Human mass balance study of the novel anticancer agent ixabepilone using accelerator mass spectrometry

Summary
Introduction
The taxanes paclitaxel and docetaxel are important anti-cancer drugs [1]. However, their use is complicated by low oral bioavailability and the development of resistance due to MDR1 over-expression or β-tubulin mutation [2, 3]. A search for microtubule stabilizing agents with improved characteristics compared to taxanes led to the discovery of epothilone A and B in extracts of the myxobacterium Sorangium cellulosum [3], which were subsequently shown to bind to tubulin at the same binding site as paclitaxel [4]. In vitro, epothilone B was more active than in vivo (mouse models) [5], and in vivo hydrolysis of the lactone ring was suspected of producing inactive metabolites. To improve the metabolic stability of the naturally occurring epothilones, these were modified by replacing the ester bond with an amide bond [6,7]. In general, lactam analogues displayed tubulin-polymerizing and cytotoxic potencies inferior to their naturally occurring counterparts. The exception to this rule was the lactam analogue of epothilone B, ixabepilone (BMS-247550, NSC 71028, EpoB-lactam, aza-EpoB, 15-desoxy-15-aza-epothilone B), see Fig. 1 for structure [6]. Ixabepilone has undergone a number of phase II clinical trials with varying schedules at doses from 6 to 40 mg/m2 administered as an intravenous infusion over 1 to 3 h [8–11]. Response rates in metastatic breast (12–44%), non-small cell lung (14%), hormone-refractory metastatic prostate cancer (33%), and metastatic renal cell carcinoma (14%) were modest to promising and warrant further investigation [8]. The pharmacokinetics of ixabepilone have been described [9,12,13] and the contribution of two known chemical degradation products was shown to be negligible [14], however, other than that it is a CYP3A4/5 substrate [15], little is known about the excretion and metabolic fate of ixabepilone.
Fig. 1Chemical structures of epothilone B and its lactam analogue ixabepilone
The present investigation was aimed at determining the pharmacokinetics and excretory pathways of [14C]ixabepilone. To this end, we initiated a mass balance study of ixabepilone in humans. A mass balance is an elaborate pharmacokinetic investigation employing a radioactive tracer. It investigates the plasma pharmacokinetics and excretion of both the unchanged drug and the total radioactivity (drug and metabolites), and allows elucidation of the metabolic fate of a drug. The main objective is the maximum recovery of the radioactive dose in urine and faeces [16]. Due to autoradiolysis, [14C]ixabepilone at a typical specific activity of 1.5 μCi/mg was unstable. However, [14C]ixabepilone at a specific activity of less than 10 nCi/mg was found to be stable. Therefore a study was conducted with a low radioactive dose of 80 nCi of 14C-ixabepilone, as opposed to a more typical human radio-tracer dose of circa 100 μCi dosed in study. For detection of these very low levels of radioactivity we used an ultra-sensitive detection method, Accelerator Mass Spectrometry (AMS) [17–19], which measures the amount of 14C as if it were a stable isotope label instead of measuring its decay. AMS separates elemental isotopes through differences in mass, charge, and energy, resulting in a [14C]/[12C] isotope ratio. Subtracting the natural [14C] background level and taking into account the total carbon content of the sample results in a total [14C] amount that is converted to a conventional unit for radioactivity (DPM, Bq, or Ci). Given the very low dose of radioactivity administered (80 nCi compared to 100 μCi in a typical radio-tracer study), administration would result in negligible radiation exposure.
Materials and methods
Study design
This was an open-label, single-dose study in which eight patients with histologically confirmed solid tumours received an intravenous fixed dose of 70 mg, 80 nCi [14C]ixabepilone over 3 h. The protocol and informed consent form were reviewed and approved by the Institutional Review Board of the Antoni van Leeuwenhoek Hospital/The Netherlands Cancer Institute, and each patient gave written informed consent before receiving the study medication. Eligibility criteria most relevant to the mass balance study were: availability for in-house admittance during the first 8 days, adequate hepatic and renal function, a performance status (PS) of 0–2, and a life expectancy of at least 12 weeks.
Patients
Eight patients (three male and five female) with a median age of 54.5 years and a WHO performance status of 0–2 were enrolled in the study. All patients had advanced cancer for which no standard anti-cancer therapy was available. The primary tumours were: squamous cell carcinoma, colon cancer (n = 2), gastric carcinoma, ovarian cancer, non-small cell lung cancer, sigmoid adenocarcinoma, and pancreas carcinoma.
Study medication
Individual vials of radiolabeled ixabepilone contained 20 mg, 22.8 nCi [14C]ixabepilone (specific activity 1.14 nCi/mg). The [14C]ixabepilone was synthesized by Bristol-Myers Squibb by growing the myxobacterium Sorangium cellulosum in 14C containing medium. This resulted in labeling throughout the ixabepilone molecule. Radiochemical and chemical purity was confirmed at >99%. Vials were reconstituted using 10 ml Cremophor®EL:ethanol (1:1, v/v) each. A 35 ml volume of the reconstituted [14C]ixabepilone was added to 140 ml Lactated Ringer’s Infusion (LRI). After mixing, a 2 ml sample was taken for quantitation of radioactivity by liquid scintillation counting (LSC). LSC radioactivity values were recalculated to amounts of [14C]ixabepilone for use in calculating recovery from feces and urine. After the 3-h intravenous administration, infusion lines were flushed with 500 ml LRI. Gravimetric measurements of the infusion flask prior to, and after administration enabled calculation of the amount of radioactivity actually administered. To prevent Cremophor®EL-related hypersensitivity reactions, patients were pre-medicated with oral H1 and H2 blockers.
Sample collection
Blood was collected at 0 (pre-dose), 1.5, 3 (prior to end of infusion), 3.25, 3.5, 3.75, 4, 5, 6, 8, 12, 24, 48, 72, 120, and 168 h after start of infusion. An indwelling catheter contra-lateral to the administration site was used for serial blood sampling. Separate, but simultaneous, blood samples were taken for the quantitation of total radioactivity and unchanged ixabepilone, respectively. Plasma was obtained by immediate centrifugation of the blood (10 min, 1000 × g, 4°C). The plasma layer was aspirated and stored below −20°C until analysis. We collected complete urinary and faecal output up to 7 days after administration. Urine was collected over 24-h intervals in refrigerated collection jugs. At the end of each collection interval, the respective urine samples were mixed and total volume was recorded. Aliquots were stored below −20°C until analysis. Faecal samples were collected and stored at −20°C per portion and combined per 24-h interval. Faeces were homogenized after addition of water (2:1, w/w). Aliquots were stored below −20°C until analysis.
AMS analysis of total radioactivity (TRA) in plasma, urine and faeces
TRA in plasma, urine and faeces was determined using AMS (Exceleron, York, UK) [20, 21]. Briefly, sample pre-treatment consisted of the conversion of the carbon within the samples to graphite via a two-step process of oxidation and reduction [22]. Oxidation (combustion) was performed by heating the dried sample with copper oxide under vacuum at 900°C for 2 h in a heat-sealed tube. Reduction (graphitisation) was performed by heating the carbon dioxide formed from the oxidised sample with zinc, titanium hydride and cobalt at 500°C for 4 h, followed by a further 6 h at 550°C. After the graphitisation process, the graphite was pressed into a cathode. In the AMS-procedure, this cathode was bombarded with high-energy caesium (Cs+) ions. The resulting negative carbon ion beam was introduced into the AMS instrument, accelerated by an electric field of 4.5 MV, and the carbon atoms were stripped of their valency electrons. This resulted in separation of the carbon isotopical elements as C4+ ions. The carbon isotopes 12C and 13C were quantitated using Faraday cups, while 14C was quantitated by a gas ionization detector. Because AMS provides an isotope ratio that is [14C]/[12C] and not an absolute value, it is necessary to know the carbon content of the sample plus any added carrier. This was determined using a carbon, hydrogen, nitrogen analyser (Elemental Microanalysis Ltd., Okehampton, UK). The natural background [14C]/[12C] level per matrix was subtracted to obtain the amount of ixabepilone-related 14C. The total carbon content and the specific activity of [14C]ixabepilone was used to convert the [14C]/[12C] ratio to ixabepilone equivalents (ng/ml sample or % of dose). Using this sophisticated technique, it was possible to quantitate <4 DPM/ml plasma, 0.01 DPM/ml urine and 0.1 DPM/g faeces (1 DPM corresponds to 395 ng [14C]ixabepilone).
