and treatment of colorectal cancer. However, there is no information about outcomes of DAC in Australia. AIM: To determine if DAC in North West Tasmania expedited colorectal diagnosis and treatment. METHODS: Pre-post intervention study evaluating time from referral to diagnosis and definitive treatment. Patient demographic characteristics, referral, colonoscopy and treatment information was retrieved from hospital records. Timelines were investigated in standard referrals (SR), emergency department/inpatient referrals and DAC using survival analysis. RESULTS: Two hundred and six colorectal cancer cases were identified (117 SR, 26 DAC, 48 emergency department/inpatient and 15 unknown pathways). Median time to colonoscopy/diagnosis (DAC 6 weeks vs SR 7 weeks, P = 0.55) or definitive treatment (surgery/chemoradiation) (DAC 8 weeks vs SR 9 weeks, P = 0.81) was not significantly improved with DAC. Among SR only, time to diagnosis was 9 weeks pre-intervention versus 5 weeks post-intervention (P = 0.13), and time to treatment was 11 weeks pre-intervention versus 6 weeks post-intervention (P = 0.07). CONCLUSION: There was no statistically significant improvement in time to colorectal cancer diagnosis or treatment among patients referred through DAC compared to SR. There was a trend towards improved waiting times for SR concurrent with the introduction of the DAC pathway, indicating improvement of all referral processes. DAC may not be effective at expediting colorectal cancer diagnosis if it is not accompanied by strict referral guidelines. Larger evaluations of DAC are required in the Australian context.

A Phase II trial of alternating osimertinib and gefitinib therapy in advanced EGFR-T790M positive non-small cell lung cancer: OSCILLATE.

In this phase II, single arm trial (ACTRN12617000720314), we investigate if alternating osimertinib and gefitinib would delay the development of resistance to osimertinib in advanced, non-small cell lung cancer (NSCLC) with the epidermal growth factor receptor (EGFR) T790M mutation (n = 47) by modulating selective pressure on resistant clones. The primary endpoint is progression free-survival (PFS) rate at 12 months, and secondary endpoints include: feasibility of alternating therapy, overall response rate (ORR), overall survival (OS), and safety. The 12-month PFS rate is 38% (95% CI 27.5-55), not meeting the pre-specified primary endpoint. Serial circulating tumor DNA (ctDNA) analysis reveals decrease and clearance of the original activating EGFR and EGFR-T790M mutations which are prognostic of clinical outcomes. In 73% of participants, loss of T790M ctDNA is observed at progression and no participants have evidence of the EGFR C797S resistance mutation following the alternating regimen. These findings highlight the challenges of treatment strategies designed to modulate clonal evolution and the clinical importance of resistance mechanisms beyond suppression of selected genetic mutations in driving therapeutic escape to highly potent targeted therapies.

A Phase Ib/II Trial of Combined BRAF and EGFR Inhibition in BRAF V600E Positive Metastatic Colorectal Cancer and Other Cancers: The EVICT (Erlotinib and Vemurafenib In Combination Trial) Study.

PURPOSE: BRAF V600E mutant metastatic colorectal cancer represents a significant clinical problem, with combination approaches being developed clinically with oral BRAF inhibitors combined with EGFR-targeting antibodies. While compelling preclinical data have highlighted the effectiveness of combination therapy with vemurafenib and small-molecule EGFR inhibitors, gefitinib or erlotinib, in colorectal cancer, this therapeutic strategy has not been investigated in clinical studies. PATIENTS AND METHODS: We conducted a phase Ib/II dose-escalation/expansion trial investigating the safety/efficacy of the BRAF inhibitor vemurafenib and EGFR inhibitor erlotinib. RESULTS: Thirty-two patients with BRAF V600E positive metastatic colorectal cancer (mCRC) and 7 patients with other cancers were enrolled. No dose-limiting toxicities were observed in escalation, with vemurafenib 960 mg twice daily with erlotinib 150 mg daily selected as the recommended phase II dose. Among 31 evaluable patients with mCRC and 7 with other cancers, overall response rates were 32% [10/31, 16% (5/31) confirmed] and 43% (3/7), respectively, with clinical benefit rates of 65% and 100%. Early ctDNA dynamics were predictive of treatment efficacy, and serial ctDNA monitoring revealed distinct patterns of convergent genomic evolution associated with acquired treatment resistance, with frequent emergence of MAPK pathway alterations, including polyclonal KRAS, NRAS, and MAP2K1 mutations, and MET amplification. CONCLUSIONS: The Erlotinib and Vemurafenib In Combination Trial study demonstrated a safe and novel combination of two oral inhibitors targeting BRAF and EGFR. The dynamic assessment of serial ctDNA was a useful measure of underlying genomic changes in response to this combination and in understanding potential mechanisms of resistance.

