The prognostic value of cancer stem-like cell markers SOX2 and CD133 in stage III colon cancer is modified by expression of the immune-related markers FoxP3, PD-L1 and CD3.

Cancer stem-like cells are highly tumourigenic cells that can repopulate entire tumours after apparent successful treatment. Recent evidence suggests they interact with other cells in the tumour microenvironment, including immune cell subsets, to enhance their survival. The aim of this study was to determine whether the expression of immune cell markers in primary colon cancer impacts the prognostic significance of cancer stem-like cell marker expression. Immunohistochemistry was used to assess the expression of putative stem cell markers (ALDH1, CD44v6, CD133, Lgr5, SOX2) and immune cell related markers (CD3, CD8, FoxP3, PD-L1) in 104 patients with stage III colon cancer. Associations of marker expression with overall and cancer-specific survival were determined using Kaplan-Meier analysis. High SOX2 expression in the central tumour area was found to be an independent factor for poor cancer-specific survival [hazard ratio (HR) 6.19; 95% confidence interval (CI) 2.24-17.14; p=0.001]. When immune-related factors were taken into account, patients categorised as SOX2low/FoxP3high had good outcome (HR 0.164; 95%CI 0.066-0.406; p<0.0001) whereas patients categorised as SOX2high/PD-L1low had poor outcome (HR 8.992; 95%CI 3.397-23.803; p<0.0001). The prognostic value of the SOX2 cancer stem-like cell marker in colon cancer is modified by expression of immune-cell related factors FoxP3 and PD-L1.

Objective analysis of cancer stem cell marker expression using immunohistochemistry.

Analysis of immunohistochemical expression is often a subjective and semiquantitative process that can lead to the inconsistent reporting of results. To assess the effect that region selection and quantification method have on results, five different cancer stem cell markers were used in this study to compare tissue scoring with digital analysis methods that used three different tissue annotation methods. Samples of tumour and normal mucosa were used from 10 consecutive stage II colon cancer patients and stained for the putative cancer stem cell markers ALDH1, CD44v6, CD133, Lgr5 and SOX2. Tissue scoring was found to have considerably different results to digital analysis with the three different digital methods harbouring concordant results overall. However, SOX2 on normal tissue and CD133 on tumour and normal tissue produced discordant results which could be attributed to the different regions of tissue that were analysed. It is important that quantification method and selection of analysis areas are considered as part of study design to ensure that reproducible and consistent results are reported in the literature.

Low stromal Foxp3+ regulatory T-cell density is associated with complete response to neoadjuvant chemoradiotherapy in rectal cancer.

BACKGROUND: Foxp3+ regulatory T cells (Tregs) play a vital role in preventing autoimmunity, but also suppress antitumour immune responses. Tumour infiltration by Tregs has strong prognostic significance in colorectal cancer, and accumulating evidence suggests that chemotherapy and radiotherapy efficacy has an immune-mediated component. Whether Tregs play an inhibitory role in chemoradiotherapy (CRT) response in rectal cancer remains unknown. METHODS: Foxp3+, CD3+, CD4+, CD8+ and IL-17+ cell density in post-CRT surgical samples from 128 patients with rectal cancer was assessed by immunohistochemistry. The relationship between T-cell subset densities and clinical outcome (tumour regression and survival) was evaluated. RESULTS: Stromal Foxp3+ cell density was strongly associated with tumour regression grade (P=0.0006). A low stromal Foxp3+ cell density was observed in 84% of patients who had a pathologic complete response (pCR) compared with 41% of patients who did not (OR: 7.56, P=0.0005; OR: 5.27, P=0.006 after adjustment for presurgery clinical factors). Low stromal Foxp3+ cell density was also associated with improved recurrence-free survival (HR: 0.46, P=0.03), although not independent of tumour regression grade. CONCLUSIONS: Regulatory T cells in the tumour microenvironment may inhibit response to neoadjuvant CRT and may represent a therapeutic target in rectal cancer.

A phase 1b clinical trial of the CD40-activating antibody CP-870,893 in combination with cisplatin and pemetrexed in malignant pleural mesothelioma.

