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Hair nicotine concentration of cats with gastrointestinal lymphoma and unaffected control cases
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  1. Victoria Smith1,
  2. Clare Knottenbelt1,
  3. David Watson2,
  4. Dominic J Mellor1,
  5. Alexandra Guillen Martinez3,
  6. Helen Philp1,
  7. Sarah Keegan4,
  8. Mary Marrington5,
  9. Chiara Giannasi6,
  10. Tom Cave6 and
  11. Alix Rebecca McBrearty1
  1. 1 Small Animal Hospital, Veterinary School, University of Glasgow, Glasgow, UK
  2. 2 Strathclyde Institute of Pharmacy and Biomedical Sciences, Glasgow, Glasgow, UK
  3. 3 University of Liverpool Faculty of Veterinary Science, Liverpool, Merseyside, UK
  4. 4 Langford Veterinary Services, Bristol, Bristol, UK
  5. 5 Department of Oncology, North West Veterinary Specialists, Runcorn, UK
  6. 6 Department of Oncology, Cave Veterinary Specialists, West Buckland, UK
  1. Correspondence to Victoria Smith, Internal Medicine, North Downs Specialist Referrals, Bletchingley RH1 4QP, UK; v-smith{at}hotmail.co.uk

Abstract

Background A previous study showed an association between owner-reported exposure to environmental tobacco smoke (ETS) and lymphoma in cats. This study aimed to investigate the association between ETS exposure and gastrointestinal lymphoma in cats, using hair nicotine concentration (HNC) as a biomarker.

Methods This was a prospective, multi-centre, case–control study. Gastrointestinal lymphoma was diagnosed on cytology or histopathology. Hair samples were obtained from 35 cats with gastrointestinal lymphoma and 32 controls. Nicotine was extracted from hair by sonification in methanol followed by hydrophilic interaction chromatography with mass spectrometry. Non-parametric tests were used.

Results The median HNC of the gastrointestinal lymphoma and control groups was not significantly different (0.030 ng/mg and 0.029 ng/mg, respectively, p=0.46). When the HNC of all 67 cats was rank ordered and divided into quartiles, there was no significant difference in the proportion of lymphoma cases or controls within these groups (p=0.63). The percentage of cats with an HNC≥0.1 ng/mg was higher for the lymphoma group (22.9%) than the control group (15.6%) but failed to reach significance (p=0.45).

Conclusion A significant association was not identified between HNC (a biomarker for ETS) and gastrointestinal lymphoma in cats; however, an association may exist and further studies are therefore required.

  • Cats
  • hair nicotine concentration
  • environmental tobacco smoke
  • gastrointestinal
  • lymphoma
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Introduction

Gastrointestinal lymphoma is the most common anatomical form of lymphoma in cats.1 It is characterised by infiltration of the gastrointestinal tract with neoplastic lymphocytes, with or without mesenteric lymph node involvement.2 3 It has long been known that there is an association between infection with feline leukaemia virus (FeLV) and the development of lymphoma in cats; however, gastrointestinal lymphoma is said to have the weakest association with FeLV antigenaemia.1 4 The aetiology of non-retroviral-induced lymphomas in cats is poorly understood2 but associations with a number of genetic, lifestyle and environmental factors have been suggested. These include the observation that feline lymphoma may develop at sites of chronic inflammation such as that of chronic inflammatory bowel disease,2 5 and the suggestion that cats exposed to environmental tobacco smoke (ETS) are at an increased risk of developing several anatomical forms of lymphoma, including gastrointestinal lymphoma.5

ETS, also known as second-hand smoke, contains more than 40 mutagens and carcinogens, many of which are associated with carcinogenesis in humans.6–9 Cats often maintain close physical relationships with owners, resulting in direct exposure to inhaled ETS but also exposure to contaminated skin and clothing. Given the frequent and extensive self-grooming that is typical of cats, exposure also occurs through oral ingestion of ETS particles deposited on their skin and hair coat. In vitro studies using human tissue have demonstrated that tobacco smoke has a direct carcinogenic effect on gastric mucosal cells.10 The oral ingestion of ETS toxins and carcinogens could increase the likelihood of gastrointestinal lymphoma developing in exposed cats by a similar mechanism.