Analysis of ixabepilone in plasma and urine
Concentrations of ixabepilone parent drug in plasma and urine were determined with a validated liquid chromatography assay equipped with tandem mass spectrometric detection (Bristol-Myers Squibb, Princeton, NJ). Briefly, after addition of internal standard (BMS-212188) to 0.2 ml of each sample, calibration standard and quality control sample, the samples were precipitated with acetone. The supernatant was further extracted with 1-chlorobutane. The organic layer was removed and evaporated to dryness. The residue was reconstituted and injected into the LC-MS/MS system. Chromatographic separation was achieved, isocratically, on a YMC ODS-AQ column (4.6 × 50 mm internal diameter) at a flow rate of 0.3 ml/min with detection by electrospray tandem mass spectrometry. The mobile phase contained acetonitrile—0.01 M ammonium acetate (pH 5.0) (65:35, v/v). The standard curve, which ranged from 2 to 500 ng/ml, was constructed with a 1/x weighted quadratic regression model. The within-run precision for ixabepilone in plasma and urine was within 15 and 9%, respectively. The between-run precision for ixabepilone in plasma and urine was within 13 and 9%, respectively. The accuracy was within 11 and 6% of the nominal values in plasma and urine, respectively.
Pharmacokinetic analyses
The pharmacokinetic analyses of the ixabepilone plasma concentration versus time data were performed by non-compartmental methods using Kinetica Version 4.2 (InnaPhase Corporation, Philadelphia, PA). The peak plasma concentration (Cmax) and the time to reach maximal concentration (Tmax) represent observed values. The area under the plasma concentration versus time curve (AUC) was calculated using a combination of linear and log trapezoidal summations. The AUC was extrapolated to infinity (AUCinf) by dividing the last measurable concentration by the terminal rate constant, k. The absolute value of k was used to estimate the apparent terminal half-life, t1/2 = ln 2/ k. The total body clearance (Cl) was determined by dividing dose by AUCinf and the volume of distribution at steady-state (Vss) was calculated as dose × area under the moment curve (AUMCinf)/(AUCinf)2 with appropriate corrections for infusion duration. Renal clearance was calculated by dividing amount excreted by the AUC. Similarly, pseudopharmacokinetic parameters were calculated for TRA.
Outliers were statistically assessed using Dixon’s Q test.
Results
To evaluate the pharmacokinetics and excretory pathways of [14C]ixabepilone, we performed a mass balance study in eight patients. Figure 2 shows the mean plasma concentrations over time of both TRA and ixabepilone as determined by AMS and LC-MS/MS, respectively.
Fig. 2Mean (±SD) plasma concentration-time curves of [14C]ixabepilone derived radioactivity (filled square) and unchanged [14C]ixabepilone (open square) for eight patients
Both TRA and ixabepilone in plasma exhibited an initial distribution phase of approximately 10 h followed by a much slower terminal elimination phase with half-lives of 73.1 and 50.3 h, respectively. The time to maximum plasma concentration was typically around the end of infusion. Plasma ixabepilone concentrations were quantifiable through 168 h in all patients and plasma TRA concentrations were quantifiable through 168 h in all patients except in patient 1 in whom plasma TRA concentrations were only quantifiable for 48 h. Pharmacokinetic parameters are summarized in Table 1. The ratio of the ixabepilone AUC to the total radioactivity AUC, or AUC-ratio, is a measure of exposure to [14C]ixabepilone relative to total [14C]ixabepilone related compounds. The AUC ratio of patient 1 was larger than unity. This was caused by the underestimated TRA AUC, which was based on measurements until only 48 h, after which plasma concentrations fell below the lower limit of quantitation. Besides the TRA AUC, the t1/2 of patient 1 was also inaccurately estimated at 8.3 h. Therefore, patient 1 was omitted from calculation of mean plasma TRA parameters. Figure 3 shows the plasma concentrations of ixabepilone relative to TRA for patients 1–7 and patient 8. Patient 8 experienced a blocked bile duct (reflected by a maximum plasma bilirubin concentration of 47 μmol/l (3 × upper limit of normal) and alkaline phosphatase of 500 U/l (4 × upper limit of normal) at day 7) resulting in a higher plasma TRA, Cmax and AUC. The higher TRA plasma concentrations in patient 8 resulted in an AUC ratio of plasma ixabepilone to TRA that was lower than that of the other patients. In contrast, unchanged ixabepilone plasma pharmacokinetics were not affected in this patient.
Table 1Plasma pharmacokinetic parameters of ixabepilone and total radioactivity in cancer patients after a 3 h intravenous infusion of 70 mg, 80 nCi [14C]ixabepiloneNrIxabepiloneRadioactivity (ixabepilone equiv.)AUClast ratioaCmax (ng/ml)AUClast (μg*h/ml)AUCinf (μg*h/ml)t1/2(h)Cl (l/h)Vss (l)Cmax (ng/ml)AUClast (μg*h/ml)AUCinf (μg*h/ml)t1/2 (h)12773.063.3551.720.912753221.891.968.31.62b22872.752.9753.023.6117449711.011.848.70.2534332.832.9951.723.492968110.911.336.50.2644863.824.3267.416.2102794020.121.646.90.1952662.422.5223.527.875650320.044.3185.20.1262732.172.2936.730.591257311.916.399.80.1872012.252.4971.528.115204498.539.5961.20.2681871.932.2247.231.61635150232.534.434.40.06Mean3012.652.8950.325.3115468316.4b21.3b73.1b0.19bSD1050.600.6915.45.23083788.44b13.2b54.0b0.08baAUClast ratio is calculated by dividing AUClast ixabepilone by AUClast TRA and expresses the relative contribution of ixabepilone to TRA exposure in plasma.bPatient 1 was not included in the calculation because the radioactivity AUC and t1/2 could not be determined accurately (see text). This also resulted in the deviating AUClast ratio (outlier).Fig. 3Mean (+SD) [14C]ixabepilone to total radioactivity ratio in plasma over time for patients 1–7 (filled square) and patient 8 (open square)
Figure 4 displays the mean excretion of TRA in urine and faeces and the total, in time, and Fig. 5 displays the mean cumulative urinary excretion of [14C]ixabepilone derived radioactivity and unchanged [14C]ixabepilone. Table 2 shows individual 168 h excretions of [14C]ixabepilone and TRA in urine and faeces. The U/F ratio is the ratio of urinary recovery relative to faecal recovery. Patient 3 displayed a rather low recovery of radioactivity from both urine (14.8%) and faeces (38.3%). Plasma pharmacokinetics were not different compared to the other patients. A low total recovery of 37.8% was also obtained for patient 4, attributable to a very low faecal recovery (12.7%), while urinary recovery was normal (25.1%). This patient only defecated at 24 h and at 168 h (a few grams), which almost certainly explains the observed low recovery. Although the plasma pharmacokinetics and urinary excretion did not deviate extremely from the other patients, patient 4 displayed a somewhat elevated plasma exposure to both radioactivity and ixabepilone, and the lowest clearance for unchanged ixabepilone. If we now look at the U/F ratios (a measure of urinary versus faecal elimination), patient 4, who had the low fecal output, is an outlier (Q = 0.86, critical value p = 0.05: 0.57) compared to patients 1, 2, 3, 5, 6 and 7. Patient 8, who experienced the blocked bile duct stent, is also an outlier (Q = 0.91, critical value p = 0.05: 0.52) compared to the same patients. The urinary recovery of patient 8 was highest at 47.7% causing the large standard deviation of urinary recovery. However, the urinary recovery of unchanged ixabepilone of 4.0% was within the normal range. The faecal recovery of this patient was the lowest (excluding patient 4) at 15.6%. Apparently, there was no accumulation of the active drug despite a decreased biliary excretion.
Fig. 4Mean urinary (open square, −SD), faecal (open circle, +SD) and total (filled square, +SD) cumulative excretion (168 h) of [14C]ixabepilone derived radioactivity for eight patientsFig. 5Mean (±SD) cumulative urinary excretion of [14C]ixabepilone derived radioactivity (filled square) and unchanged [14C]ixabepilone (open square) for eight patientsTable 2Urinary and faecal recovery (0–168 h) of ixabepilone and total radioactivity in cancer patients after a 3 h intravenous infusion of 70 mg, 80 nCi [14C]ixabepiloneNraRecovery (% of dose)Radioactivity (U/F ratioc)Ixabepilone (Clrenal (l/h))IxabepiloneTotal radioactivityUrineUrineFaecesTotal15.0714.5659.6974.250.241.1624.6623.2874.7898.060.311.1937.7614.7738.2953.060.391.9243.3225.0812.7137.791.970.6155.4619.6665.0284.680.301.5866.1230.7864.5995.370.481.9876.0325.0586.72111.770.291.8784.0247.6515.6263.273.051.46Mean5.3025.1052.1877.280.33b1.47SD1.3810.6327.1724.940.08b0.47aPatients 1, 2, and 8 were male.bPatient 4 was not included because of the extremely low production of faeces and patient 8 was not included in the calculation because his bile duct obstruction (relevance illustrated by increasing plasma bilirubin concentrations) may have reduced faecal excretion.cThe U/F ratio is the ratio of urinary recovery relative to faecal recovery.
