Delise H. Herron

Characterizing the patterns of clonal selection in circulating tumor DNA from patients with colorectal cancer refractory to anti-EGFR treatment

INTRODUCTION KRAS and EGFR ectodomain-acquired mutations in patients with metastatic colorectal cancer (mCRC) have been correlated with acquired resistance to anti-EGFR monoclonal antibodies (mAbs). We investigated the frequency, co-occurrence, and distribution of acquired KRAS and EGFR mutations in patients with mCRC refractory to anti-EGFR mAbs using circulating tumor DNA (ctDNA). PATIENTS AND METHODS Sixty-two post-treatment plasma and 20 matching pretreatment archival tissue samples from KRAS (wt) mCRC patients refractory to anti-EGFR mAbs were evaluated by high-sensitivity emulsion polymerase chain reaction for KRAS codon 12, 13, 61, and 146 and EGFR 492 mutations. RESULTS Plasma analyses showed newly detectable EGFR and KRAS mutations in 5/62 [8%; 95% confidence interval (CI) 0.02-0.18] and 27/62 (44%; 95% CI 0.3-0.56) samples, respectively. KRAS codon 61 and 146 mutations were predominant (33% and 11%, respectively), and multiple EGFR and/or KRAS mutations were detected in 11/27 (41%) cases. The percentage of mutant allele reads was inversely correlated with time since last treatment with EGFR mAbs (P = 0.038). In the matching archival tissue, these mutations were detectable as low-allele-frequency clones in 35% of patients with plasma mutations after treatment with anti-EGFR mAbs and correlated with shorter progression-free survival (PFS) compared with the cases with no new mutations (3.0 versus 8.0 months, P = 0.0004). CONCLUSION Newly detected KRAS and/or EGFR mutations in plasma ctDNA from patients refractory to anti-EGFR treatment appear to derive from rare, pre-existing clones in the primary tumors. These rare clones were associated with shorter PFS in patients receiving anti-EGFR treatment. Multiple simultaneous mutations in KRAS and EGFR in the ctDNA and the decline in allele frequency after discontinuation of anti-EGFR therapy in a subset of patients suggest that several resistance mechanisms can co-exist and that relative clonal burdens may change over time. Monitoring treatment-induced genetic alterations by sequencing ctDNA could identify biomarkers for treatment screening in anti-EGFR-refractory patients.

Reproducibility of CT Perfusion Parameters in Liver Tumors and Normal Liver

PURPOSE To assess the reproducibility of computed tomographic (CT) perfusion measurements in liver tumors and normal liver and effects of motion and data acquisition time on parameters. MATERIALS AND METHODS Institutional review board approval and written informed consent were obtained for this prospective study. The study complied with HIPAA regulations. Two CT perfusion scans were obtained 2-7 days apart in seven patients with liver tumors with two scanning phases (phase 1: 30-second breath-hold cine; phase 2: six intermittent free-breathing cines) spanning 135 seconds. Blood flow (BF), blood volume (BV), mean transit time (MTT), and permeability-surface area product (PS) for tumors and normal liver were calculated from phase 1 with and without rigid registration and, for combined phases 1 and 2, with manually and rigid-registered phase 2 images, by using deconvolution modeling. Variability was assessed with within-patient coefficients of variation (CVs) and Bland-Altman analyses. RESULTS For tumors, BF, BV, MTT, and PS values and reproducibility varied by analytical method, the former by up to 11%, 23%, 21%, and 138%, respectively. Median PS values doubled with the addition of phase 2 data to phase 1 data. The best overall reproducibility was obtained with rigidly registered phase 1 and phase 2 images, with within-patient CVs for BF, BV, MTT, and PS of 11.2%, 14.4%, 5.5% and 12.1%, respectively. Normal liver evaluations were similar, except with marginally lower variability. CONCLUSION Absolute values and reproducibility of CT perfusion parameters were markedly influenced by motion and data acquisition time. PS, in particular, probably requires data acquisition beyond a single breath hold, for which motion-correction techniques are likely necessary.