A previously documented association between exposure to ETS and all anatomic forms of feline lymphoma was based on a retrospective study, whereby owners documented historic exposure in the home environment via a questionnaire.5 Such questionnaires can be simple to use; however, they are subject to recall and selection biases.11 To confirm that ETS is actually absorbed by a person or their pet, and to more accurately quantify the level of exposure, the measurement of a biomarker is required.12 Hair nicotine concentration (HNC) has been extensively studied in people and has been found to correlate well with ETS exposure.13–17 In both dogs and cats, HNC has been strongly and positively associated with owner-reported exposure to ETS.18 19 In a recent study in cats, an HNC of ≥0.1 ng/mg was found to be an appropriate cut-off to suggest exposure to ETS with a specificity of 98% and a sensitivity of 69%.19

The principal aim of this study was to further investigate the association between ETS exposure and gastrointestinal lymphoma, using HNC as a biomarker.

Materials and methods

Ethical approval was granted by the School of Veterinary Medicine’s Ethics and Welfare Committee.

Gastrointestinal cases and controls

This was a prospective study with cats being recruited from five veterinary referral centres within the UK over a 2-year period from March 2015 until March 2017. Owners were asked for consent for study inclusion and hair sample collection.

Cats over 6 months of age that had lived with the same owner for at least 6 months were eligible for inclusion. Only newly diagnosed cases of gastrointestinal lymphoma were eligible. A brief clinical history was requested to allow documentation of clinical signs including vomiting and diarrhoea. Hair samples were collected on a clinical suspicion of gastrointestinal lymphoma and subsequently excluded if this was not confirmed. A diagnosis was confirmed either (a) by consistent cytology from ultrasound-guided fine-needle aspirations of abnormal areas of the gastrointestinal tract or enlarged local lymph nodes or (b) by consistent histopathology of the gastrointestinal tract or local lymph nodes. Histopathological samples were collected endoscopically, via Tru-Cut biopsy, surgically or during post-mortem examinations. The method of diagnosis was decided by the attending clinician and recorded, as were results of PCR for Antigen Receptor Rearrangement or immunohistochemistry. Hair samples were only included if collected within 30 days of confirming the diagnosis.

Contributing institutions were asked to provide a control case for each gastrointestinal lymphoma case recruited. Cats with no clinical suspicion of gastrointestinal lymphoma and no history of vomiting or diarrhoea within the previous six months were enrolled as control cases. The reason for presentation and the final diagnosis recorded.

Hair samples

Hair clipped from over the jugular vein for blood sampling was collected. The hair sample was categorised as black, white or other and placed in a sealed paper envelope within a sealed plastic wallet. Samples were stored in a smoke-free environment until processing.

Measurement of HNC

​Preparation of a calibration curve

Using a standard stock nicotine solution of 0.2 µg/mL, 10 solutions of different nicotine concentrations were prepared (0, 0.2, 1, 2, 4, 8, 16, 32, 64 and 128 ng/mL). Each was additionally spiked with the internal standard of deuterated nicotine (50 µL of a 2 ng/µL solution of 2H4-nicotine).

The samples were placed into 1.8 mL glass autosampler vials (Kinesis, UK) for automatic injection into an Orbitrap Exactive mass spectrometer (Thermoelectron, UK). The mass spectrometer was operated in positive/negative ion switching electrospray ionisation (ESI) mode with a needle voltage of 4.5 kV in positive mode and 4.0 kV in negative ion mode, a heated capillary temperature of 320°C, sheath gas flow of 50 arbitrary units and auxillary gas flow of 17 arbitrary units. The instrument was operated at 50 000 resolution and scanned from 75 to 1000 amu.