BACKGROUND: Data from murine models suggest that CD40 activation may synergize with cytotoxic chemotherapy. We aimed to determine the maximum tolerated dose (MTD) and toxicity profile and to explore immunological biomarkers of the CD40-activating antibody CP-870,893 with cisplatin and pemetrexed in patients with malignant pleural mesothelioma (MPM). PATIENTS AND METHODS: Eligible patients had confirmed MPM, ECOG performance status 0-1, and measurable disease. Patients received cisplatin 75 mg/m(2) and pemetrexed 500 mg/m(2) on day 1 and CP-870,893 on day 8 of a 21-day cycle for maximum 6 cycles with up to 6 subsequent cycles single-agent CP-870,893. Immune cell subset changes were examined weekly by flow cytometry. RESULTS: Fifteen patients were treated at three dose levels. The MTD of CP-870,893 was 0.15 mg/kg, and was exceeded at 0.2 mg/kg with one grade 4 splenic infarction and one grade 3 confusion and hyponatraemia. Cytokine release syndrome (CRS) occurred in most patients (80%) following CP-870,893. Haematological toxicities were consistent with cisplatin and pemetrexed chemotherapy. Six partial responses (40%) and 9 stable disease (53%) as best response were observed. The median overall survival was 16.5 months; the median progression-free survival was 6.3 months. Three patients survived beyond 30 months. CD19+ B cells decreased over 6 cycles of chemoimmunotherapy (P < 0.001) with a concomitant increase in the proportion of CD27+ memory B cells (P < 0.001) and activated CD86+CD27+ memory B cells (P < 0.001), as an immunopharmacodynamic marker of CD40 activation. CONCLUSIONS: CP-870,893 with cisplatin and pemetrexed is safe and tolerable at 0.15 mg/kg, although most patients experience CRS. While objective response rates are similar to chemotherapy alone, three patients achieved long-term survival. AUSTRALIA NEW ZEALAND CLINICAL TRIALS REGISTRY NUMBER: ACTRN12609000294257.

Post-chemotherapy T-cell recovery is a marker of improved survival in patients with advanced thoracic malignancies.

BACKGROUND: There is increasing interest in combining chemotherapy with immunotherapy. However, the effects of chemotherapy on the human immune system are largely unknown. METHODS: Longitudinal changes in peripheral T-cell subsets in 40 patients with malignant mesothelioma (MM) or advanced non-small cell lung cancer (NSCLC) receiving platinum-based chemotherapy were assessed by flow cytometry and evaluated for associations with clinical outcome. RESULTS: Proliferating T cells of all subsets were almost entirely depleted at day 8 following chemotherapy, but rapidly recovered above baseline levels. Regulatory T cells (Treg) were most profoundly depleted at this time point. A greater increase in CD8(+) T-cell proliferation following one treatment cycle was associated with improved overall survival in univariate (hazard ratio (HR)=0.40; P<0.05) and multivariate (HR=0.17; P<0.01) analyses. A greater increase in the ratio of CD8(+) T cell to Treg proliferation was also predictive of better prognosis. CONCLUSION: Chemotherapy potentially provides a favourable environment for the development of anti-tumour immunity through transient Treg depletion and regeneration of the T-cell pool. Change in CD8(+) T-cell proliferation after one cycle of chemotherapy may represent a useful prognostic indicator in patients with MM and NSCLC.

Beneficial effects of a 3-week course of intramuscular glucocorticoid injections in patients with very early inflammatory polyarthritis: results of the STIVEA trial.

OBJECTIVE: To evaluate whether treating patients with very early inflammatory polyarthritis (IP) with a 3-week course of intramuscular (IM) methylprednisolone acetate may postpone the need for disease-modifying antirheumatic drugs (DMARDs) and prevent IP from evolving into rheumatoid arthritis (RA). METHODS: Patients with very early IP (4-10 weeks' duration) were randomised to receive three injections of either 80 mg IM methylprednisolone acetate or placebo, given at weekly intervals. Assessments were monthly until 6 months after the first injection, and then concluded at 12 months. The primary outcome was the need to start DMARDs by the 6-month assessment. Secondary outcomes included disease activity and final clinical diagnosis by the rheumatologist at 12 months. RESULTS: Patients in the placebo group (76%) were more likely to need DMARDs during the first 6 months of the trial than patients in the glucocorticoid group (61%) (adjusted OR = 2.11, 95% CI 1.16 to 3.85, p = 0.015). Disease activity did not differ between the two groups at 12 months, probably because many patients in the placebo group started DMARDs early in the study. After 12 months, the arthritis had resolved without the need for DMARDs in 9.9% (11/111) of the patients in the placebo group and in 19.8% (22/111) in the glucocorticoid-treated group (adjusted OR = 0.42, 95% CI 0.18 to 0.99, p = 0.048). CONCLUSION: Treatment of patients with very early IP with IM methylprednisolone acetate appears to postpone the prescription of DMARDs and prevent one in 10 patients from progressing into RA.

