REVIEW ARTICLES

Evaluarea imagistică modernă a colului uterin. Review al literaturii privind microscopia confocală şi colposcopia digitală automatizată

 Modern cervical imaging assessment. Review of literature on confocal microscopy and digital automated colposcopy

Ana-Maria Petrescu, Dan Ruican, George Lucian Zorilă, Nicolae Cernea, Ştefania Tudorache, Gabriel Dominic Iliescu

First published: 29 octombrie 2019

Editorial Group: MEDICHUB MEDIA

DOI: 10.26416/ObsGin.67.3.2019.2599

Abstract

Colposcopy is today the gold standard investigation to diagnose cervical intraepithelial neoplasia (CIN) following a modified Pap smear. It is used for cervical image magnification and to guide cervical biopsy. Still, there can be other techniques that could facilitate the diagnosis of cervical neoplastic lesions and cervical cancer. This paper aims to analyze the advantages of modern cervical imaging techniques, namely confocal microscopy and digital automated colposcopy, in detecting and staging cervical neoplasia lesions. Confocal microscopy has a wide applicability as an endoscopic imaging technique and already proved useful in cases with cervical lesions. While colposcopy focuses on the macroscopic aspect of the cervical lesions, the advantage of confocal microscopy is that it provides information regarding the microscopic histological changes: architectural changes, increased volume or an increased ratio between the nuclear and cytoplasmic volumes in serial planes tangential to the epithelium, that can be reconfigured 3D. These changes are routinely diagnosed by histopathological examination of a biopsy specimen; however, the confocal microscopy has the ability to visualize these changes in vivo. This helps the diagnosis process and therapeutic decision by eliminating the time required for biopsy and histopathological examination. However, confocal microscopy is an expensive method, therefore digital colposcopy is more appropriate for the screening and diagnosis process, especially in low-income countries. In conclusion, confocal microscopy has the potential to become an important diagnostic and staging tool in cervical neoplastic lesions. The greatest benefit of this method is the ability to visualize in vivo changes that are currently detected by histopathological examination and thus to favorize a faster therapeutic management. However, because its limited accessibility, colposcopy remains the standard imaging method in the diagnosis of cervical lesions.

Keywords
confocal microscopy, colposcopy, digital colposcopy, cervical lesions

Rezumat

Colposcopia reprezintă în prezent standardul de aur în diagnosticarea neoplaziei cervicale intraepiteliale (CIN), după un examen Babeş-Papanicolau cu modificări. Investigaţia este utilizată pentru mărirea imaginii cervicale şi pentru a ghida biopsiile de la nivelul colului uterin. Totuşi, pot exista şi alte tehnici care să faciliteze diagnosticul leziunilor neoplazice cervicale şi al cancerului de col uterin. Această lucrare urmăreşte să analizeze avantajele tehnicilor moderne de imagistică de la nivelul colului uterin – şi, anume, microscopia confocală şi colposcopia digitală automatizată – în procesele de detecţie şi stadializare a leziunilor neoplazice şi preneoplazice cervicale. Microscopia confocală are o aplicabilitate largă ca tehnică imagistică endoscopică şi deja s-a dovedit utilă şi în cazul leziunilor cervicale. În timp ce colposcopia clasică se axează pe aspectul macroscopic al leziunilor cervicale, avantajul microscopiei confocale este acela că furnizează informaţii privind modificările microscopice histologice: modificări arhitecturale, creşterea în volum sau creşterea raportului nucleo-citoplasmatic, în planuri seriate tangenţiale la epiteliu, care pot fi reconstruite 3D. Aceste modificări sunt diagnosticate de rutină la examenul histopatologic al piesei de biopsie; totuşi, microscopia confocală are abilitatea de a vizualiza aceste modificări in vivo. Acest aspect facilitează procesul de diagnosticare şi de decizie terapeutică, eliminând timpul necesar biopsierii şi interpretării histopatologice. Cu toate acestea, microscopia confocală este o metodă costisitoare, prin urmare colposcopia digitală este mai accesibilă în procesele de diagnosticare şi screening, mai ales în ţările în curs de dezvoltare. În concluzie, microscopia confocală are potenţialul de a deveni un instrument de diagnostic şi stadializare important pentru leziunile neoplazice cervicale. Cel mai mare avantaj al acestei metode este de a vizualiza in vivo modificări care sunt actualmente detectabile doar prin examinare histopatologică şi, astfel, să favorizeze un management terapeutic mai rapid. Cu toate acestea, din cauza accesibilităţii reduse, colposcopia rămâne metoda standard de diagnostic al leziunilor cervicale. 