Hydrophilic interaction chromatography was performed using a Surveyor High Performance Liquid Chromatography (HPLC) pump fitted with a Supelco F5 PFP column (150mm × 4.6mm, 3 µm particle size; Sigma-Aldrich, UK). An isocratic method was used with a flow rate of 0.4 mL per minute. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The system was run in isocratic mode with 55% A: 45% B with 10 min for each run.

From the chromatogram created, the areas under the peak of the ion traces for both the internal standard (2H4-nicotine) and for the nicotine were measured.

A calibration curve was created (online supplementary figure 1), by plotting the ratio of the nicotine: 2H4-nicotine signal as a function of the nicotine concentrations of the standards. The correlation coefficient (R2) was 0.996. The calibration curve was used to generate an equation to enable the nicotine content of the hair samples to be calculated and to allow calculation of the limit of detection (LOD).

Supplemental material

Extraction of nicotine from the cat hair

Samples of cat hair (~30 mg) were weighed and the exact weight was noted. The hair was sonicated for 30 min at room temperature with 950 µL of methanol with 0.1% formic acid, and 50 µL of a 2 ng/µL solution of 2H4-nicotine (the internal standard). The latter was included to control variability in the quantitative assay. Following sonification, the supernatant was filtered (Acrodisc syringe filters; Sigma-Aldrich, UK) and the samples were analysed as described above. The area under the peak of the ion traces for both the nicotine and the internal standard were calculated. The ratio of these two values was input into the equation of the line from the calibration curve, allowing calculation of the amount of nicotine present in nanograms. This was converted to ng/mg by dividing the result by the exact weight of hair used.

Intra-day instrument precision (repeatability)

To assess the precision of the instrument, two extracts were subjected to repeat analysis within the same batch of samples being run through the instrument. Six analyses were performed on each of these extracts.

Intra-day within-assay variability

Two samples of cat hair were selected for assessment of within-assay variability; one with a low HNC and one with a high HNC. Each selected hair sample had to be of sufficient size to enable the preparation of 10 extract solutions, each requiring approximately 30 mg of hair. The extract solutions were analysed as previously described.

Statistical analysis

Statistical analysis was performed using Minitab Statistical Software (V.17.1.0). p<0.05 was considered significant. Variables were tested for normality using the Kolmogorov–Smirnov normality test.

To determine the LOD, the six lowest points on the calibration curve were used to prevent weighting towards the higher values. The SD of the response of the curve and the slope of the calibration curve were determined, allowing calculation of the LOD.

Data for the intra-day instrument precision (repeatability) and intra-day within-assay variability were normally distributed. The mean, SD and coefficient of variation of each were calculated. The modified Thomson Tau test was used to identify outliers, which were excluded.

The HNC data for cases and controls were not normally distributed; therefore, non-parametric tests were used. CIs were determined using non-parametric one-sample sign tests.

Mann–Whitney and Kruskal–Wallis tests were used to assess associations between the median HNC of gastrointestinal lymphoma cases and controls. χ2 by association was used to assess the association between cats with an HNC≥0.1 ng/mg and the proportion of cats diagnosed with gastrointestinal lymphoma. This statistical test was also used to assess for associations between rank ordered HNC divided into quartiles, and the proportion of cats diagnosed with gastrointestinal lymphoma.

Results

Gastrointestinal lymphoma and unaffected control cases

In all, 35 cats with gastrointestinal lymphoma and 32 controls were eligible for inclusion during the collection period. Signalment and coat colour data of the two groups are presented in table 1. Median time from diagnosis of gastrointestinal lymphoma to hair sample collection was 6 days (range 1–30).

Table 1

Descriptive details of the gastrointestinal lymphoma cases and controls

The reason for presentation of the gastrointestinal lymphoma cases is shown in tables 2 and 3. In all, 21 (60%) were diagnosed on cytology of fine-needle aspirates and 14 on histopathology. Histological samples were obtained by laparotomy (11 cases), endoscopy (1 case), ultrasound guided Tru-Cut biopsy (1 case) or post-mortem examination (1 case).