The comparative responsiveness of the EQ-5D and SF-6D to change in patients with inflammatory arthritis.

PURPOSE: Comparative evidence regarding the responsiveness of the EQ-5D and SF-6D in arthritis patients is conflicting and insufficient across the range of disease severity. We examined the comparative responsiveness of the EQ-5D and SF-6D in cohorts of patients with early inflammatory disease through to severe rheumatoid arthritis (RA). METHODS: Responsiveness was tested using the effect size (ES) and standardised response mean (SRM). Correlation of change in EQ-5D and SF-6D with disease specific measures was tested using Pearson correlations and the Steiger's Z test. Treatment response and self-reported change were used as anchors of important change. RESULTS: The EQ-5D was more responsive to deterioration (ES ratio (EQ-5D/SF-6D): 1.6-3.0) and the SF-6D more responsive to improvement (ES ratio (SF-6D/EQ-5D): 1.1-1.8) in health. The SF-6D did not respond well to deterioration in patients with established severe RA (ES and SRM 0.08). The EQ-5D provided larger absolute mean change estimates but with greater variance compared to the SF-6D. CONCLUSIONS: The comparative responsiveness of the EQ-5D and SF-6D differs according to the direction of change. The level of mean change of the EQ-5D relative to the SF-6D has implications for cost-effectiveness analysis. Use of the SF-6D in patients with severe progressive disease may be inappropriate.

Chemoimmunotherapy: an emerging strategy for the treatment of malignant mesothelioma.

Whether the immune system can recognize malignant and premalignant cells and eliminate them to prevent the development of cancer is still a matter of open debate, but in our view, the balance of evidence favours this concept. Nonetheless, the International Agency for Research on Cancer has now predicted that cancer will overtake heart disease as the leading cause of death worldwide by 2010, showing that this protective mechanism often fails. Malignant mesothelioma has traditionally been considered a relatively non-immunogenic cancer. However, mesothelioma cells do express a set of well-defined tumour antigens that have been shown to engage with the host immune system. Mesothelioma should therefore be considered a target for immunotherapy. A variety of anticancer immunotherapies have been investigated in mesothelioma and in other malignancies, although these have been largely ineffective when used in isolation. Over recent years, there has been increasing interest in the possibility of combining immunotherapy with chemotherapy in the fight against cancer. Here, we discuss the rationale behind combining these two, long considered antagonistic, treatment options in the context of malignant mesothelioma.

Computer-aided design and modeling of workstations and radiology reading rooms for the new millennium.

Three-dimensional (3D) computer modeling, simulation, and rendering techniques were used to redesign the diagnostic workstations and radiology reading rooms for a proposed hospital with particular attention given to lighting conditions, noise reduction, and optimal use of limited workspace. The results were presented to a panel of multidisciplinary experts and iteratively improved and redesigned with the development or addition of new design criteria or requirements. These 3D techniques allowed faster, more efficient design and presentation of multiple options than is possible with traditional two-dimensional drawings, thereby expediting decision making and resulting in significant savings. The current workstation designs can easily be developed and implemented with available technology at a reasonable cost. They can also accommodate anticipated advances in computer and display technology as well as new imaging paradigms (eg, changes in keyboard and control ergonomics such as adjustable virtual keys on touch-sensitive screens, digital drawing tablets for annotations and controls, direct film digitizing, personal identification devices, offline media readers such as compact disks and digital videodisks, and speech recognition and voice activation). Use of 3D techniques in designing other parts of the radiology department (eg, examination rooms, technologists' areas, physicians' offices) could greatly improve and facilitate the design and implementation of complex settings in these work areas.