On average, more than 77% of the drug was excreted over the 7-day period with the majority of 52% being excreted in faeces and 25% in urine. Excretion in faeces occurred at a relatively constant rate over the entire collection period, with only 10 and 12% of dose recovered in the 0–24 and 24–48 h collection intervals, respectively. In contrast, urinary excretion displayed two distinct phases with 65% of total urinary radioactivity (corresponding to 17% of dose) and 76% of total urinary ixabepilone being excreted in the first 24 h. From 24 to 168 h, urinary excretion occurred at a relatively slow constant rate. This may reflect the biphasic plasma curve, with high concentrations in the distribution phase and lower concentrations in the terminal elimination phase. At first urinary excretion predominates and after 24 h, faecal excretion becomes the dominant route of excretion.
Discussion
The present investigation was aimed at determining the pharmacokinetics and excretory pathways of [14C]ixabepilone by performing a mass balance study.
In conventional human mass balance studies, approximately 100 μCi of [14C] is typically used to trace the fate of a drug [16]. Due to autoradiolysis of ixabepilone, this level of labeling was not possible. The level of radioactive labeling we used was more than 1,000-fold lower at 80 nCi. The most commonly used method for detection of radiotracers in mass balance studies is liquid scintillation counting (LSC). This method however, relies on the detection of β-emissions and therefore can only exploit a very small proportion of the isotope. 14C has a half-life of 5740 years and therefore only about 0.012% of the 14C atoms in a sample decay over the course of a year. AMS however, is a nuclear physics technique that detects the individual 14C atoms, as if they were stable isotopes, and is therefore much more sensitive than LSC [19]. Originally, AMS technology was developed in the 1970s for radiocarbon dating, but has recently found novel uses in biomedical research. It has been used to study DNA adducts of carcinogens, and it can replace decay counting as a method of analysis in biological samples [17, 23–25]. A first human mass balance using this ultra-sensitive technique has already been reported and is expected to be followed by many others [26].
The ixabepilone plasma pharmacokinetic parameters observed in the present investigation are similar to the values previously reported for a standard phase I dose escalation study in which 11 patients were administered 40 mg/m2 of ixabepilone in an expansion of the recommended phase II dose [9]. However, in our study, the coefficient of variation for AUCinf and Cl of ixabepilone was 24 and 21% as compared to 46 and 38% in the phase I dose escalation study. The lower inter-patient variability may result from the greater rigor toward sample collection and processing that is possible in a mass balance study during which the patient is hospitalized. It should also be noted that the administration of ixabepilone as a flat 70 mg dose in the present study did not result in any increase in inter-patient variability compared with the dose, based on body surface area, which was administered during the phase I study.
Our results show that ixabepilone is extensively metabolised. This is indicated by the plasma AUC ratio of 0.19 and confirmed by the recovery of ixabepilone relative to TRA in urine of approximately 0.21. Patient 8 experienced a blocked bile duct resulting in a higher plasma TRA, Cmax and AUC, but interestingly, unchanged ixabepilone plasma pharmacokinetics were not affected and excretion of ixabepilone metabolites was re-routed to urine.
The calculated half-life of TRA was longer than that of ixabepilone (t1/2 of 73.1 and 50.3 h respectively), however, we do not conclude that TRA was eliminated more slowly than ixabepilone. Comparison of ixabepilone and TRA half-lives per patient revealed that in 5 cases (patient 2, 3, 4, 7, and 8) the TRA half-life was shorter than the ixabepilone half-life. The mean TRA half-life was disproportionally influenced by the long half-life observed in patient 5 (185 h). The very long half-life of TRA in this patient is not likely to be clinically relevant because this patient excreted 85% of the dose within 7 days. Based on the half-lives for plasma radioactivity observed in the other patients, in the once every 3 weeks administration schedule, accumulation of metabolites should not occur. The seemingly long ixabepilone half-life of over 50 h is not unusual when compared to other tubulin-interacting drugs. Half-lives of e.g. vinca-alkaloids range from 12–42 h for vindesine and up to a half-life of several days for vincristine.
With an average recovery of 77% of the drug excreted over the 7-day period, the recovery of radioactivity of this mass balance study is reasonably good [16]. The incomplete recovery is explained by the still ongoing excretion at the end of the 7 day collection period, and the long plasma half-life of TRA. Immediate assessment of the still appreciable amounts of radioactivity excreted on day 7 to possible extend the period of collecting excreta, as suggested in the literature [16], was not possible. The shipping and lag-time in quantitating radioactivity by AMS precluded this. Another contributing route of elimination not monitored in the current study is loss of radioactivity through expiration of 14C O2 [16]. At 52% of the dose versus 25%, faecal excretion is a quantitatively more important route of elimination than urinary excretion. Biliary excretion is governed by a molecular weight limit. In humans, polar compounds with a molecular weight above approximately 500 g/mol are predominantly excreted via bile [27, 28]. Ixabepilone metabolites will probably be more polar (phase I metabolism, oxidation, hydrolysis etc.) and have higher masses (conjugates or phase II metabolites) than ixabepilone (Mwt 506.7 g/mol, and rather hydrophobic). Whether this is the case for ixabepilone remains to be investigated by metabolic profiling of the excreta.
Given the normal ixabepilone plasma concentrations of patient 8 while experiencing a blocked bile duct, biliary excretion of unchanged ixabepilone into faeces may not be a very important elimination route, and the results suggest that ixabepilone was equally well metabolised but that the biliary excretion of these metabolites was hindered by the blocked bile duct stent, resulting in a shift to urinary excretion of total radioactivity.
The importance of metabolism in the elimination of ixabepilone has been confirmed by this study as apparent from the low contribution of unchanged ixabepilone to TRA in plasma and urine. Ixabepilone related radioactivity is predominantly excreted in the faeces. Future investigations must be aimed at elucidating the metabolic fate of ixabepilone, and determining the activity of the metabolites. Subsequent identification of drug metabolizing enzymes involved in ixabepilone metabolism may result in explaining pharmacokinetic variability of ixabepilone in individual patients.

mass balance
ixabepilone
accelerator mass spectrometry
epothilone
anti-cancer agent


Increased epithelial cell proliferation in the ileal pouch mucosa of patients with familial adenomatous polyposis

To eliminate the risk of colorectal cancer in patients with familial adenomatous polyposis (FAP), reconstructive proctocolectomy is performed. Although most colonic mucosa is resected during the ileal pouch anal anastomosis, adenomas and carcinomas may develop in the pouch. This may be caused by altered cell kinetics due to intraluminal changes in the pouch. In 32 patients with FAP, biopsy specimens from the mucosa of the pouch and also of the afferent ileal loop were taken. Tissue sections were immunohistochemically processed with the monoclonal antibodies M30 and MIB-1 to assess apoptotic and proliferative indices, respectively. Cell proliferation was also assessed by a modified sign test. There were no significant differences in apoptotic rates between the mucosa of the pouch and the mucosa of the afferent ileal loop. However, cell proliferation was significantly higher in the mucosa of the pouch vs afferent ileal loop, both by using the quantitative (68.3% vs 61.6%, p = 0.001) and semiquantitative methods (p < 0.05). Our newly developed semiquantitative approach outperformed previously described methods. The higher cell proliferation in the pouch as compared to the afferent ileal loop may contribute to the increased risk for adenomas and carcinomas in the pouch of patients with FAP and emphasizes the need for regular endoscopic surveillance.
Introduction
Familial adenomatous polyposis (FAP) is an autosomal-dominant disease with an estimated prevalence of 1 in 5,000 to 1 in 7,500 [5]. It is characterized by the development of hundreds of adenomas in the large intestine. Without surgical intervention, virtually all patients will develop colorectal cancer at relatively young age.
To eliminate the risk of colorectal cancer, a restorative proctocolectomy with ileal pouch anal anastomosis (IPAA) is accepted as one of the surgical treatments of choice in these patients.