Table 2

Summary of lymphoma cases diagnosed by cytology

Table 3

Summary of lymphoma cases diagnosed by histopathology

Hair nicotine concentrations

Limit of detection

The LOD of hair nicotine was calculated as 0.425 ng. As hair nicotine levels were subsequently corrected for the weight of the hair samples used (approximately 30 mg per sample), the LOD could be alternatively considered as approximately 0.014 ng/mg.

Intra-day instrument precision (repeatability)

The mean HNCs for six times repeat analyses of the two extracts were 0.028 ng/mg and 0.022 ng/mg, with coefficients of variation of 6.1% and 3.3%, respectively.

Intra-day within-assay variability

Two extracts from the cat hair sample with a relatively low HNC failed to run due to machine error. The HNC of the remaining eight repeats ranged from 0.028 to 0.034 ng/mg, with a mean of 0.031 ng/mg and no outliers were identified. The SD was 0.002 ng/mg with a coefficient of variation of 5.9%.

All 10 extracts from the cat hair sample with a relatively high HNC were run successfully. One outlier was identified and removed from the dataset. The HNC of the remaining nine repeats ranged from 1.8 to 2.683 ng/mg, with a mean of 2.229 ng/mg. The SD was 0.364 ng/mg with a coefficient of variation of 16.3%.

HNCs in lymphoma and control cases

The HNC was successfully measured from all 67 cats and ranged from 0 to 2.694 ng/mg (median 0.030 ng/mg; 95% CI: to 0.028 to 0.035). Three samples had an HNC below the LOD as they had no detectable nicotine present and therefore an HNC of 0 ng/mg. In all, 13 out of 67 cases (19.4%) had HNC≥0.1 ng/mg.

The HNC of the lymphoma cats ranged from 0.021 to 2.269 ng/mg (median 0.030 ng/mg; 95% CI: 0.028 to 0.045) and the HNC of control cats ranged from 0 to 1.511 ng/mg (median 0.029 ng/mg; 95% CI: 0.027 to 0.038). The difference in median HNC between lymphoma and controls cases was not significant (p=0.46) (figure 1).

Figure 1

Hair nicotine concentration (ng/mg) of cats diagnosed with gastrointestinal lymphoma compared to control cats. The circles represent the individual values and the solid lines represent the median value for each group.

The percentage of cats with an HNC≥0.1 ng/mg was similar in the lymphoma group and the control group: 22.9% (8/35) and 15.6% (5/32), respectively (p=0.45).

When the HNC of all 67 cats was rank ordered and divided into quartiles, there was no significant difference in the proportion of gastrointestinal lymphoma cases or controls within these groups (p=0.63). The lowest quartile (≤0.025 ng/mg) included eight lymphoma cases (47.1%) and nine controls; the second quartile (>0.025 to≤0.030 ng/mg) included nine lymphoma cases (52.9%) and eight controls; the third quartile (>0.030 to≤0.063 ng/mg) included seven lymphoma cases (41.2%) and nine controls; and the highest quartile (>0.063 to≤2.269 ng/mg) included 11 lymphoma cases (64.7%) and 6 controls.

HNC in the 25 control cats without neoplasia

The median HNC of the 25 control cats without neoplasia was 0.030 ng/mg (95% CI: 0.026 to 0.038; range: 0 to 0.224), which was the same as that of the cases with gastrointestinal lymphoma.