Time-windowing of click-evoked otoacoustic emissions to increase signal-to-noise ratio.

OBJECTIVE: To investigate the effects of decreasing the response-window duration on the signal-to-noise ratio (S/N) of click-evoked otoacoustic emissions (CEOAEs). DESIGN: The ILO88 (Otodynamics, Ltd.) was used to measure CEOAEs from 149 normal adult ears, and 75 adult ears with high-frequency sensorineural hearing loss. Data were collected using the default response window of 2.5 to 20.5 msec post-click. Each response was rewindowed, post-hoc, from 2.5 to 7.5 msec, 2.5 to 9 msec, 7.75 to 14.25 msec, and 13 to 19.5 msec post-click. For each window, spectra of the CEOAE and of the background noise were determined. The S/N was estimated by subtracting the noise level from the CEOAE amplitude. RESULTS: The 13- to 19.5-msec window contained little CEOAE energy relative to earlier windows. Relative to the 2.5- to 20.5-msec window, the 2.5- to 7.5- and 2.5- to 9-msec windows reduced noise levels more than CEOAE amplitudes, yielding increased S/N, and greater "reproducibility" values. The increased S/N of the 2.5- to 7.5- and 2.5- to 9-msec windows allowed measurement of greater CEOAE-amplitude reductions in the impaired ears relative to the normal ears. With short-duration windows, click-presentation rate could be increased, allowing more responses to be averaged in a given time, thus further decreasing noise levels. Although click rate was not varied in the present study, the decrease of noise levels is predictable. Accounting for this factor, it is expected that a specified S/N would be obtained about five times faster using the 2.5- to 7.5-msec window with a 7.5-msec interstimulus interval, than when using the default window. CONCLUSIONS: Decreasing the response-window duration substantially increases the measurement efficiency of CEOAEs in adults, and thus may enhance clinical-test performance.

Monitoring cochlear function intraoperatively using distortion product otoacoustic emissions.

Distortion product otoacoustic emissions (DPOAEs) elicited by a bitonal stimulus complex are low-level sounds that are generated within the cochlea, and are easily measured by a miniature microphone system placed in the external auditory canal. Under conditions of rapid measurement, DPOAEs have been shown in an animal model to be exquisitely sensitive to interruption of the blood supply to the inner ear. Currently, there is no real-time procedure available for monitoring hearing function intraoperatively during neurotologic surgery. The goal of the present study was to determine the utility of DPOAEs in monitoring cochlear status intraoperatively, particularly during procedures commonly used to remove acoustic neuromas. Distortion product otoacoustic emissions were measured pre- and intraoperatively in 11 patients, who were under general anesthesia for a variety of otolaryngologic procedures, including acoustic neuroma resection. High frequency emissions (i.e., > or = 4 kHz) were less affected by the elevated levels of acoustic noise present in the operating-room environment, thus permitting emitted responses to be updated as quickly as every 2 seconds. However, a number of technical problems were encountered and addressed during development of the emission monitoring technique. Despite these difficulties, overall, DPOAEs appear to be a promising adjunct to the intraoperative monitoring of auditory function.

Dependence of distortion-product otoacoustic emissions on primary levels in normal and impaired ears. I. Effects of decreasing L2 below L1.

