MSACL 2017 US Abstract

Applicability of Rapid Evaporative Ionization Mass Spectrometry (REIMS) in Uterine Cervical Pathology — a Proof of Principle Study

Menelaos Tzafetas (Presenter)
Imperial College London

Bio: Dr Menelaos Tzafetas is a Clinical Research Fellow in Gynecology at Imperial College London, with a particular interest in the use of mass spectrometry technologies for improving clinical practice and patient experience satisfaction in cervical cancer and pre-cancer treatment services.

Authorship: Anita Mitra1, Ilkka Kalliala1, Menelaos Tzafetas1, Zsolt Bodai2, Francesca Rosini3, David Phelps1, Louise Gildea2, Adele Savage2, Rashpal Flora3, Sadaf Ghaem-Maghami1, Zoltan Takats2, Maria Kyrgiou1.
(1) Department of Surgery and Cancer Imperial College London, (2) Department of Computational and Systems Biology Imperial College London, (3) Department of Histopathology Imperial College Healthcare NHS Trust

Short Abstract

Cervical cancer and its precancerous form cervical intraepithelial neoplasia (CIN) are common diseases in women of reproductive age. Fertility-sparing treatments are available, however they require precise disease clearance at the margins to balance future oncological and reproductive outcomes for these patients. In our pilot study, we showed REIMS was able to quickly and accurately differentiate between cancerous and normal cervical tissue. We plan to develop this technique in this field and believe it has great potential to improve patient satisfaction and balance oncological and reproductive outcomes.

Long Abstract

Background

Cervical cancer is the fourth most common female cancer worldwide. It is caused by a persistent infection of oncogenic human papilloma virus (HPV) and the invasive cancer develops during the course of 10 to 15 years, progressing through detectable and treatable precancerous lesions, cervical intraepithelial neoplasias (CIN). Cervical screening programmes in the developed world have a significant impact on the reduction in the number of cases through identification and treatment of CIN. Precancerous lesions detected through abnormal cytological screening results can be difficult to manage and often require repeat visits, further diagnostic punch biopsies with increased patient anxiety, risk of non-compliance and overload of services. Both CIN and cervical cancer are common amongst young women to whom future fertility and reproductive wishes are very important. Local excision of CIN and early invasive cervical cancer is highly effective particularly if the excisional margins are clear. However, in women in reproductive age an important aim is to remove the minimal amount of healthy tissue in order to conserve maximal cervical length for future pregnancy, because excisional treatment has been associated with preterm birth and second trimester miscarriage, along with increased risk of neonatal mortality and morbidity (1,2). This can present a challenge for the surgeon when weighing up future oncological and obstetric outcomes.

Furthermore, in place of more radical hysterectomy, trachelectomy is a fertility sparing treatment for early cervical cancer and again clear margins are essential to ensure optimal future oncological outcomes and prevent the need for subsequent repeat treatment involving radical surgery to remove the uterus.

Current diagnostic techniques are slow, technically demanding and require highly-skilled personnel to carry out the analysis of cytological and histological specimens. Positive margins may be found in 23% of excisional specimens for treatment of CIN (3), and up to 33% of cases of trachelectomy for cervical cancer (4). There is a need for a rapid, consistent method of near-patient tissue identification for use in the colposcopy and gynaecology theatre to improve patient satisfaction and to enable the clinician to ensure maximum disease clearance with removal of the smallest amount of tissue to reduce the impact on future reproductive outcomes.

Rapid Evaporative Ionization Mass Spectrometry (REIMS) is an ambient ionization technique using electrosurgery-generated aerosols, which are analysed by time-of-flight mass spectrometry (TOF MS) to provide tissue identification. The spectra can be generated directly from tissue surfaces, without any need for sample preparation, presenting the possibility for this technology to give an intra-operative diagnosis (3).

Dysregulation of lipid synthesis is associated with many cancers, and REIMS has been used in several other tissues to characterise the lipid fingerprint of a tissue for subsequent tissue identification. Liquid chromatography mass spectrometry (LCMS) has previously been used to show a significant difference in the lipid profiles of normal and cancerous cervical tissue, with phosphatidylcholine found to be the predominant membrane lipid in cancerous tissue, compared to sphingomyelin in normal cervical tissue (4).

We therefore conducted a pilot study to test the hypothesis that malignant cervical cancer tissues exhibit different metabolic profiles compared to normal, healthy cervical tissue, and that the REIMS technique can be used to distinguish such differences. The future clinical application would be in-vivo real time tissue identification, which would lead to better patient satisfaction and outcomes.