Reproducibility of Perfusion Parameters Obtained From Perfusion CT in Lung Tumors

OBJECTIVE The purpose of this article is to assess the variability of perfusion CT measurements in lung tumors and the effects of motion and duration of data acquisition on perfusion CT parameter values. SUBJECTS AND METHODS Two perfusion CT scans were obtained in 11 patients with lung tumors, 2-7 days apart, using phase 1 scans (30-second breath-hold cine) followed by phase 2 scans (six intermittent helical breath-holds), spanning 125 seconds. Tumor blood flow (BF), blood volume (BV), mean transit time (MTT), and permeability were calculated for phase 1 using all-cine and motion-corrected (rigidly registered) images, both with and without matching phase 2 images (manually or rigidly registered). Variability was assessed by the within-patient coefficient of variation (CV) and Bland-Altman analyses. RESULTS BF, BV, MTT, and permeability values varied widely by method of analysis (median BF, 45.3-65.1 mL/min/100 g; median BV, 2.6-3.8 mL/100 g; median MTT, 3.6-4.1 seconds, and median permeability, 13.7-39.3 mL/min/100 g), as did within-patient CVs (10.9-114.4%, 25.3-117.6%, 22.3-51.5%, and 29.6-134.9%, respectively). Parameter values and variability were affected by motion and duration of data analyzed: permeability values doubled when phase 2 images were added to phase 1 data. Overall, the best reproducibility was obtained with registered phase 1 and 2 data, with within-patient CVs of 11.6%, 26.5%, 45.4%, and 30.2%, respectively. CONCLUSION The absolute values and reproducibility of perfusion parameters in lung tumors are markedly influenced by motion and duration of data acquisition. Permeability, in particular, probably requires data acquisition beyond a single breath-hold. The smallest variability in parameter values was obtained with motion correction and extended acquisition durations.

Subtraction CT colonography: Feasibility in an animal model

AIM To determine, in an in vitro animal model, the feasibility of subtracting residual faecal material, during virtual colonoscopy, by uniform mixing of the colonic contents with barium. MATERIALS AND METHODS Segments of animal colon containing simulated soft-tissue polyps, measuring 3-10 mm, were filled with a faecal substitute consisting of a barium and peanut butter mix that had an attenuation value greater than 200 Hounsfield units. The colonic segments were then examined by CT using 3-10 mm beam collimations, and at pitch factors of 1 and 2. Using subtraction cut-off levels varying from 100-250 Hounsfield units, the barium and peanut butter mix was then subtracted to reveal the underlying polyps. RESULTS Our optimal technical parameters required 3-mm beam collimation, a helical pitch factor of 2, reconstruction intervals of 3 mm, and a subtraction cut-off level of 150 Hounsfield units. The subtraction technique, in this animal model, had a sensitivity of 94% and a specificity of 80% for polyps measuring greater than 3 mm. CONCLUSION In this limited animal model, the subtraction technique utilized was technically feasible and had an acceptable sensitivity.

Metastases to the Liver from Neuroendocrine Tumors: Effect of Duration of Scan Acquisition on CT Perfusion Values

PURPOSE To assess the effects of acquisition duration on computed tomographic (CT) perfusion parameter values in neuroendocrine liver metastases and normal liver tissue. MATERIALS AND METHODS This retrospective study was institutional review board approved, with waiver of informed consent. CT perfusion studies in 16 patients (median age, 57.5 years; range, 42.0-69.7 years), including six men (median, 54.1 years; range, 42.0-69.7), and 10 women (median, 59.3 years; range 43.6-66.3), with neuroendocrine liver metastases were analyzed by means of distributed parametric modeling to determine tissue blood flow, blood volume, mean transit time, permeability, and hepatic arterial fraction for tumors and normal liver tissue. Analyses were undertaken with acquisition time of 12-590 seconds. Nonparameteric regression analyses were used to evaluate the functional relationships between CT perfusion parameters and acquisition duration. Evidence for time invariance was evaluated for each parameter at multiple time points by inferring the fitted derivative to assess its proximity to zero as a function of acquisition time by using equivalence tests with three levels of confidence (20%, 70%, and 90%). RESULTS CT perfusion parameter values varied, approaching stable values with increasing acquisition duration. Acquisition duration greater than 160 seconds was required to obtain at least low confidence stability in any of the CT perfusion parameters. At 160 seconds of acquisition, all five CT perfusion parameters stabilized with low confidence in tumor and normal tissues, with the exception of hepatic arterial fraction in tumors. After 220 seconds of acquisition, there was stabilization with moderate confidence for blood flow, blood volume, and hepatic arterial fraction in tumors and normal tissue, and for mean transit time in tumors; however, permeability values did not satisfy the moderate stabilization criteria in both tumors and normal tissue until 360 seconds of acquisition. Blood flow, mean transit time, permeability, and hepatic arterial fraction were significantly different between tumor and normal tissue at 360 seconds (P < .001). CONCLUSION CT perfusion parameter values are affected by acquisition duration and approach progressively stable values with increasing acquisition times. Online supplemental material is available for this article.