In the IPAA procedure, almost all colonic mucosa is resected, thus reducing cancer risk significantly. However in patients with FAP, adenomas are also present in the upper gastrointestinal tract; approximately 5% of the patients having gastric, 60 to 90% duodenal [4], 50% jejunal [14], and 9 to 25% ileal adenomas [14, 23, 34]. Occurrence of ileal adenomas is of special interest because the terminal ileum, which is used to construct the pouch, may already contain adenomas. There is accumulating evidence that adenomas develop in the pouch. The reported incidence after a follow-up of at least 5 years is 8 to 60%, increasing up to 75% in certain subgroups [14, 26, 33, 38], which is much higher as compared to the reported incidence of 9–25% in the preoperative ileum of patients with FAP [14, 23, 34]. Therefore, it seems that in the ileal pouch of patients with FAP, the development of adenomas is accelerated. At present, the malignant potential of the pouch adenomas is unclear. However, 13 patients with a carcinoma in the pouch have been recently described in the literature [2, 3, 7, 9, 13, 24, 25, 29, 35–37].
Stasis in the pouch causes a change in the luminal content, which is in close contact with the ileal mucosa. There is an increase in the concentration of short-chain fatty acids to colonic levels [10], an increase in anaerobic bacterial counts resulting in a more colonic type of flora, and an increased deconjugation and dehydroxylation of bile acids by anaerobic bacteria [20, 21]. The intestinal flora could play a role in initiation and promotion of colon cancer by activation of various classes of carcinogens, such as nitrosamines, polycyclic aromatic hydrocarbons, and glycosides [17]. A direct link between intestinal bacterial flora and tumor promotion in the APCMin/+ mouse model for FAP has already been established [22].
Therefore, we hypothesize that the intraluminal ecological changes in the pouch are responsible for enhanced tumor formation in the mucosa of the pouch. Alteration of cell kinetics in the mucosa, including a lower rate of apoptosis and a higher rate of cell proliferation, is associated with tumor initiation and promotion. In colorectal cancer, the balance between apoptosis, cell proliferation, and cell shedding is disturbed, and such changes may favor the development of adenomas [30]. Up to now, no data are available on changes in proliferation and apoptosis rates of the ileal mucosa of the pouch after IPAA in patients with FAP.
The aim of this study is to investigate changes in apoptosis and cell proliferation rates, occurring in the mucosa of the pouch of patients with FAP, in comparison with the ileum of the afferent loop. The results may contribute to a better understanding of the enhanced adenoma formation in the pouch compared to normal ileum.
Materials and methods
Patients and tissues
The study was approved by the regional medical ethical commission, and informed consent was obtained from all patients.
Patients with FAP and an IPAA, who were under surveillance in the Radboud University Nijmegen Medical Centre or regional affiliated hospitals, were invited to participate in this study. Thirty-two patients with FAP were included. The diagnosis FAP was based on either a clinical presentation of at least 100 colonic adenomas or a mutation in the APC gene. Data concerning the surgical procedures were obtained from medical records.
From each patient, mucosal biopsy specimens of both the pouch and the afferent ileal loop were obtained during a regular surveillance endoscopy, in the period January 2002 until April 2004. Patients were fasted overnight. On the day of examination, patients were encouraged to drink liberally. No laxatives or cathartic enemas were given. To clear the pouch of fecal ruminants, two 250-ml water enemas were given before the endoscopy.
The endoscopy was performed with an Olympus GIF-1T140 video endoscope. From January 2002 until August 2003, a 2.8-mm diameter biopsy forceps (FB 13K-1 Olympus, Tokyo, Japan) was used, and from September 2003 until April 2004, a 3.0-mm diameter biopsy forceps (B102-C1-30.160 MedWork/Treier Endoscopie GA, Beromünster, Switzerland) was used. The afferent loop was introduced up to 20 cm proximal of the pouch. The mucosa was sprayed with 1% indigo carmine dye (Laboratoires SERB, Paris) at 1:1 dilution with water, where after photographs were taken to evaluate number and size of adenomas present.
For pathological examination, at least four biopsies were taken at random locations from the afferent ileal loop (10 to 20 cm proximal from the pouch), four biopsies from the pouch mucosa (at least 5 cm proximal from the anal verge), and four biopsies from adenomas if present. The biopsies from adenomas were used only for pathological evaluation, i.e., to exclude serious dysplasia in pouch adenomas, and were not used for research purposes. The biopsies were stretched on filter paper to maintain correct orientation of crypts, fixated in formalin, and embedded in paraffin.
Immunohistochemistry
Cell proliferation and apoptosis were measured in the stretched formalin-fixed nonadenomatous tissue. The crypt cell proliferation activity was assessed after staining with the monoclonal antibody MIB-1 (Dako A/S, Glostrup, Denmark), which recognizes the Ki-67 nuclear antigen of dividing cells in formalin-fixed paraffin-embedded tissue [8]. Apoptosis was assessed by staining with the monoclonal antibody M30 (Roche Diagnostics, Mannheim, Germany), which recognizes cleaved cytokeratin 18. Immunoreactivity of M30 is confined to the cytoplasm of apoptotic epithelial cells and is expressed during early apoptosis [18].
Tissue sections of 4-μm thickness were cut from paraffin blocks, mounted on electrostatic slides (Super Frost Plus, Menzel-Gläser, Germany), and dried overnight, followed by drying in a stove at 50°C for 15 min.
Sections were put in xylol for 10 min and taken from xylol through 100% alcohol to water. After deparaffinization, endogenous peroxidase was blocked by treatment with 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 30 min. Pretreatment was performed by heating the tissue sections in citrate buffer (10 mmol/l, pH 6.0) at 180-W power in a microwave oven for 10 min. After cooling at room temperature for 1.5 h, sections were rinsed with PBS. Then, 20% normal horse serum (Vector Laboratories, Burlingame, CA, USA) was applied for 10 min. The sections were then incubated overnight at 4°C with either the mouse monoclonal antibodies MIB-1 at 1:1,000 dilution or M30 at 1:100 dilution.
Thereafter, the sections were rinsed in PBS for 10 min and incubated with biotinylated horse anti-mouse IgG (Vector) for 30 min. After rinsing for 10 min in PBS, incubation with ‘avidin and biotinylated horseradish peroxidase macromolecular complex’ (Vector) was performed for 45 min. Finally, 5 mg/10 ml diaminobenzidine was used as chromogen, and the sections were put in 0.5% CuSO4/0.9% NaCl for 5 min to intensify the staining. Mayer hematoxylin counterstaining was applied. Sections were dehydrated through 100% alcohol, cleared in xylol, and coverslipped with permount (Fisher Scientific, Fiar Lawn, NJ, USA).
Tissue sections of rectal carcinoma were used as positive controls.
Evaluation of immunostaining results
Investigators were blinded for the origin of the tissue sections, regarding patient and biopsy location.
For evaluation of M30 staining, tissue sections were examined by light microscopy. M30 positivity was identified as brown cytoplasmic staining. M30-positive cells were marked by a first investigator (BvH) and reevaluated by an expert pathologist (IN). In all cases, the complete section was evaluated, and all M30-positive epithelial cells were counted. The apoptotic index was expressed as the number of M30-positive cells per tissue area in square millimeters. Tissue area was assessed by using a Zeiss KS400 computer-aided system.
In each MIB-1-immunostained tissue section, crypts whose entire length could be visualized were photographed under ×400 magnification using a Zeiss KS400 computer-aided system. Crypts were excluded when they did not reach the muscularis mucosae or had multilayered bases.
MIB-1 positivity was identified as brown nuclear staining. The number of MIB-1-positive epithelial cells and the total number of epithelial cells in up to five crypts per tissue section were counted from screen. The labeling index for each crypt was given by the ratio of MIB-1-positive cells and the total number of crypt epithelial cells and is expressed as percentage of total. For each patient, the labeling indices of pouch and afferent ileal loop were expressed as means of three to five counted crypts. If less than three crypts could be photographed for either pouch or ileal loop, the patient was excluded from analysis. The photographed crypts of five randomly selected patients were counted twice by one investigator (BvH) to determine intraobserver variability.
Although great effort was made to obtain well-orientated mucosal crypts when using the quantitative method, however, not all biopsies reached the criteria mentioned above, and therefore, could not be examined. This problem mainly occurred in the biopsies taken from the pouch and might be due to friability of the pouch mucosa. We therefore developed a new semiquantitative scoring system. A representative part of the biopsies showing several complete crypt/villous axes was photographed under ×100 magnification. The photographs were judged pair-wise (pouch vs afferent loop) during which the investigators had to choose from four possible outcomes; one of the two locations showed most MIB-1 positivity. MIB-1 positivity did not differ or no judgement could be made. Judgement was based on relative length of the area of positive cells and the relative size of the stem cell compartment. Five investigators, two pathologists (IN, HvK), two gastroenterologists (PF, FN), and one junior investigator (BvH) independently compared the paired photographs of biopsies of pouch and afferent ileal loop of all patients. When three or more observers agreed in their judgement, this judgement was denoted as consensus judgement. If this criterion was not met, no consensus was reached. If the quality of the tissue sections was poor, no judgment was made. One investigator (BvH) judged the whole series twice for evaluation of intraobserver reliability.