Materials and methods

Fresh frozen cervical biopsies stored at -80 °C were obtained from the Imperial College Healthcare NHS Trust Tissue Bank and collected prospectively within Imperial College Healthcare NHS Trust. Samples were thawed at room temperature and cut using a Covidien diathermy hand-piece. The surgical aerosol produced by the diathermy was transferred into a Waters Xevo G2-S mass spectrometer. Sampling was performed in a random order of tissue diagnosis.

Following REIMS processing, the tissue samples were fixed in formalin and paraffin embedded for routine haematoxylin and eosin (H&E) staining. A histopathologist analysed the samples giving a final diagnosis for the sample and further information regarding the percentage cancer content of the sample (were relevant) and the diagnosis based on the tissue type surrounding the diathermy sampling holes. The histopathologist was blinded to previous tissue diagnosis from the original surgery. Samples containing cancer were only included in further data analysis if there was ≥ 75% cancer in the sample.

These final histological diagnoses were used in multivariate statistical analysis of mass spectroscopic spectra, including principal components analysis (PCA) and linear discriminant analysis (LDA) performed using Online Model Builder (OMB) software (Waters Research Centre, Budapest, Hungary). Internal lock mass (699.947m/z) correction was employed, with m/z bining set to 0.1. After the model building leave one patient out cross-validation was performed to test the correct classification rate.

Results and discussion

Samples from 24 patients met the inclusion criteria (9 with cervical cancer and 15 healthy controls), and sampled in the cut diathermy mode to give 86 spectra; 33 spectra from cancerous tissues and 53 from normal tissue, between March and April 2016. Spectral differences were observed between normal and cancerous cervical tissues in 500-1000m/z range. Data was imported to OMB and combined PCA/LDA model was used for tissue classification. To test the classification rate of the built model, leave one patient out cross validation was applied. Leave one patient out cross-validation resulted in correct detection rate of 91% of cervical cancers and 92% in normal tissue.

Frozen section is the current method for intraoperative assessment of margin status at the time of trachelectomy, and the concordance between intraoperative frozen section and final histology has been quoted as 84% (4). In addition to providing real-time information, thus reducing anaesthetic time, REIMS has the potential to improve the accuracy of intraoperative margin detection.

Conclusion

This proof of concept study demonstrates that REIMS can be used to differentiate between normal and cancerous cervical tissue with a relatively high degree of accuracy. A larger sample size is required to increase the accuracy of the technique, and for metabolite identification of spectral peaks that are identified to differ significantly between normal and cancerous cervical tissue. This will allow the development of REIMS-based technologies as a tool for intraoperative margin detection. We further plan to expand our investigation into the ability of the technique to differentiate between grades of CIN for further application in the colposcopy clinic as a real-time diagnosis to prevent the need for extra visits and to perform successful local excisional treatment with removal of the smallest amount of tissues as necessary. The advantages of this technology are that there is no disruption to routine surgical techniques, and that the near real time results offers the possibility to alter the path of tissue excision at the time of operation, which has great potential to improve patient satisfaction and balance oncological and reproductive outcomes.


References & Acknowledgements:

References

1. Kyrgiou et al. (2016) Adverse obstetric outcomes after local treatment for cervical preinvasive and early invasive disease according to cone depth: systematic review and meta-analysis. BMJ;354:i3633.

2. Kyrgiou et al. (2014) Fertility and early pregnancy outcomes after treatment for cervical intraepithelial neoplasia: systematic review and meta-analysis. BMJ;349:g6192.

3. Ghaem-Maghami et al. (2007) Incomplete excision of cervical intraepithelial neoplasia and risk of treatment failure: a meta-analysis. Lancet Oncology;8(11):985-993.

4. Park et al. (2010) Frozen-section evaluation of cervical adenocarcinoma at time of radical trachelectomy: pathologic pitfalls and the application of an objective scoring system. Gynecol Oncol; 110(3):316–323

5. Balog et al. (2013) Intraoperative tissue identification using rapid evaporative ionization mass spectrometry. Sci Transl Med 17;5(194):194ra93.

6. Preetha et al. (2005) Surface activity, lipid profiles and their implications in cervical cancer. J Cancer Res Ther;1(3):180-6.

Acknowledgements

This study was supported by Imperial Experimental Cancer Medicine Centre, Imperial NIHR Biomedical Research Centre, Cancer Research UK Imperial Centre, Imperial College Healthcare Charity, The British Society of Colposcopy and Cervical Pathology and tissue samples were provided by the Imperial College Healthcare NHS Trust Tissue Bank.


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