Semiautomated Motion Correction of Tumors in Lung CT-perfusion Studies

RATIONALE AND OBJECTIVES To compare the relative performance of one-dimensional (1D) manual, rigid-translational, and nonrigid registration techniques to correct misalignment of lung tumor anatomy acquired from computed tomography perfusion (CTp) datasets. MATERIALS AND METHODS Twenty-five datasets in patients with lung tumors who had undergone a CTp protocol were evaluated. Each dataset consisted of one reference CT image from an initial cine slab and six subsequent breathhold helical volumes (16-row multi-detector CT), acquired during intravenous contrast administration. Each helical volume was registered to the reference image using two semiautomated intensity-based registration methods (rigid-translational and nonrigid), and 1D manual registration (the only registration method available in the relevant application software). The performance of each technique to align tumor regions was assessed quantitatively (percent overlap and distance of center of mass), and by a visual validation study (using a 5-point scale). The registration methods were statistically compared using linear mixed and ordinal probit regression models. RESULTS Quantitatively, tumor alignment with the nonrigid method compared to rigid-translation was borderline significant, which in turn was significantly better than the 1D manual method: average (± SD) percent overlap, 91.8 ± 2.3%, 87.7 ± 5.5%, and 77.6 ± 5.9%, respectively; and average (± SD) DCOM, 0.41 ± 0.16 mm, 1.08 ± 1.13 mm, and 2.99 ± 2.93 mm, respectively (all P < .0001). Visual validation confirmed these findings. CONCLUSION Semiautomated registration methods achieved superior alignment of lung tumors compared to the 1D manual method. This will hopefully translate into more reliable CTp analyses.

Effect of Sampling Frequency on Perfusion Values in Perfusion CT of Lung Tumors

OBJECTIVE The purpose of this study was to assess as a potential means of limiting radiation exposure the effect on perfusion CT values of increasing sampling intervals in lung perfusion CT acquisition. SUBJECTS AND METHODS Lung perfusion CT datasets in patients with lung tumors (> 2.5 cm diameter) were analyzed by distributed parameter modeling to yield tumor blood flow, blood volume, mean transit time, and permeability values. Scans were obtained 2-7 days apart with a 16-MDCT scanner without intervening therapy. Linear mixed-model analyses were used to compare perfusion CT values for the reference standard sampling interval of 0.5 second with those of datasets obtained at sampling intervals of 1, 2, and 3 seconds, which included relative shifts to account for uncertainty in preenhancement set points. Scan-rescan reproducibility was assessed by between-visit coefficient of variation. RESULTS Twenty-four lung perfusion CT datasets in 12 patients were analyzed. With increasing sampling interval, mean and 95% CI blood flow and blood volume values were increasingly overestimated by up to 14% (95% CI, 11-18%) and 8% (95% CI, 5-11%) at the 3-second sampling interval, and mean transit time and permeability values were underestimated by up to 11% (95% CI, 9-13%) and 3% (95% CI, 1-6%) compared with the results in the standard sampling interval of 0.5 second. The differences were significant for blood flow, blood volume, and mean transit time for sampling intervals of 2 and 3 seconds (p ≤ 0.0002) but not for the 1-second sampling interval. The between-visit coefficient of variation increased with subsampling for blood flow (32.9-34.2%), blood volume (27.1-33.5%), and permeability (39.0-42.4%) compared with the values in the 0.5-second sampling interval (21.3%, 23.6%, and 32.2%). CONCLUSION Increasing sampling intervals beyond 1 second yields significantly different perfusion CT parameter values compared with the reference standard (up to 18% for 3 seconds of sampling). Scan-rescan reproducibility is also adversely affected.