Human nonverbal discrimination of relative and absolute number.

The nonverbal discrimination of relative and absolute number of sequential visual stimuli was investigated with humans in bisection, reproduction, and report tasks. Participants viewed a sequence of 40 red and black objects on each trial, randomly intermixed, and had to identify the number of red objects, which varied from 1 to 20. To prevent the use of a verbal-counting strategy, participants were required to name the objects as they appeared. The characteristics of human performance resembled those of pigeons in analogous procedures (Tan & Grace Learning and Behavior 38:408-417, 2010; Tan, Grace, Holland, & McLean Journal of Experimental Psychology 33:409-427, 2007): Average response number increased systematically with sample number, and bisection points were located at the arithmetic, not the geometric, mean. Additionally, in both the reproduction and report tasks, coefficients of variation decreased for values less than 6 but increased or remained constant for larger values, suggesting that different representations were used for small and large numbers.

Circulating Tumor DNA: An Emerging Tool in Gastrointestinal Cancers.

Circulating tumor DNA (ctDNA) is tumor-derived fragmented DNA in the bloodstream that has come from primary or metastatic cancer sites. Neoplasm-specific genetic and epigenetic abnormalities are increasingly being identified through liquid biopsy: a novel, minimally invasive technique used to isolate and analyze ctDNA in the peripheral circulation. Liquid biopsy and other emerging ctDNA technologies represent a paradigm shift in cancer diagnostics because they allow for the detection of minimal residual disease in patients with early-stage disease, improve risk stratification, capture tumor heterogeneity and genomic evolution, and enhance ctDNA-guided adjuvant and palliative cancer therapy. Moreover, ctDNA can be used to monitor the tumor response to neoadjuvant and postoperative therapy in patients with metastatic disease. Using clearance of ctDNA as an endpoint for escalation/de-escalation of adjuvant chemotherapy for patients considered to have high-risk disease has become an important area of research. The possibility of using ctDNA as a surrogate for treatment response-including for overall survival, progression-free survival, and disease-free survival-is an attractive concept; this surrogate will arguably reduce study duration and expedite the development of new therapies. In this review, we summarize the current evidence on the applications of ctDNA for the diagnosis and management of gastrointestinal tumors. Gastrointestinal cancers-including tumors of the esophagus, stomach, colon, liver, and pancreas-account for one-quarter of global cancer diagnoses and contribute to more than one-third of cancer-related deaths. Given the prevalence of gastrointestinal malignancies, ctDNA technology represents a powerful tool to reduce the global burden of disease.

Social preference in rats.

Rats were given repeated choices between social and nonsocial outcomes, and between familiar and unfamiliar social outcomes. Lever presses on either of 2 levers in the middle chamber of a 3-chamber apparatus opened a door adjacent to the lever, permitting 45-s access to social interaction with the rat in the chosen side chamber. In Experiment 1, rats preferred (a) social over nonsocial options, choosing their cagemate rat over an empty chamber, and (b) an unfamiliar over a familiar rat, choosing a non-cagemate over their cagemate. These findings were replicated in Experiment 2 with 2 different non-cagemate rats. Rats preferred both non-cagemate rats to a similar degree when pitted against their cagemate, but were indifferent when the 2 non-cagemates were pitted against each other. Similar preference for social over nonsocial and non-cagemate over cagemate was seen in Experiment 3, with new non-cagemate rats introduced after every third session. Response rates (for both cagemate and non-cagemate rats) were elevated under conditions of nonsocial (isolated) housing compared to conditions of social (paired) housing, demonstrating a social deprivation effect. Together, the experiments contribute to an experimental analysis of social preference within a social reinforcement framework, drawing on methods with proven efficacy in the analysis of reinforcement more generally.

RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies.