Statistical analyses
Values for apoptosis and cell proliferation in the quantitative study were not expected to be normally distributed; therefore, they were presented as median and range. The Wilcoxon matched-pairs signed-ranks test was used to compare the paired observations in the apoptosis staining and the paired mean labeling indices in the cell proliferation study.
Consensus judgements on the semiquantitative assessment of cell proliferation, favoring either pouch or ileal afferent loop, were compared with a Sign test. To evaluate the reliability of this semiquantitative method to assess cell proliferation, Cohen’s kappa was calculated for the first and second series of judgements by the prime investigator to determine intraobserver reliability. Also, for each pair of investigators, a Cohen’s kappa was calculated. The mean Cohen’s kappa was taken as value for interobserver reliability.
Consensus judgments were compared to the difference in mean labeling index between pouch and ileal afferent loop for each evaluable patient.
A p value less than 0.05 was considered as significant (SPSS for Windows 11.0.1, 2001).
Results
Patient characteristics
Patient characteristics are given in Table 1. The median age of the 32 patients (19 men, 13 women) included in the study was 32 (range 16–72) years.
Table 1Patient characteristics ApoptosisCell proliferationNumber of patients studied3220Male/Female19/1312/8Median age in years (range)32 (16–72)29 (16–62)Median age at surgery in years (range)24 (10–55)20 (10–52)Median age pouch in months (range)96 (9–216)105 (9–216)IPAA: hand-sewn/double-stapled/ unknown9/21/25/14/1Carcinoma at surgery40Patients with adenomas at biopsy:yes/no24/815/5For quantitative cell proliferation analysis, 12 patients had to be excluded from analysis (right column) due to absence of sufficient evaluable crypts (see under “Materials and methods”).
Twenty-three patients were operated in the Radboud University Nijmegen Medical Centre. The median age at the time of reconstructive colectomy was 24 (range 10–55) years. A mucosectomy with hand-sewn IPAA was performed in 9 patients, and a double-stapled IPAA was performed in 21 patients. For two patients, the information about the performed technical procedure could not be retrieved. At the moment of colectomy, four patients had a colorectal adenocarcinoma localized in the rectum, sigmoid, hepatic flexure, or appendix, respectively.
At the time of endoscopy, the median age of the pouch was 96 (range 9–216) months. The medication used was loperamide by 18 patients, psyllium fibres by 3 patients, and iron, metoclopramide, colestyramine, omeprazole, sulindac, tramadol, tamoxifen, gosereline, nifedipine, metoprolol, furosemide, or losartan each by 1 patient. Thirteen patients were not on medication 3 months before endoscopy.
One patient used sulindac, a nonspecific cyclooxygenase inhibitor, which is thought to influence cell proliferation and especially apoptosis [15, 27]. Exclusion of this patient from analyses had no effect on the results.
Histological examination revealed pouch adenomas in 24 patients (75%).
Apoptosis
In both pouch and afferent ileal loop, M30-positive cells were predominantly detected in the mucosal villi (Fig. 1).
Fig. 1Apoptotic epithelial cells (encircled) using M30 immunohistochemistry. Original magnification ×200 (left) and ×400 (right)
In the 32 pairs of biopsies from pouch and afferent ileal tissue investigated, the median absolute number of apoptotic cells in the pouch mucosa was two per tissue section (range 0–9), which was identical to the values in the afferent ileal loop (2, range 0–19). The median apoptotic index (expressed as number of M30-positive cells per mm2 tissue section area) did not differ between pouch (median 0.4/mm2, range 0–2.9) and afferent ileal tissue (median 0.3, range 0–2.7; see Fig. 2).
Fig. 2Box-Whisker plots of apoptosis expressed as number of M30-positive epithelial cells per mm2 tissue section area of pouch and afferent ileum of 32 patients with FAP. Difference between groups was not significant. Values are given as median (fat line), lower to upper quartile (green box), and minimum and maximum values (error bars)
Cell proliferation
Quantitative comparison
The intraobserver reliability for counting of the photographed crypts (Fig. 3) was rs = 0.855, p = 0.002.
Fig. 3Detection of proliferating crypt cells with MIB-1 immunohistochemistry. Brown stained nuclei are positive. Nonproliferative crypt stem cells are visualized at the base of the crypt. Original magnification ×400
In 12 pouch tissue sections, less than three crypts were available for counting, and these samples were therefore excluded from analysis. For the same reason, two afferent ileal tissue sections were excluded. In the 20 pairs of pouch and afferent ileum tissue sections left for comparison, median labeling index (expressed as percentage of MIB-1-positive epithelial crypt cells) was significantly higher in the pouch as compared to the afferent ileum (median 68.3%, range 52.9–79.6% vs median 61.6%, range 38.0–73.9%; Wilcoxon signed-ranks test, p = 0.001; Fig. 4).
Fig. 4Box-Whisker plots of cell proliferation expressed as median labeling index (MIB-1-positive crypt cells per total number of crypt cells) of 3–5 crypts in tissue sections of pouch and afferent ileum of 20 patients with FAP. Difference between groups was significant (p = 0.001). Values are given as median (fat line), lower to upper quartile (red box), and minimum and maximum values (error bars)
Semiquantitative comparison
The results of the semiquantitative comparison of the photographed tissue sections from the pouch and afferent ileum mucosae (Fig. 5) are visualized in Fig. 6. In 15 patients (47%), the proliferation was higher in the pouch; in 7 patients (22%), there was no difference; in 5 patients (16%), proliferation was higher in the afferent ileal loop; in 2 patients (6%), no consensus could be achieved; and in 3 patients (9%), no judgement was possible due to poor quality of the tissue sections. These differences were significant (p < 0.05).
Fig. 5Photographs used for semiquantitative comparison of cell proliferation in MIB-1 immunohistochemically stained tissue sections of pouch (left) and afferent ileal mucosae (right; original magnification ×100)Fig. 6Histogram of semiquantitative comparison; 0 ileal mucosa shows more cell proliferation than pouch mucosa, 1 no difference in cell proliferation between pouch and ileal mucosae, 2 pouch mucosa shows more cell proliferation than ileal mucosa, 3 no judgement possible, 4 no consensus could be reached
Intraobserver reliability was κ = 0.87. Cohen’s kappa for each pair of observers ranged from 0.26 to 0.59 with a mean Cohen’s kappa of 0.38, which corresponds with “fair agreement” following Byrt’s guidelines [6]. Thus, interobserver reliability was acceptable.
In cases in which a labeling index could be assigned to both pouch and afferent ileal loop, consensus judgment was available in 18 cases (Fig. 7). In all cases in which the semiquantitative analyses showed more proliferation in the pouch (n = 11), this was confirmed by the quantitative method; the median value of the difference in proliferation between pouch and ileum was 0.107 (range 0.032–0.175). In the five cases where no difference was observed with the semiquantitative approach, the median value of the difference in proliferation between pouch and ileum was 0.023 (range −0.096 to 0.120). There were only three cases in which the semiquantitative method showed more proliferation in the afferent loop, and the median value of the difference in proliferation between pouch and ileum in these cases was 0.098 (range −0.021 to 0.262). The Spearman’s correlation coefficient between the semiquantitative and the quantitative methods was 0.273 (p = 0.244).
Fig. 7Semiquantitative comparison vs difference in mean labeling index of pouch minus afferent ileal mucosa; 0 ileal mucosa shows more cell proliferation compared to the pouch mucosa, 1 no difference in cell proliferation between pouch and ileal mucosae, 2 pouch mucosa shows more cell proliferation compared to the ileal mucosa
Discussion
Although both the pouch and its afferent loop contain the same preexisting ileal mucosa, adenomas occur more frequently in the pouch of patients with FAP than in the afferent ileal loop, suggesting an accelerated adenoma formation in the pouch [14, 26, 33, 38]. To investigate the role of cell kinetics as a possible explanation for this observation, apoptosis and cell proliferation rates in the mucosa of the pouch were compared with those of the afferent ileal loop from the same patient to eliminate bias caused by interindividual differences. Cell proliferation was significantly higher in the pouch mucosa in comparison to mucosa of the afferent ileal loop. No significant difference in apoptosis was found in the mucosa of the pouch and afferent ileal loop.