Two out of 25 (8.0%) of the control cases without neoplasia had HNC≥0.1 ng/mg. When the HNC of the 60 cats was rank ordered and divided into quartiles, there was no significant difference in the proportion of lymphoma cases or controls within these groups (p=0.49). The lowest quartile (≤0.0253 ng/mg) included eight lymphoma cases (53.3%) and seven controls; the second quartile (>0.0253 to≤0.030 ng/mg) included nine lymphoma cases (60.0%) and six controls; the third quartile (>0.030 to≤0.052 ng/mg) included seven lymphoma cases (46.7%) and eight controls; and the highest quartile (>0.052 to≤2.269 ng/mg) included 11 lymphoma cases (73.3%) and 4 controls.

Discussion

Nicotine concentrations were successfully measured in all hair samples by a similar technique to that recently described.19 Repeat analyses were performed to assess intra-day within-assay variability which demonstrated an acceptable coefficient of variation for both low and high HNCs. A good coefficient of variation for intra-day instrument precision (repeatability) was documented at a low HNC; however, similar repeated analyses of extracts with a high HNC were not performed.

No significant difference was found between the median HNC of cats with gastrointestinal lymphoma and that of control cats, regardless of whether the cases with neoplasia were excluded from the control group, and the median HNC were almost the same values. As HNC is an accepted biomarker of exposure to ETS in people and increased HNC concentrations have been detected in cats with greater ETS exposure,15–17 19 this study has failed to identify a difference in exposure to ETS between cats with or without gastrointestinal lymphoma.

Bertone et al 5 identified a significant association between ETS exposure and the development of lymphoma in cats. In this questionnaire-based, case–control study, the relative risk of developing malignant lymphoma for cats with any exposure to ETS in the home environment was estimated to be 2.4, with a 95% CI of 1.2 to 4.5. In addition, Bertone et al 5 found that the risk increased with the quantity of exposure. This study does not support the findings of the Bertone et al 5 study; however the failure to identify a difference in the HNC between the two groups does not mean there is no difference and an equivalence study would have to be performed to test the hypothesis that there is no difference between HNC (and therefore ETS exposure) in the two groups.

In addition, there are several differences between this study and the Bertone et al study which should be highlighted. First, Bertone et al 5 examined the association with all forms of lymphoma not only gastrointestinal lymphoma and it is possible that it is other forms of lymphoma that are associated with ETS exposure, not as we hypothesised gastrointestinal lymphoma.

Second, the studies used different methodologies to determine ETS exposure. The Bertone et al 5 study was a questionnaire-based study; therefore, recall bias and selection bias could have affected the findings, particularly the former as respondents were asked to recall ETS exposure over the pets’ lifetime until 2 years prior to diagnosis. HNC likely represents ETS exposure over the preceding few months as hairs are gradually replaced following a quiescent phase (telogen) of several months’ duration.20 If a cat were therefore exposed to ETS for several years but this ceased 6 months prior to the study, HNC measurements would result in classification of the cat as having low ETS exposure whereas the epidemiological study of Bertone et al 5 would result in a classification of high ETS exposure. In addition, owner-reported ETS exposure might not correspond to the actual exposure of the cat, for example if the cat spent much of its time outside or in a distant part of the house. In this respect, HNC likely reflects the actual ETS exposure of the cat more accurately.

Finally, the Bertone et al 5 study was a much larger study and therefore had more power to detect a difference in ETS exposure between groups.

Carcinogenesis is a complex and multifactorial process, involving the accumulation of genetic mutations including activation of oncogenes and inactivation of tumour suppressor genes, ultimately leading to a loss of normal cellular homeostasis.21 Many environmental and genetic factors, including ETS exposure and chronic mucosal inflammation2 19 may therefore play a role in the development of gastrointestinal lymphoma in any individual patient. The number of interacting factors involved and the protracted time over which the mutations accumulate makes it more difficult to identify individual risk factors and associations and increases the number of cases required to identify an association.

In this study, although no significant difference in the proportion of affected and unaffected cats was found when the cats were divided into four groups on the basis of the magnitude of the HNC, the biggest difference was however, seen in the highest HNC quartile (>0.063 to≤2.269 ng/mg), which contained 64.7% lymphoma cases. Additionally, a higher proportion of lymphoma cats had HNC≥0.1 ng/mg than controls (22.9% vs 15.6%), although this was not statistically significantly different. A much larger study with more power would be more likely to identify an association between HNC and gastrointestinal lymphoma if one exists. This study does, however, show that this methodology works and could be used in future, larger studies.