INTRODUCTION: Novel rearranged in transfection (RET)-specific tyrosine kinase inhibitors (TKIs) such as selpercatinib (LOXO-292) have shown unprecedented efficacy in tumors positive for RET fusions or mutations, notably RET fusion-positive NSCLC and RET-mutated medullary thyroid cancer (MTC). However, the mechanisms of resistance to these agents have not yet been described. METHODS: Analysis was performed of circulating tumor DNA and tissue in patients with RET fusion-positive NSCLC and RET-mutation positive MTC who developed disease progression after an initial response to selpercatinib. Acquired resistance was modeled preclinically using a CCDC6-RET fusion-positive NSCLC patient-derived xenograft. The inhibitory activity of anti-RET multikinase inhibitors and selective RET TKIs was evaluated in enzyme and cell-based assays. RESULTS: After a dramatic initial response to selpercatinib in a patient with KIF5B-RET NSCLC, analysis of circulating tumor DNA revealed emergence of RET G810R, G810S, and G810C mutations in the RET solvent front before the emergence of clinical resistance. Postmortem biopsy studies reported intratumor and intertumor heterogeneity with distinct disease subclones containing G810S, G810R, and G810C mutations in multiple disease sites indicative of convergent evolution on the G810 residue resulting in a common mechanism of resistance. Acquired mutations in RET G810 were identified in tumor tissue from a second patient with CCDC6-RET fusion-positive NSCLC and in plasma from patients with additional RET fusion-positive NSCLC and RET-mutant MTC progressing on an ongoing phase 1 and 2 trial of selpercatinib. Preclinical studies reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft (from a patient with NSCLC) model of acquired resistance to selpercatinib. Structural modeling predicted that these mutations sterically hinder the binding of selpercatinib, and in vitro assays confirmed loss of activity for both anti-RET multikinase inhibitors and selective RET TKIs. CONCLUSIONS: RET G810 solvent front mutations represent the first described recurrent mechanism of resistance to selective RET inhibition with selpercatinib. Development of potent inhibitor of these mutations and maintaining activity against RET gatekeeper mutations could be an effective strategy to target resistance to selective RET inhibitors.

An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer.

Loss of MHC class I (MHC-I) antigen presentation in cancer cells can elicit immunotherapy resistance. A genome-wide CRISPR/Cas9 screen identified an evolutionarily conserved function of polycomb repressive complex 2 (PRC2) that mediates coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP), promoting evasion of T cell-mediated immunity. MHC-I APP gene promoters in MHC-I low cancers harbor bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine-induced upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological MHC-I silencing highlights a conserved mechanism by which cancers arising from these primitive tissues exploit PRC2 activity to enable immune evasion.

Effects of retention interval on performance in a numerical reproduction task.

The retention interval (RI) between the sample and production phase in a numerical reproduction task was varied to determine whether a "produce-small" effect would be obtained with increased delays. Four pigeons were trained with a retention interval of 2s, and then tested with intervals of 0.5s and 8s. Results showed a number-dependent, "produce-large" effect-response number increased when RI was increased-analyses of average response number and accuracy suggested RI affected responding most on the 2-flash trials with an 8-s RI. Additionally, discrimination between trial types decreased as RI increased. Existing explanations for the "choose-short/small" effect appear unable to account for these results; however the "produce-large" effect may be attributed to a disruption in stimulus control over responding.

Genomic Characterization of Lung Cancer and Its Impact on the Use and Timing of PET in Therapeutic Response Assessment.

Significant advances in understanding the genomic landscape of non-small cell lung cancer (NSCLC) together with the coupling discovery of key oncogenic drivers and the development of effective targeted and immunotherapeutic agents have revolutionized the management of this malignancy. Although these therapies have resulted in improved outcomes for a subgroup of patients, their benefit may not necessarily be reflected by conventional response assessment criteria, because these therapeutic agents differ in their mechanism of action and response time compared with cytotoxic chemotherapy. Here the authors review available therapies in NSCLC and the utility of PET in therapeutic response assessment.

To free, or not to free: Social reinforcement effects in the social release paradigm with rats.

The present research measured social reinforcement in rats, using a social-release procedure in which lever presses permitted 10-s access to a familiar social partner. The work requirements for reinforcement increased systematically according to progressive-ratio (PR) schedules. Social and food reinforcement value were compared across blocks of sessions (Experiment 1) and concurrently within the same sessions (Experiment 2). To assess motivational effects, response and reinforcer rates for both reinforcer types were studied under food restriction, social restriction, and combined food and social restriction. Responding was maintained by both reinforcers, albeit at substantially higher levels for food than for social access. Responding for social access decreased to low levels under extinction conditions, demonstrating functional control by the social-reinforcement contingency. Sensitivity to social restriction was seen in some conditions in Experiment 2, in which social reinforcers were earned earlier in the session (at lower food prices) under social restriction than under the other deprivation conditions. Altogether, results are consistent with a social reinforcement conceptualization, and demonstrate an important role for social contact in social release behavior. The study demonstrates a promising set of methods for analyzing and quantifying social reinforcement.