A low amount of apoptotic cells in the pouch as well as in the ileal mucosa was found. This might be caused by the APC mutation-induced apoptotic resistance [16]. Only a trend but no significant difference in apoptosis was found. Taking into account the broad range of apoptotic rates as found in our study samples, a significant difference might be found when a larger group of patients could be studied. There are no clear guidelines for estimation of ileal proliferation. For estimation of colonic proliferation, an accepted method is to count proliferating cells in five colonic crypts. Although most biopsy specimens were stretched and orientated directly after endoscopy, only a lower number of crypts could be counted completely along the longitudinal axis in most patients. For this reason, we accepted three full crypts as the minimal number to assess proliferation. Using this criterion, tissue sections from 12 patients still could not be used to determine the cell proliferation and had to be excluded from the study. Especially in the tissue sections of the pouch, this problem was evident and is possibly caused by a higher fragility of this tissue. In the remaining 20 pairs of tissue sections left for comparison, we found a significantly higher median labeling index in the pouch compared to the ileal afferent loop.
To overcome the problem of the relative low number of assessable crypts, another quicker but less quantitative method was applied in which five investigators compared photographs of tissue sections of pouch and ileal mucosae. In accordance with the results of the first method, we found significantly higher cell proliferation in the mucosa of the pouch compared to that of the ileal mucosa. Although no significant correlation could be found in a case-by-case comparison between both methods, in all cases in which the semiquantitative analyses showed more proliferation in the pouch, this was confirmed by the quantitative analyses of crypts. Furthermore, the inter- and intraobserver variability was good.
In addition, the semiquantitative method is far less time-consuming and can therefore give a relatively fast and easy impression of eventual differences in cell proliferation. Evaluation of this method in a larger study may further demonstrate its value.
Data on cell proliferation in ileal mucosa in patients with FAP are limited. Previously, de Silva et al. [11] reported a labeling index of 19.8% in afferent ileal loop mucosa of patients with FAP or ulcerative colitis (UC), far lower than the 61.6% that was found here. Their labeling index for cell proliferation in ileal pouch mucosa (33.6%) was also much lower than in the present study. Only the labeling index of 51.7% as found by them in pouches with pouchitis nears the values we obtained. However, the majority of patients included in the study of de Silva et al. were patients with UC (23 of the 26 patients), so direct comparison with our results therefore seems inappropriate. In addition, Goldberg et al. [12] reported a median labeling index of 34.9% in the ileal pouch of patients with FAP, which is much lower than the 68.3% we found. However, their labeling indices were based on a minimum of three counted crypts, and if this number could not be reached, halves of crypts were included in the analyses. Moreover, their study group consisted of only 5 patients with FAP, whereas in the present study, 3 to 5 whole crypts of 20 patients were counted.
Several studies indicated that cell proliferation of normal-appearing colorectal mucosa of patients with adenomas or carcinomas was 19 to 86% higher compared to colorectal mucosa of healthy persons [1, 28, 30, 32]. These findings strongly suggest an association between the presence of adenomas or carcinomas and an increased mucosal cell proliferation. However, the difference in cell proliferation between pouch and afferent ileal mucosae of 6.7% as found in this study is less pronounced in comparison to the above referred findings. A possible explanation for this relatively small difference in cell proliferation between pouch and ileal mucosae in our study may be that intestinal epithelial cell proliferation is already very high in patients with FAP [19, 31], and these high cell proliferation rates make a further increase less pronounced.
The higher proliferation found in the pouch mucosa in comparison to mucosa of the afferent ileal loop can only be explained by intraluminal changes that occur after construction of the pouch. Whether changes in bacterial flora, bile acid composition, short-chain fatty acids, or other compounds are responsible for this finding remains unclear, but a better understanding of this process is necessary to find a possible treatment for this group of patients.
In conclusion, the increased cell proliferation in the ileal pouch mucosa compared to the mucosa of the afferent ileal loop may contribute to the enhanced risk for adenomas and carcinomas in the pouch of patients with FAP and emphasizes the need for regular endoscopical surveillance of the pouch in these patients. In addition, cell proliferation can be used as an early endpoint marker in chemopreventive studies in these patients.
The applied new method for semiquantitative evaluation of cell proliferation in immunohistochemically stained tissue sections seems promising, as it offers a relatively fast and easy means of assessment.

cell proliferation
familial adenomatous polyposis
restorative proctocolectomy
apoptosis and carcinogenesis


Cancer Internet Search Activity on a Major Search Engine, United States 2001-2003

Background To locate online health information, Internet users typically use a search engine, such as Yahoo! or Google. We studied Yahoo! search activity related to the 23 most common cancers in the United States.
Introduction
Health care providers [1-3] and their patients [4-7] regularly search for health information online. Internet users generally begin looking for health information using a search engine [8-12], an Internet tool that searches for Web pages containing terms specified by users [13]. In February 2004, Google and Yahoo! were the most popular search engines in the United States, with 38% and 32% of market share, respectively [14].
To date, few studies of Internet search activity related to health topics have been published. Most analyzed the proportion of health and non-health searches on various search engines and found that health searches constituted a small percentage of total searches [15-18]. This finding is not surprising given how infrequently Internet users search for health information compared with how often they look for news reports, product information, and other topics [19]. In any case, a small percentage of total Internet searches translates into thousands of health searches each day [16], and an estimated 95 million Americans have used the Internet at least once to search for health information [20].
Cancer appears to be a health topic of some interest to Internet users. Eysenbach and Köhler [16] found that searches for cancer information accounted for 5% of health-related searches on the search engine MetaCrawler. Phillipov and Phillips [18] found that “skin cancer” was one of only 17 health-related search terms among the most popular 300 Internet keywords identified by Wordtracker, a private research company. Bader and Theofanos [21] studied cancer searches conducted using the search engine AskJeeves during a 3-month period and found the most commonly searched cancers were digestive/gastrointestinal/bowel, breast, and skin. This study also compared the incidence of selected cancers with their associated search activity. While this relationship was not statistically tested, the authors observed that some rarer cancers constituted a higher proportion of total searches than their proportion of total cancer incidence. In addition, Bader and Theofanos, as well as Phillipov and Phillips, noted that media coverage appeared to prompt Internet searches for health topics, but they did not systematically investigate or test the relationship.
The study reported here builds on this prior work by analyzing cancer-related searches conducted in the United States from 2001 to 2003 using the search engine Yahoo! Specifically, we investigated three potential correlates of Yahoo! cancer search activity—estimated cancer incidence, estimated cancer mortality, and the volume of cancer news coverage. Cancers that afflicted more individuals, claimed more lives, and generated more news coverage were expected to be associated with more Internet search activity than other cancers, given the interest generated by relevance and publicity. In addition, we assessed the periodicity of Yahoo! cancer search activity and examined sharp increases in Yahoo! search activity related to specific cancer types.
Methods
This analysis included three types of 2001–2003 US data: Yahoo! cancer search activity, cancer burden (estimated incidence and mortality), and cancer news coverage. The study protocol was reviewed by the Institutional Review Board of the National Center for Chronic Disease Prevention and Health Promotion and was designated as “research not involving human subjects.”
Yahoo! Cancer Search Activity
During 2001 (the beginning of the study period), Yahoo! was the most popular US search engine, with a 49% market share; however, in 2003 (the end of the study period), Google surpassed Yahoo! as the leading US search engine [22]. Yahoo! remains a widely used search engine; more than 45 million US Internet users conducted Yahoo! searches in February 2004 [14].
Yahoo! maintains a database of search activity called the Yahoo! Buzz Index [23]. This index provides a search activity score for individual search terms—the words or characters that users type into the Yahoo! search box. Each point of a Yahoo! Buzz Index score equals 0.001% of users searching Yahoo! during the time period of interest (day, week, or month). For example, if 250 out of a total of 1 million users searched for “breast cancer” on January 1, 2001, the Yahoo! “breast cancer” search activity score on this day would be 25 (250/1 million × 100000). For a search term to register a search activity score, it must generate at least 50 searches during the time period for which the score is calculated. Yahoo! search activity generated by search terms can be segmented by country, US state, or selected US cities. This study used daily US Yahoo! search activity data from January 1, 2001 (the earliest date for which Yahoo! search activity data are available) to December 31, 2003. We limited our analysis to Yahoo! searches because at the time of this study no other Internet search engine offered a dataset like the Yahoo! Buzz Index, which provides search activity scores adjusted for the size of the population searching for online information.
Yahoo! employs professional “surfers” or content indexers who manually classify Web pages into one of more than 2000 content categories, such as “movies,” “footwear,” “astrology,” or “cancer or neoplasms.” The Yahoo! Buzz Index classifies search terms in the same content category as the first Web page link that a user “clicks” or activates after conducting a search. For instance, if a user entered the search term “colon” and then clicked on a cancer website, “colon” would be classified as a “cancer or neoplasms” search term. If the user clicked on a grammar website, however, “colon” in that instance would be classified as an “education” search term. When a user does not click on a Web page link or when a user clicks on a Web page link that has not been classified, the Yahoo! Buzz Index categorizes the search term using a variety of algorithms that analyze recent content viewed by the user.