Feline HNC has previously been shown to be strongly associated with owner-reported exposure by Smith et al, with an HNC of ≥0.1 ng/mg being suggestive of exposure. The methodology used in that study was similar; however, the instrument and column used were different and therefore this cut-off may not be directly applicable to the current study population. It is interesting to note that only 13 out of all 67 cases (19.4%) in this study would have been classified as exposed on that basis, a relatively low proportion. Furthermore, when comparing the HNC measured in these two studies, the median HNC of all 67 cats in this study was 0.030 ng/mg (range: 0–2.694 ng/mg; 95% CI: 0.028 to 0.035) which is considerably lower than the reported median HNC of 0.281 ng/mg (range 0.055–5.968; 95% CI: 0.133 to 0.766) of the 37 exposed cats in the Smith et al 19 study and is less than half the reported median HNC of 0.064 ng/mg (range 0–0.269; 95% CI: 0.055 to 0.074) in the 40 unexposed cats in that study. This may suggest that the cats in the current study had a relatively low level of ETS exposure making an association with smoking-related disease less likely to be identified.

A potential cause of misclassification of ETS exposure in this study could be the use of electronic cigarettes (e-cigarettes). These battery-powered devices do not involve tobacco combustion but instead aerosolise nicotine prior to inhalation.22 The possibility that some cats in the current study were exposed to e-cigarettes, and therefore nicotine but not traditional tobacco smoke carcinogens, cannot be excluded. This could alter the relationship between the HNC and the concentration of carcinogens on the cats’ coats.

In this study, a control population of unhealthy (rather than healthy) cats was used, in part because recruiting healthy cats from a veterinary referral hospital population is extremely difficult and selection of cases from a different population would have risked altering the demographics of the population and therefore the exposure to ETS. Cases with neoplasia were eligible for inclusion in the control population as we aimed to investigate the association between feline HNC and gastrointestinal lymphoma specifically as it has been suggested that oral ingestion of carcinogens during grooming has a local carcinogenic effect on the gastrointestinal tract. However, as there is a widely recognised association between ETS and some forms of neoplasia, the analyses were also performed with the seven control cases with neoplasia removed and again an association between HNC and gastrointestinal lymphoma was not found. These repeated analyses will have less power to detect a difference and again the failure to identify an association is not evidence that there is no association.

In this study, histopathology of full-thickness gastrointestinal biopsies was not a requirement for inclusion in the gastrointestinal lymphoma group and 21 of the 35 gastrointestinal lymphoma cases were diagnosed by aspiration cytology. Of these, 20 were classified as intermediate and/or large cell lymphoma. These cases were considered eligible for inclusion as this is an accepted diagnostic technique, especially for intermediate-grade and high-grade alimentary lymphoma.23 The only case diagnosed as small to intermediate cell lymphoma based on cytology, had a large multi-lobulated mass consistent with marked mesenteric lymphadenopathy as well as a thickened area of small intestine, making inflammatory bowel disease extremely unlikely and inclusion of this case was therefore considered reasonable.

In conclusion, this study failed to identify a significant difference in HNC between cats with gastrointestinal lymphoma and control cases. As HNC has previously been shown to be a biomarker of ETS exposure, this study failed to identify an association between ETS and gastrointestinal lymphoma; however, an association may still exist. This is one of very few studies using biomarkers for the detection of ETS in cats and as such further demonstrates the methodology and potential for such techniques. Further larger studies using biomarkers of ETS exposure in larger numbers of cats are warranted.

References

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Footnotes

  • Funding This study was funded by British Small Animal Veterinary Association, Grant No: PetSavers Clinical Research Project 04.14.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.

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