Introduction

World Health Organization (WHO) confirms an incidence of 570,000 new cases of cervical cancer diagnosed in 2018 and a total of 270,000 deaths in 2015. This data classifies cervical cancer as the fourth leading cause of death among female population, representing 6.6% of all cancers in women. Approximately 90% of these deaths occurred in developing countries(1), and 95-98% of these lesions are caused by human papillomavirus (HPV) infection, cases with increased oncological potential(2,3). In Romania, cervical cancer represents 10.1% of all female cancers(1), with 3,308 new cases being diagnosed every year and 1,743 women dying of this disease annually(4). Screening programs have been developed for cervical cancer and nowadays they are one of the best screening processes available worldwide. However, in underdeveloped countries there is no general access to these programs and the mortality rate is very high.

An accurate diagnosis of cervical intraepithelial neoplasia (CIN) is important, and also the early detection of the cervical cancer, because therapy can cure the disease. In early stages, patients have a five-year survival rate of 90%(5). Pap smear, and testing for HPV infection followed by colposcopy and biopsy are the gold standard in the screening and diagnosis of cervical lesions(3).

This paper analyzes cervical imaging alternatives to classic colposcopy that have the potential to reduce the number of unnecessary biopsies and eludes the time required for pathology diagnosis that often, especially in developing countries, is a source for lost to follow-up patients. The current screening/diagnosis method has certain limitations: minimum three visits to the specialist and a long period in the diagnosis process (about four weeks until the implementation of the specific therapy). A modern technique to facilitate the screening process and speed up the diagnosis process would be of great use.

Another important aspect for the developing countries is the lack of medical specialists and busy work intervals for practitioners. The new technologies thus enclose more advantages, such as speed and automation. The two methods reviewed in this paper are the confocal endomicroscopy and the digital colposcopy. They fulfill the requirements necessary for an effective diagnostic test: accurate, safe, fast and convenient for the patient; the see-and-treat method is a principle that specialists try to implement through confocal endomicroscopy and digital colposcopy.

In the transformation process, from normal to dysplastic and neoplastic cells, certain modifications happen at the cell level, and these alterations might change the optic properties of the cervical cells(5). The confocal microscopy is an optical technique that can identify the optical properties of the cells and classify these lesions as low-risk or high-risk.

Confocal endomicroscopy

The confocal microscopy is a high-resolution microscopy currently used for diagnosing stomach lining lesions, lung nodules, pancreatic and biliary strictures, pancreatic cysts, inflammatory diseases or intestinal lesions and urological diseases(6). There was a limited use for confocal microscopy in gynaecology, but in cases of non-invasive cervical melanoma, this method demon­strated its diagnostic value(7). The advantages of confocal microscopy are: obtaining microscopic images in vivo, targeted biopsies, accuracy improvement of diagnosis and reducing the time needed for therapy initiation(8,9). It offers the possibility of real-time assessment of the tissue(9) and it can help implementing the “see-and-treat” strategy. This can be a new gold standard in underdeveloped countries, where the follow-up is nearly impossible. Avoiding loop excisions in patients without obstetrical history is another advantage of the endomicroscopic assessment of the cervix. Literature shows a higher risk of preterm labor for patients who undergo a conization before pregnancy(10). Therefore, cervical integrity is very important for these patients and the use of in vivo histological diagnosis provided by confocal endomicroscopy technique might reduce this obstetrical risk by avoiding unnecessary conizations and loop excisions. Besides these advantages, this method provides much faster results, in a more comfortable manner for the patient, and permits to establish much faster the proper therapy.

The principle of this technique is very simple: instead of a biopsy and histological examination, this method assesses the cervix in real time under the microscope, with a great resolution, down to 1 µm(9,11). In one of the first cervical applications of this technique, Zhang et al.(12) studied the cervix pregnancy modifications using endomicroscopy.

This method has also the advantage of creating 3D images that can facilitate the biopsy guidance and follow-up the healing process. The images obtained are tangential on the lesion surface, an approach that cannot be obtained during histopathological examination on routine sectioning, where the section plans are perpendicular to epithelium surface(2).

The applicability of confocal microscopy in cervical cancer is suggested by the morphological changes. Intraepithelial cervical lesions are characterized by an increased nucleus volume, size, eccentricity, and an increased nuclei-cytoplasmic ratio. The gold standard for diagnosing these lesions is biopsy and histopathological examination. Confocal high-resolution microscope can diagnose these changes in vivo, shortening the standard diagnosis process. The nuclear changes are not the only cellular modifications that occur in cervical lesions, but these are the key signs to the in vivo diagnosis, as they are characteristic to high-grade lesions and do not appear in other types of lesions, such as in inflammation. False positive results due to nuclear crowding in inflammation are avoided by this technique(2,13). Even small changes of nuclei-cytoplasmic ratio have been noted through the entire depth of the epithelium in dysplastic lesions(2,8). Tan et al.(8) used confocal microscopy and observed the existence of cellular atypia, significant nuclear changes in shape and increased density and volume in CIN lesions.