The 2f1-f2 distortion-product otoacoustic emission (DPOAE) is evoked by two primary tones of frequencies f1 < f2, and levels L1 and L2. Previous reports indicate that decreasing L2 below L1 = L2 can; (1) increase DPOAE amplitude in normal ears, and (2) increase the degree to which DPOAE amplitudes are reduced by cochlear trauma. Although both of these factors could be advantageous for clinical applications of DPOAEs, neither has been explored in detail. In the present study, 2f1-f2 DPOAE-amplitude frequency functions were collected from normal and impaired ears of rabbits and humans, with L1 = L2, and with L2 < L1, at each of three values of L1. In rabbits, controlled tonal or noise overexposures were used to produce permanent reductions of DPOAE amplitudes. Comparison of pre- and postexposure DPOAE-amplitude frequency functions demonstrated that the frequency-specific reductions of DPOAEs were enhanced by decreasing L2 below L1. In humans, DPOAE-amplitude frequency functions obtained with the various L1 and L2 combinations were collected from 16 normal ears to provide preliminary normative data for each stimulus-level condition. The L1-L2 that produced the maximum DPOAE amplitude in normal ears was systematically dependent on L1. Thus at most frequencies, decreasing L2 below L1 = L2 substantially reduced mean DPOAE amplitude when L1 > or = 75 dB SPL, but increased mean DPOAE amplitudes at L1 = 65 dB SPL. However, the increase of mean DPOAE amplitude obtained by decreasing L2 below L1 = 65 dB SPL was small, being less than 3.5 dB at most frequencies. More importantly, at L1 = 65 dB SPL, L2 could be decreased considerably below L1 = L2 without reducing mean DPOAE amplitude relative to that at L1 = L2. Inspection of DPOAE-amplitude frequency functions obtained from subjects with mild or moderate sensorineural hearing losses indicated that, in frequency regions of hearing impairment, decreasing L2 below L1 can enhance the degree of reduction of DPOAEs below the corresponding normative amplitudes, without reducing the normative amplitude. It is concluded that decreasing L2 below L1 = L2 has the potential to enhance the performance of DPOAEs in clinical applications.

Dependence of distortion-product otoacoustic emissions on primary levels in normal and impaired ears. II. Asymmetry in L1,L2 space.

Previous studies indicate that the amplitude of 2f1-f2 distortion-product otoacoustic emissions (DPOAEs), evoked by two tones of frequencies f1 < f2, demonstrates a complex dependence on the levels (L1 and L2) of the primary tones. In the present study, 2f1-f2 DPOAE amplitudes were measured over a wide range of L1 and L2 in normal human ears, allowing a systematic, level-dependent asymmetry of DPOAE amplitude in L1,L2 space to be characterized. The L1,L2 at which DPOAEs were largest was close to L1 = L2 at high stimulus levels, but moved monotonically toward L1 > L2 as stimulus levels decreased. A related observation was that DPOAE amplitude had a greater dependence on L1 and on L2. These asymmetries were quantified in normal human ears, and compared to the corresponding asymmetries apparent in data from animal models. Recent studies have demonstrated that the reduction of DPOAE amplitude by cochlear trauma is greater when L1 > L2 than when L1 = L2, suggesting that the reduction of DPOAEs by trauma demonstrates an asymmetry in L1,L2 space that is qualitatively similar to that of normative DPOAE amplitude. To investigate this issue, 2f1-f2 DPOAE amplitudes were measured over a wide range of L1 and L2 in rabbit ears pre- and postinjection of the ototoxic loop-diuretic ethacrynic acid. The results indicate that the asymmetry in L1,L2 space of the reduction of DPOAEs by trauma is both qualitatively and quantitatively similar to the asymmetry in L1,L2 space of normative DPOAE amplitude. Specifically, the L1 values that maximized normative DPOAE amplitudes for any specified L2 (or, equivalently, the L1 values that allowed L2 to be minimized for any specified normative DPOAE amplitude) also yielded the greatest reduction of DPOAEs by the diuretic. In humans, the L1 values that maximize normative DPOAE amplitudes for any specified L2 are well approximated by a simple equation, with parameters that vary with frequency and f2/f1. It is suggested that the L1,L2 values defined by this equation may be optimum for use in clinical applications.

Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation.

Vascular cell adhesion molecule-1 (VCAM-1) is expressed on vascular endothelium in a variety of inflammatory conditions and mediates leukocyte recruitment from blood into tissues. In this study we report a novel role for VCAM-1 in the formation of the umbilical cord and placenta during development. The murine VCAM1 gene was disrupted by targeted homologous recombination, and a distinct phenotype was found in VCAM-1-deficient embryos. At 8.5 days of gestation, the allantois failed to fuse to the chorion, resulting in abnormal placental development and embryonic death within 1-3 days. In addition, a role for VCAM-1 in early placental formation after chorioallantoic fusion was observed. In a minority of VCAM-1-deficient embryos, the allantois was able to fuse with the chorion, but the allantoic mesoderm was abnormally distributed over the chorionic surface. A small number of VCAM-1-deficient embryos survived, presumably by circumventing the placentation defects. They became viable and fertile adult mice with lack of VCAM-1 expression, normal organs, and an elevated number of circulating blood mononuclear leukocytes.