Numerical reproduction in pigeons.

Pigeons were trained to match the number of responses made during a production phase to the number of keylight flashes (2, 4, or 6) in a previous sample phase. In Experiment 1, there were 2 conditions in which the flashes were programmed to occur at a constant rate or within a constant overall duration. For both conditions, although accuracy was relatively low, responding increased linearly with flash number and coefficients of variation decreased. Positive transfer to novel numbers was obtained only when test and baseline trials had the same temporal organization, but multiple regressions revealed significant control by number independently of temporal cues. In Experiment 2, flashes were programmed to occur pseudorandomly to degrade the validity of temporal cues. Results were similar to in Experiment 1. A prototype response class model accounted for the major features of the data. According to the model, responses during the production phase are shaped into higher order units that are associated with different stimulus numbers and function as a rough category scale of numerosity.

Functional analysis of mutual behavior in laboratory rats (Rattus norvegicus).

Three pairs of rats were trained to synchronize their lever pressing according to a mutual reinforcement contingency, in which alternating lever presses that fell within a 500-ms window were reinforced with food. In Experiment 1, rats worked in adjacent chambers separated by a transparent barrier, and the effects of the mutual reinforcement contingency were compared with those under yoked-control conditions that provided the same rate of food reinforcement but without the temporal coordination response requirement. In Experiment 2, coordinated behavior was compared with and without a barrier, and across different barrier types: transparent, opaque, wire mesh. In Experiment 3, the effects of social familiarity were assessed by switching partners, enabling a comparison of coordinated behavior with familiar and unfamiliar partners. The overall pattern of results shows that the coordinated behavior of 2 rats was (a) maintained by mutual reinforcement contingencies, (b) unrelated to the type or presence of a barrier separating the rats, and (c) sufficiently flexible to adjust to the presence and behavior of an unfamiliar partner. Taken as a whole, the study illustrates a promising approach to conceptualizing and analyzing behavioral mechanisms of mutual behavior, an important component of an integrated study of social behavior.

Pigeons' demand and preference for specific and generalized conditioned reinforcers in a token economy.

Pigeons' demand and preference for specific and generalized tokens was examined in a token economy. Pigeons could produce and exchange different colored tokens for food, for water, or for food or water. Token production was measured across three phases, which examined: (1) across-session price increases (typical demand curve method); (2) within-session price increases (progressive-ratio, PR, schedule); and (3) concurrent pairwise choices between the token types. Exponential demand curves were fitted to the response data and accounted for over 90% total variance. Demand curve parameter values, Pmax , Omax and α showed that demand was ordered in the following way: food tokens, generalized tokens, water tokens, both in Phase 1 and in Phase 3. This suggests that the preferences were predictable on the basis of elasticity and response output from the demand analysis. Pmax and Omax values failed to consistently predict breakpoints and peak response rates in the PR schedules in Phase 2, however, suggesting limits on a unitary conception of reinforcer efficacy. The patterns of generalized token production and exchange in Phase 3 suggest that the generalized tokens served as substitutes for the specific food and water tokens. Taken together, the present findings demonstrate the utility of behavioral economic concepts in the analysis of generalized reinforcement.

Effects of predictability and competition on group and individual choice in a free-ranging foraging environment.

The present study examined the social foraging of rats in an open arena. The relative quantity of food varied across two food sources, or "patches." Five food quantity ratios (1:1, 1:2, 1:8, 8:1, 2:1) were presented in a series of 30-min sessions. Ratios varied randomly across 6-min components within sessions (Phase 1), or in a consistent order across sessions (Phase 2). Group and individual preferences were well described by the ideal free distribution and the generalized matching law, respectively, with evidence of undermatching at both group and individual levels. Sensitivity of individual and collective behavior to the relative quantities of food was higher in Phase 2 than in Phase 1. Competitiveness rankings, assessed before and after experimental sessions by delivering food in rapid succession from a single feeder, was positively related to sensitivity values in Phase 1, but less consistently so in Phase 2. This study illustrates a promising experimental method for investigating foraging in a social context.

Mortality by ethnic group to 2006: is extending census-mortality linkage robust?