One of the substances used to stain the nuclei of the epithelium was acriflavine, as a fluorescent contrast agent. However, deep penetration in the epithelium layers was low. On the other hand, the stain was much more evident in high-grade lesions, because of a greater density of the nuclei at superficial levels. These changes were described through the optical endomicroscope and the cases were classified from normal cells to CIN3 or cancer. Histopathological analysis confirmed the confocal microscopy diagnosis with a 97% sensitivity for the CIN lesions detection and a specificity of 80% for CIN1 and 93% for CIN2 and CIN3(8). Other researchers used fluorescein(9), injected intravenously to stain the cytoplasmic components, extravascular matrix and the microvascular structures, but not the nuclei(9,14).

Grant et al.(13) used proflavine solution, which is also an antiseptic, to investigate the cervix and stain the cell nuclei, and developed a low-cost, high-resolution endomicroscope (HRME) using optical fiber. Through HRME the patients may be classified in low risk (<CIN2) and high risk (CIN2+), based on the nuclear aspect: small, well-shaped and uniformed in normal cells, and large, irregularly and crowded in high-grade dysplasia. Mean nuclear area and the median nuclear eccentricity increase from low-grade to high-grade dysplasia and may be added in the scoring analysis to reduce the number of false positive cases. Also, prominent vessels were highlighted in some cases with CIN3 or cancer. This protocol achieved a 92% sensitivity and a 77% specificity, with an accuracy that increased with the severity of the biopsy results.

Digital colposcopy

In our opinion, based on data from literature, digital colposcopy has the potential to ease the work of the practitioners in low-income countries as a screening and diagnosis alternative.

Some strategies were suggested in developing countries, trying to replace the Pap smear and colposcopic examination, relatively expensive, with cervical direct visual inspection using acetic acid and/or Lugol’s iodine that is cheaper and more accessible(15). Sankaranarayanan et al. compared these direct inspection methods with Pap smear and HPV testing, and found a satisfactory specificity of 86% and a 74-94% lesion-dependent sensitivity(16). However, a professional with specific training is needed to analyze the clinical aspects. Also, the interpretation depends on one’s experience and expertise. 

Different researchers implemented different strategies to obtain automatic colposcopic images, but the interpretation of these images needed specialists to manually analyze and diagnose them. Cervicography, with images captured before and after the application of acetic acid, was used by Bomfim-Hyppolito et al.(17), but this method still requires time for a specialist to interpret the images.

A modern cervical work-flow assessment method would imply real-time images captures seconded by an automatically diagnostic. There are many studies that tried to demonstrate the importance and benefits of screening and diagnosis methods for cervical preneoplastic and neoplastic lesions by means of optical spectroscopy and digital colposcopy(15). The idea of digital colposcopy using a camera and a software capable of pattern recognition to detect cervical neoplasia was promising, with a sensitivity of 79% and a specificity of 88%(14,17).

A computer program that can be connected to a cervical images acquisition system and automatically detect the lesion in the image and differentiate normal cervix epithelium from low-grade lesions, high-grade lesions and neoplasia is a revolutionary tool in the cervical cancer screening process, especially in underdeveloped countries. For example, Li et al.(18) implemented a Computer-Aided Diagnosis (CAD) system that automatizes the work-flow, diagnoses the cervical lesions and also offers recommendations based on the particularities of each case.

The detection and diagnosis of these lesions are based on the characteristics of the images, such as the margins of the lesions, the blood vessels and the acetowhite regions. The authors also used special filters to enhance the images and increase the accuracy, for example the green filter that accentuates the vasculature. The results of this research were: a sensitivity of 60.77%, and a specificity of 93.41% in a group of 40 patients(19).

Another group a researchers (Park et al.(15)) developed a system that was able to acquire images of the cervix based on white-light illumination after acetic acid application. Their system had two main steps: the regions with similar optical properties were identified and the regions were classified to determine whether neoplastic tissue was present, all based on color patterns. The result of their study was promising, with a sensitivity of 79% and a specificity of 88%.

Lowers et al.(20), in an extended research on the aceto-whiting reaction and its digital interpretation, which was finalized by producing a high resolution colposcope with an adjunctive intuitive map that assists with interpretation and offers accurate guidance for biopsy, obtained a sensitivity of 83% and a specificity of 75% for the digital colposcope examination. They also compared the digital examination with the classic examination and the digital and classic examinations combined, and obtained for classic colposcopy a sensitivity of 55% and a specificity of 84%, and for the digital and classic examinations combined, a sensitivity of 89% and a specificity of 68%.