New approaches to the evaluation of the auditory system and a current analysis of otoacoustic emissions.

Healthy ears generate low-level sounds known as otoacoustic emissions that are produced by the normal workings of the inner ear. By placing a specially constructed probe containing an assembly of miniature microphones in the ear canal, hearing investigators can listen to these sounds. Before emissions were discovered, the only methods available to explore the ordinarily inaccessible structures of the cochlea involved invasive and, thus damaging, experiments, which could only be performed on animals. With the discovery of otoacoustic emissions, noninvasive research on the inner ear became possible, thus allowing study of the fundamental processes that determine the excellent sensitivity and fine frequency tuning that are uniquely associated with human hearing. The results of these basic experiments have made it possible to develop a number of useful clinical applications based on emissions testing. One noteworthy benefit is the use of emissions as a screening test that objectively assesses the functional integrity of peripheral processing in patients who are difficult to examine, such as infants and young children. Other applications take advantage of the test's diagnostic strength as an indicator of the sensory component of a sensorineural hearing loss. Finally, because emissions testing can be conducted rapidly and accurately under computer control, it has proved useful in the serial monitoring of ear performance in instances where a progressive hearing impairment is suspected.

Angina and myocardial infarction with use of leuprolide acetate.

Use of leuprolide acetate (Lupron) has been associated with angina in men, but there have been no previous reports of angina or myocardial infarction associated with its use in women. The first case of angina and myocardial infarction occurring in a woman undergoing treatment with leuprolide acetate depot is reported.

Clinical testing of distortion-product otoacoustic emissions.

Otoacoustic emissions have great promise for use in clinical tests of the functional status of outer hair cells, which represent cochlear structures that make a major contribution to the hearing process. A substantial literature is available concerning the evaluation of outer hair cell function by transiently evoked otoacoustic emissions. However, relatively little attention has been focused on the benefits of testing with distortion-product otoacoustic emissions. The purpose of this presentation is to provide knowledge of the principal advantages offered by distortion-product emissions testing.

Otoacoustic emissions in children with normal ears, middle ear dysfunction, and ventilating tubes.

The clinical utility of otoacoustic emissions (OAEs) has been well established in adults. The purpose of this investigation was to determine the efficacy of OAE testing in children. Distortion-product OAE (DPOAE) audiograms, response/growth functions, and transiently evoked OAEs elicited with clicks were measured from the ears of both healthy volunteers, aged 4 to 13 years, and children with confirmed middle ear disorders. These measures established the means and variabilities for DPOAE and noise-floor amplitudes of normal and diseased young ears. Compared with adult emissions, the healthy young ears exhibited greater mean DPOAE and noise-floor amplitudes. In contrast, ears with type B and type C tympanogram patterns showed absent or markedly reduced OAE amplitudes, when compared with emissions measured in their control counterparts. Finally, ears with ventilating tubes exhibited OAE amplitudes lower than amplitudes from healthy ears, but higher than those of the untreated diseased ears. Although these findings imply that using OAEs to test the outer hair-cell reserve of infected ears is problematic, emitted responses provide useful information concerning the normalcy of middle ear function.

Test/retest reliability of distortion-product and transiently evoked otoacoustic emissions.

Otoacoustic emissions can be used to study cochlear function in an objective and noninvasive manner. These features of emitted responses have stimulated a great deal of investigation into the utility of evoked emissions as clinical tests of hearing. One practical and essential aspect of any clinical measure is the consistency of its result upon repeated testing of the same individual (i.e., its test/retest reliability). The goal of the present work was to conduct a systematic study of the test/retest reliability of the two evoked emission types, the transiently evoked and the distortion-product otoacoustic emissions, that have the greatest promise of becoming clinically useful. Toward this end, the short- and long-term reliabilities of these two response measures were examined in 12 normally hearing adults. The results of these experiments showed that the consistency of both measures of evoked otoacoustic emissions was generally excellent.