To identify commonly used Internet search terms related to specific cancers, we reviewed the search terms classified in the “cancer or neoplasms” category of the Yahoo! Buzz Index, which generated at least 50 searches in any month from January 2001 to December 2003. This search strategy identified 76 unique search terms, of which 23 were included in the analysis (Table 1). The remaining 53 terms were discarded because they did not relate to a specific cancer or included non-English words. Discarded terms included drug names or treatment modalities, such as “chemotherapy” (n = 19); the names of organizations or events, such as “Race for the Cure” (n = 16); general cancer or anatomy terms, such as “oncology” (n = 11); names of celebrities, such as “Gilda Radner” (n = 5); and the carcinogen “DES” (n = 1). Also, “leucemia” (n = 1), the Spanish word for “leukemia,” was discarded because the Yahoo! Buzz Index does not consistently track foreign words, as it excludes search terms that contain non-English characters. For instance, the Yahoo! Buzz Index would not capture a search term with an accent mark, such as “cáncer colorectal” (Spanish for “colorectal cancer”).
Cancer Burden
The estimated incidence and mortality for 23 cancers during the study period were obtained from annual burden reports published by the American Cancer Society [24-26]. All cancers with at least 7500 new cases in 2001, 2002, or 2003 were included in the analysis (n = 23) whether or not they were associated with Yahoo! search activity.
Cancer News Coverage
News reports associated with specific cancer types were identified using the LexisNexis “US News” database, which includes more than 400 national and regional newspapers, such as the Wall Street Journal and the Baltimore Sun, and a variety of newswire services, such as the Associated Press and UPI (United Press International). News reports published from January 1, 2001, to December 31, 2003, related to specific cancer types were found by locating reports with the identified Yahoo! search activity terms (eg, “breast cancer”) in their headlines. In the case of cancers located in the esophagus and oral cavity, for which no Yahoo! search activity terms were associated, the terms “esophageal cancer” and “oral cancer” were used as the headline search terms.
Analysis
Descriptive statistics were calculated for the Yahoo! search activity score, estimated incidence, estimated morality, and news coverage volume associated with the cancers included in the study. Spearman rank correlations were used to establish the consistency of these variables across the study period, and the data were aggregated. Next, the relationships between Yahoo! search activity and the potential correlates of interest were tested using Spearman rank correlations.
The relationship between Yahoo! search activity and news coverage was further analyzed for the five cancers with the highest daily Yahoo! search activity. The number of news reports published each day was transformed into a categorical variable with four levels (0 news reports, 1–2 news reports, 3–4 news reports, and 5 or more news reports), and one-way analysis of variance (ANOVA) was used to compare mean daily Yahoo! search activity scores at increasing levels of news coverage. To detect possible periodicity effects, Yahoo! search activity data were visually inspected. Three possible periodicity effects were noted: a rise during weekdays (Monday–Friday) compared with weekends; a rise during national cancer awareness months compared with other months; and a decline during summer months (June–August) compared with other months. These possible effects were tested using t tests. Finally, the Yahoo! search activity associated with several cancers was marked by sharp increases of 100% or more from one day to the next. For these cancers, we identified the three highest peaks in 2003 Yahoo! search activity and investigated corresponding news events.
Results
We found Internet search terms generating Yahoo! search activity associated with 21 of the 23 cancers included in the study (Table 1). Of these, 19 cancers were associated with only one Yahoo! search term each. The 2 remaining cancers were associated with two search terms each: cancers of the brain (“brain tumor” and “brain cancer”) and multiple myeloma (“multiple myeloma” and “myeloma”). In these cases, the daily Yahoo! search activity scores associated with each term were summed into a composite score for these cancers.
The highest mean daily Yahoo! search activity scores were generated by breast cancer (mean = 14.37), lung cancer (mean = 9.08), and leukemia (mean = 7.15). Cancers with the highest US 2001–2003 incidences were breast (n = 611300), prostate (n = 608000), and lung (n = 510800). For cancer mortality, lung (n = 469500), colorectal (n = 170400), and breast (n = 120800) cancer were the leading causes of death. Breast cancer (n = 5840), leukemia (n = 2143), and prostate cancer (n = 1822) were associated with the most US news reports from 2001 to 2003. Some cancers, such as leukemia, ovarian, and testicular, appeared to be associated with more Internet search activity than their burden would dictate.
Cancers were ranked by mean daily Yahoo! search activity score, estimated incidence, estimated mortality, and number of related news reports for each year in the study period (2001 to 2003). To explore the consistency of ranks over the study period within each variable, Spearman rank correlations were determined for each pair of years (2001 and 2002, 2002 and 2003, 2001 and 2003). We found statistically significant correlations (P < .001) between all year pairs tested (data not shown). Because the ranks associated with these variables were highly consistent from 2001 to 2003, we aggregated the data across the study period.
Table 1
Mean daily Yahoo! search activity score (United States, 2001–2003), estimated incidence, estimated mortality, and number of news reports, by cancer
Cancer
Yahoo! Search Terms
Mean Daily Yahoo! Search Activity Score*
(Rank)
Estimated
Incidence
(Rank)
Estimated
Mortality
(Rank)
Number of News
Reports (Rank)
Breast
“breast cancer”
14.37 (1)
611300 (1)
120800 (3)
5840 (1)
Lung
“lung cancer”
9.08 (2)
510800 (3)
469500 (1)
918 (5)
Leukemia
“leukemia”
7.15 (3)
92900 (10)
65100 (7)
2143 (2)
Colorectal
“colon cancer”
7.08 (4)
43120 (4)
170400 (2)
617 (6)
Prostate
“prostate cancer”
6.13 (5)
608000 (2)
90600 (4)
1822 (3)
Ovary
“ovarian cancer”
3.71 (6)
72100 (13)
42100 (9)
458 (8)
Lymphoma
“lymphoma”
3.54 (7)
185500 (5)
78100 (6)
480 (7)
Uterine, cervix
“cervical cancer”
2.53 (8)
38100 (20)
12600 (19)
392 (9)
Melanoma
“melanoma”
2.25 (9)
159200 (7)
22800 (16)
376 (10)
Brain
“brain tumor”“brain cancer”
1.52 (10)
52500 (16)
39300 (10)
925 (4)
Liver
“liver cancer”
0.70 (11)
50100 (17)
42600 (8)
110 (14)
Testis
“testicular cancer”
0.62 (12)
22300 (23)
1200 (23)
50 (17)
Pancreas
“pancreatic cancer”
0.23 (13)
90200 (11)
88600 (5)
185 (11)
Multiple myeloma
“multiple myeloma”“myeloma”
0.11 (14)
43600 (18)
32900 (15)
185 (11)
Stomach
“stomach cancer”
0.08 (15)
65700 (14)
37300 (13)
50 (17)
Uterine, corpus
“uterine cancer”
0.012 (16)
117700 (8)
20000 (18)
17 (22)
Larynx
“throat cancer”
0.012 (16)
28400 (21)
11500 (21)
30 (20)
Bladder
“bladder cancer”
0.010 (18)
168200 (6)
37500 (12)
118 (13)
Soft tissue
“sarcoma”
0.009 (19)
25300 (22)
12200 (20)
25 (21)
Thyroid
“thyroid cancer”
0.002 (20)
62200 (15)
4000 (22)
40 (19)
Kidney
“kidney cancer”
0.001 (21)
94500 (9)
35600 (14)
77 (15)
Oral cavity
-
0.000 (22)
86700 (12)
22400 (17)
69 (16)
Esophagus
-
0.000 (22)
40200 (19)
38100 (11)
13 (23)
* Each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day.
Correlates of Yahoo! Cancer Search Activity
We tested the relationships between variables by determining Spearman rank correlations between each pair. Statistically significant correlations were found between all variable pairs (Table 2).
Table 2
Spearman rank correlations between mean daily Yahoo! search activity score (United States, 2001–2003), estimated incidence, estimated mortality, and number of news reports
Spearman Rank Correlation*
Mean Daily Yahoo! Search Activity Score
Estimated Incidence
Estimated Mortality
Number of news reports
.88†
.62‡
.74†
Estimated mortality
.66†
.71†
-
Estimated incidence
.50§
-
-
* Spearman rank correlations were done on the rankings reported in Table 1.
†
P ≤ .001
‡
P = .002
§
P = .015
The relationship between Yahoo! search activity and its most statistically significant correlate—news coverage—was further analyzed for the five cancers with the highest daily Yahoo! search activity (breast, lung, leukemia, colorectal, and prostate). For these cancers, the number of news reports published each day was transformed into a categorical variable with four levels. The mean daily Yahoo! search activity at each level was compared using ANOVA, and all tests were statistically significant (P ≤ .001). For all five cancers, the mean daily search activity score was higher at each increasing level of news coverage (Table 3).