Effect of dual vascular input functions on CT perfusion parameter values and reproducibility in liver tumors and normal liver

Objective To assess the impact on absolute values and reproducibility of adding portal venous (PV) to arterial input functions in computed tomographic perfusion (CTp) evaluations of liver tumors and normal liver. Methods Institutional review board approval and written informed consent were obtained; the study complied with Health Insurance Portability and Accountability Act regulations. Computed tomographic perfusion source data sets, obtained from 7 patients (containing 9 liver tumors) on 2 occasions, 2 to 7 days apart, were analyzed by deconvolution modeling using dual (“Liver” protocol: PV and aorta) and single (“Body” protocol: aorta only) vascular inputs. Identical tumor, normal liver, aortic and, where applicable, PV regions of interest were used in corresponding analyses to generate tissue blood flow (BF), blood volume (BV), mean transit time (MTT), and permeability (PS) values. Test-retest variability was assessed by within-patient coefficients of variation. Results For liver tumor and normal liver, median BF, BV, and PS were significantly higher for the Liver protocol than for the Body protocol: 171.3 to 177.8 vs 39.4 to 42.0 mL/min per 100 g, 17.2 to 18.7 vs 3.1 to 4.2 mL/100 g, and 65.1 to 78.9 vs 50.4 to 66.1 mL/min per 100 g, respectively (P < 0.01 for all). There were no differences in MTT between protocols. Within-patient coefficients of variation were lower for all parameters with the Liver protocol than with the Body protocol: BF, 7.5% to 11.2% vs 11.7% to 20.8%; BV, 10.1% to 14.4% vs 16.6% to 30.1%; MTT, 4.2% to 5.5% vs 10.4% to 12.9%; and PS, 7.3% to 12.1% vs 12.6% to 20.3%, respectively. Conclusion Utilization of dual vascular input CTp liver analyses has substantial impact on absolute CTp parameter values and test-retest variability. Incorporation of the PV inputs may yield more precise results; however, it imposes substantial practical constraints on acquiring the necessary data.

Effect of duration of scan acquisition on CT perfusion parameter values in primary and metastatic tumors in the lung.

OBJECTIVES To assess the effect of acquisition duration (T(acq)) and pre-enhancement set points (T₁) on computer tomography perfusion (CT(p)) parameter values in primary and metastatic tumors in the lung. MATERIALS AND METHODS 24 lung CT(p) datasets (10 primary; 14 metastatic), acquired using a two phase protocol spanning 125 s, in 12 patients with lung tumors, were analyzed by deconvolution modeling to yield tumor blood flow (BF), blood volume (BV), mean transit time (MTT), and permeability (PS) values. CT(p) analyses were undertaken for the reference dataset (i.e., T₁=t₀) with varying T(acq) from 12 to 125 s. This was repeated for shifts in T₁ (±0.5 s, ±1.0 s, ±2.0 s relative to the reference at t₀). Resultant CTp values were plotted against T(acq); values at 30 s, 50 s, 65 s and 125 s were compared using linear mixed model. RESULTS All CT(p) parameter values were noticeably influenced by T(acq), with generally less marked changes beyond 50 s, and with no difference in behavior between primary and secondary tumors. Apart from BV, which attained a plateau at approximately 50s, the other three CT(p) parameters did not reach steady-state values within the available 125 s of data, with values at 30 s, 50 s and 65 s significantly different from 125 s (p<0.004). Shifts in T₁ also affected the CT(p) parameters values, with positive shifts having greater impact on CT(p) values than negative shifts. CONCLUSION CT(p) parameter values derived from deconvolution modeling can be markedly affected by T(acq), and pre-enhancement set-points. 50 s acquisition may be adequate for BV, but longer than 125 s is probably required for reliable characterization of the other three CT(p) parameters.

Effect on perfusion values of sampling interval of computed tomographic perfusion acquisitions in neuroendocrine liver metastases and normal liver

Objective This study aimed to assess the effects of sampling interval (SI) of computed tomographic (CT) perfusion acquisitions on CT perfusion values in normal liver and liver metastases from neuroendocrine tumors. Methods Computed tomographic perfusion in 16 patients with neuroendocrine liver metastases was analyzed using distributed-parameter modeling to yield tissue blood flow, blood volume, mean transit time, permeability, and hepatic arterial fraction for tumor and normal liver. Computed tomographic perfusion values for the reference SI of 0.5 s (SI0.5) were compared with those of SI data sets of 1 second, 2 seconds, 3 seconds, and 4 seconds using mixed-effects model analyses. Results Increases in SI beyond 1 second were associated with significant and increasing departures of CT perfusion parameters from the reference values at SI0.5 (P ⩽ 0.0009). Computed tomographic perfusion values deviated from the reference with increasing uncertainty with increasing SIs. Findings for normal liver were concordant. Conclusions Increasing SIs beyond 1 second yield significantly different CT perfusion parameter values compared with the reference values at SI0.5.