OBJECTIVE: To update trends in mortality by ethnic group from the New Zealand Census-Mortality Study (NZCMS), by additionally linking 2004-06 mortality records to the 2001 Census. To investigate possible bias from this extended linkage, especially for Pacific and Asian people who emigrate more frequently. METHODS: Anonymous and probabilistic record linkage of 2004-06 mortality records with the 2001 Census was undertaken. Age-standardised 1-74 year old mortality rates by sex and age group, and for all-cause and selected causes of death, were calculated using the direct method for first 30 months post 2001 Census (2001-03) and second 30 months (2003-06). RESULTS: Observed all-cause mortality rates continued to fall in 2003-06 compared to previous periods, but more so for Pacific (18.3% and 21.7% for males and females for 2003-06 compared to 2001-04, respectively) and Asian (22.2%, 16.7%), than for Maori (13.2%, 14.2%) and European/Other (13.0%, 10.4%). Observed rate ratios for Maori, compared to European/Other were 2.43 (95% CI 2.31-2.57) for males and 2.72 (2.56-2.89) for females, the same (males) and slightly less (7%, females) than in 2001-03. Declines in cardiovascular disease (CVD) and injury mortality were the main drivers of all-cause mortality rate reductions for all ethnic groups. Relative inequalities in CVD between Maori and European/Other remain high (three to four-fold relative risks), but reduced by 8% for both males and females from 2001-03 to 2003-06, which in turn means that absolute inequalities closed by as much as 20%. CONCLUSION: We suspect that analyses comparing mortality rates over time within one of the closed NZCMS cohorts (e.g. 2001-03 compared to 2003-06) is prone to bias due to our inability to censor people when they migrate out of New Zealand. This limitation means mortality rates in the NZCMS are increasingly underestimated with time since census night, particularly for Pacific and Asian people. However, previously published NZCMS trends remain valid as the duration of follow-up (3 years) is short, and cohorts were not split by time since census. Nevertheless, it is safe to conclude that mortality rates continued to decline from 2001-03 to 2003-04 for all four ethnic groups. All-cause mortality inequalities for Maori compared to European/Other over this time were probably stable in relative terms and decreasing in absolute terms, but cardiovascular disease (CVD) inequalities probably decreased in both absolute and relative terms.

Comorbidity among patients with colon cancer in New Zealand.

AIMS: To identify patient factors that are associated with a higher risk of comorbidity, and to assess the impact of comorbidity on risk of in-hospital death, length of stay and 5-year all-cause survival among a large cohort of patients with colon cancer in New Zealand. METHODS: Comorbidity data were collected from patients who were diagnosed with colon cancer and admitted to public hospitals during 1996-2003. The comorbidity measures included all conditions listed in the Charlson Comorbidity Index, as well as a predetermined list of additional conditions. We examined predictors of higher comorbidity scores. We also measured the impact of comorbidity on in-hospital death, length of stay and 5-year all-cause survival using logistic, linear and Cox proportional hazard regression models to adjust for confounding by sex, age, ethnicity, extent of disease and area level deprivation. RESULTS: There were 11,524 patients included in the study. 7.5% of females and 10.3% of men had Charlson scores of three or more. Higher comorbidity scores were associated with increasing age, and were more common among males, Maori and Pacific people, those with unknown extent of disease and those living in the most deprived quintile of New Zealand. Those with Charlson scores ≥ 3 had a higher risk of in-hospital death (OR=4.8; 95% CI 3.5-6.6), longer lengths of hospital stay (0.14 days 95% CI 0.08-0.2) and lower 5-year survival HR=2.0; 95%CI=1.8-2.3) compared with those with a score of 0. CONCLUSION: This study confirms that comorbidity is common among colon cancer patients in New Zealand, and has an adverse and independent effect on outcomes related to mortality and length of hospital stay.

Discrimination and representation of relative numerosity in a bisection task by pigeons.

We trained 4 pigeons in a numerical bisection task to discriminate between pairs of keylight flashes with a ratio of 1:3 (2 vs. 6, 4 vs. 12, and 8 vs. 24) that were presented in a sample phase. Responses to the blue key were reinforced after a sequence of a larger number of flashes, and responses to the white key were reinforced after a sequence of a smaller number of flashes. The intervals between flashes in the sample phase were randomized to attenuate the covariation of temporal cues with flash number. Pigeons responded accurately in each of the discriminations, with typically 85%-90% correct responses. Transfer tests showed that the proportion of large responses increased with number and performance generalized to larger values outside the training ranges. Psychometric functions superposed when plotted on a relative scale, and estimates of Weber fractions were approximately constant, suggesting that variability was scalar. However, contrary to previous research in nonhumans, bisection points were located at the arithmetic, not geometric, mean. Hierarchical logistic regressions confirmed significant control over responding by number beyond that attributable to temporal cues. These results show that pigeons are able to respond accurately in a relative numerosity discrimination with successively presented visual stimuli, although the nature of the numerical representation and response rule remains unclear.