Table 3
Mean daily Yahoo! search activity score (United States, 2001–2003), by number of news reports published daily and cancer
Cancer
Mean Daily Yahoo! Search Activity Score*†
(Number of News Reports )
Days With
0
News Reports
Days With
1–2
News Reports
Days With
3–4
News Reports
Days With
5+
News Reports
Breast
10.09 (81)
11.49 (278)
13.36 (252)
17.27 (484)
Lung
8.27 (633)
10.00 (362)
10.54 (71)
11.71 (29)
Leukemia
6.89 (248)
7.07 (523)
7.18 (232)
8.26 (92)
Colorectal
6.72 (739)
7.44 (297)
8.25 (43)
13.92 (16)
Prostate
5.30 (390)
6.40 (467)
6.72 (150)
7.43 (88)
* Each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day.
† ANOVA was used to compare the mean daily Yahoo! search activity at each level of news coverage. For all five cancer sites, a statistically significant difference (P ≤ .001) was found.
Periodicity of Yahoo! Cancer Search Activity and News Coverage
Three possible periodicity effects were tested: a rise during weekdays (Monday–Friday) compared with weekends; a rise during national cancer awareness months compared with other months; and a decline during summer months (June–August) compared with other months. To test for these potential effects, we used the five cancers with the highest daily mean Yahoo! search activity scores (breast, lung, leukemia, colorectal, and prostate) (Table 4). For all five cancers tested, both mean daily Yahoo! search activity scores and mean daily news reports were higher Monday–Friday than they were Saturday–Sunday (P < .001). Higher mean daily Yahoo! search activity scores were found for breast cancer (P < .001), lung cancer (P < .001), and colorectal cancer (P < .001) during their respective national awareness months. The number of mean daily news reports related to breast cancer (P < .001), colorectal cancer (P < .001), and prostate cancer (P = .007) rose during their respective national awareness months. Mean daily Yahoo! search activity scores for breast cancer (P < .001), lung cancer (P < .001), and leukemia (P < .001) were lower during the summer months than during the rest of the year. While mean daily news reports about breast cancer also decreased during the summer (P < .001), mean daily news reports about prostate cancer rose (P = .01).
Table 4
Periodicity of mean daily Yahoo! search activity score (United States 2001–2003) and mean daily number of news reports, by cancer
Cancer
Weekdays
Weekends
P value
Awareness Month
Non-Awareness Months
P value
Summer: June-August
Non-Summer
P value
Breast
Mean Daily Yahoo! Search Activity Score*
15.78
10.84
< .001
26.33
13.26
< .001
10.78
15.58
< .001
Mean Daily Number of News Reports
6.26
3.02
< .001
15.30
4.41
< .001
4.19
5.72
< .001
Lung
Mean Daily Yahoo! Search Activity Score
10.31
6.00
< .001
11.69
8.84
< .001
5.76
10.20
<.001
Mean Daily Number of News Reports
1.03
0.37
< .001
1.03
0.82
.226
0.70
0.89
.086
Leukemia
Mean Daily Yahoo! Search Activity Score
8.13
4.70
< .001
6.65
7.20
.093
5.65
7.66
< .001
Mean Daily Number of News Reports
2.20
1.34
< .001
1.51
2.00
.036
1.88
1.98
.506
Colorectal
Mean Daily Yahoo! Search Activity Score
7.73
5.44
< .001
10.46
6.77
< .001
6.83
7.17
.081
Mean Daily Number of News Reports
0.68
0.27
< .001
1.55
0.47
< .001
0.49
0.59
.214
Prostate
Mean Daily Yahoo! Search Activity Score
6.82
4.41
< .001
5.68
6.18
.044
6.14
6.13
.997
Mean Daily Number of News Reports
2.03
0.74
< .001
2.39
1.60
.007
2.14
1.50
.010
* Each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day.
Peaks in Yahoo! Cancer Search Activity and News Coverage
On several occasions, Yahoo! search activity scores associated with breast cancer, colon cancer, and prostate cancer were marked by sharp increases of 100% or more from one day to the next. We investigated news events that corresponded with the highest three spikes in 2003 Yahoo! search activity for these cancers. These peaks in “breast cancer” and “colon cancer” search activity all occurred during their respective national awareness months and appeared to be related to news coverage promoting the awareness months. The highest peak in “prostate cancer” search activity (22.34) occurred on July 17 after news reports of a study [27] exploring the association between sexual behavior and prostate cancer risk (Figure 1). These news reports generally focused on the possible protective benefit of masturbation. This study was not covered widely by the US news media, but it generated substantial news coverage in Australia and filtered onto the Internet via chat rooms, message boards, and medical news Web pages. While there was no corresponding spike in “masturbation” search activity, there was a 117% increase in the search activity score (from 61.88 on July 16 to 133.08 on July 17) for “masterbation,” a common misspelling. The second highest spike in “prostate cancer” search activity (14.59) occurred on October 21 after news broke that Academy-Award-winning actor Robert DeNiro had been diagnosed with prostate cancer. This story was widely covered by the US news media, and a 277% increase in “Robert DeNiro” search activity was observed on the same date (from 15.87 on October 20 to 59.90 on October 21). The third highest peak in “prostate cancer” search activity (12.41) occurred on December 29, when a study linking obesity with increased prostate cancer risk [28] was covered by several US news outlets. No corresponding rise in searches for the terms “obesity,” “overweight,” or “weight loss” was observed.
Figure 12003 US prostate cancer Yahoo! search activity (each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day)
Discussion
This study suggests that media coverage plays a powerful role in prompting online cancer information seeking. News coverage correlated significantly with Yahoo! search activity (P < .001). Also, Yahoo! search activity was found to rise as news coverage increased, and sharp rises in search activity from one day to the next appeared to be associated with increases in relevant news coverage. This study also suggests that the Internet can rapidly disseminate health news: the highest spike in 2003 US “prostate cancer” Yahoo! search activity seemed to be generated largely by news coverage in Australia that rapidly filtered onto the Internet via chat rooms, message boards, and medical news Web pages. Thus, it possible that a news story does not necessarily have to be covered by the US news media in order to generate US Internet search activity.
News coverage volume also correlated with estimated cancer incidence (P = .015) and mortality (P < .001). This is interesting because past studies [29-33] on this topic have not generated consistent findings, with most [30-32] finding no relationship between disease burden and news coverage volume. However, none of the past studies focused on cancer, and none used our method for identifying news reports. While the news coverage of specific cancers generally matched their burden, we noted that some cancers, such as leukemia, ovarian cancer, and testicular cancer, were associated with more Internet search activity than their burden would suggest. A similar observation was reported by Bader and Theofanos [21], who suggested that this discrepancy may result from more searches being required to locate online information about less common cancers. The high correlation between cancer-specific news coverage and associated online search activity in the present study suggests another explanation: some cancers received a disproportionate share of news coverage relative to their incidence and mortality, and online search activity, often prompted by news coverage, reflects this imbalance.
We detected several periodicity effects in US Yahoo! cancer search activity, which tended to be higher on weekdays and during national cancer awareness months but lower during the summer months. It should be noted that these observations are not artifacts of the size of the online population during these periods because Yahoo! search activity scores are based on the percentage, not the number, of total users. One explanation for these results is that the volume of cancer news coverage tended to follow these trends. It is also possible that users tend to search for online cancer information from school or work settings. As a result, Yahoo! cancer search activity would be expected to drop during weekends when people are at home and over the summer months when many students are out of school and many workers go on vacation.
Although Yahoo! is a leading US Internet search engine, the extent to which the findings of this study can be generalized to other search engines is not known. Also, we were unable to discern the motivations of Yahoo! users searching for cancer information. For instance, news coverage of a breast cancer drug might be associated with an increase in “breast cancer” search activity. While the Yahoo! Buzz Index would detect this rise, it cannot tell how many searchers were breast cancer patients or family members and how many were investors interested in buying stock in the company developing the drug.
Internet search activity offers an innovative tool for passive surveillance of health information–seeking behavior. While our work focused on cancer, Internet search activity may be useful in gauging health information seeking related to other diseases. For example, the volume of Internet searches related to symptoms or conditions might be used to predict disease outbreaks (eg, influenza) or to assess mental health following a disaster. Researchers at the Centre for Global eHealth Innovation have begun to experiment with analyses of this type [34,35]. The Yahoo! Buzz Index is unique among Internet search datasets because it provides search activity scores adjusted for the size of the population searching for online information, which has steadily grown each year [19]. Perhaps in the future, other Internet search engines will offer databases similar to the Yahoo! Buzz Index, and research could be conducted using a combination of search engines.