Conclusions

The confocal microscopy technique has many benefits: faster diagnosis, high sensitivity and specificity, it reduces the number of biopsies, precise in vivo delimitation of the affected area, facilitating the biopsy and the loop electrosurgical excision procedure. It is a reliable non-invasive method to assess the resection margins after loop excision or to verify the efficiency of other therapy during the follow-up process. However, due to the high cost, this technique is not yet generally available. Also, these technique algorithms should be revised, standardized and improved, especially regarding the low-grade/borderline lesions diagnosis.

Therefore, more accessible methods of screening and diagnosis are needed, particularly in developing countries. Digital colposcopy with automated diagnosis could improve the screening process for cervical cancer, by reducing the number of specialists required and the therapy implementation timing.

In conclusion, these two methods, which are so dif­ferent but so alike, aim to improve the cervical cancer screening and the diagnosis process, shorten the period between the first medical interaction and treatment implementation, help diagnose preneoplastic and neo­plastic cervical lesions, and decrease mortality.  

Conflict of interests: The authors declare no conflict of interests.

Bibliografie

1. WHO. Cervical cancer [Internet, cited: 2019 Mar 30]. Available from: http://www.who.int/cancer/prevention/diagnosis-screening/cervical-cancer/en/
2. Novikova T. Optical techniques for cervical neoplasia detection. Beilstein J Nanotechnol. 2017; 8:1844–62. 
3. Lowy DR, Solomon D, Hildesheim A, Schiller JT, Schiffman M. Human papillomavirus infection and the primary and secondary prevention of cervical cancer. Cancer. 2008 Oct 1; 113(7 Suppl):1980–93. 
4. Romania: Human Papillomavirus and Related Cancers, Fact Sheet 2018; 2. 
5. Yang L, Liu W-T, Wu H, Wang C, Ping B, Shi D-R. Separation of normal and premalignant cervical epithelial cells using confocal light absorption and scattering spectroscopic microscopy ex vivo. J Biomed Biotechnol. 2011; 214781. 
6. Cellvizio Optical Biopsy with Probed-Based Confocal Laser Endomicroscopy (pCLE) - Mauna Kea Technologies [Internet, cited 2019 Mar 30]. Available from: https://www.maunakeatech.com/en/cellvizio
7. Perrot JL, Labeille B, Richard Coulet E, Cochin S, Biron Schneider A-C, Rubegni P, et al. Contribution of reflectance confocal microscopy in the diagnosis of uterine cervix melanoma: First case report. Ann Dermatol Venereol. 2017 Sep; 144(8–9):567–9. 
8. Tan J, Quinn MA, Pyman JM, Delaney PM, McLaren WJ. Detection of cervical intraepithelial neoplasia in vivo using confocal endomicroscopy. BJOG Int J Obstet Gynaecol. 2009 Nov; 116(12):1663–70. 
9. Degueldre M, Vandromme J, de Wind A, Feoli F. Real-time in-vivo microscopic imaging of the cervix using confocal laser endomicroscopy: preliminary observations and feasibility study. Eur J Cancer Prev Off J Eur Cancer Prev Organ ECP. 2016; 25(4):335–43. 
10. Albrechtsen S, Rasmussen S, Thoresen S, Irgens LM, Iversen OE. Pregnancy outcome in women before and after cervical conisation: population based cohort study. BMJ. 2008 Sep 18; 337:a1343. 
11. Schlosser C, Bodenschatz N, Lam S, Lee M, McAlpine JN, Miller DM, et al. Fluorescence confocal endomicroscopy of the cervix: pilot study on the potential and limitations for clinical implementation. J Biomed Opt. 2016 01; 21(12):126011. 
12. Zhang Y, Akins ML, Murari K, Xi J, Li M-J, Luby-Phelps K, et al. A compact fiber-optic SHG scanning endomicroscope and its application to visualize cervical remodeling during pregnancy. Proc Natl Acad Sci. 2012 Aug 7; 109(32):12878–83. 
13. Grant BD, Fregnani JHTG, Possati Resende JC, Scapulatempo-Neto C, Matsushita GM, Mauad EC, et al. High-resolution microendoscopy: a point-of-care diagnostic for cervical dysplasia in low-resource settings. Eur J Cancer Prev Off J Eur Cancer Prev Organ ECP. 2017; 26(1):63–70. 
14. Paramsothy S, Leong RWL. Endoscopy: Fluorescein contrast in confocal laser endomicroscopy. Nat Rev Gastroenterol Hepatol. 2010 Jul; 7(7):366–8. 
15. Park SY, Follen M, Milbourne A, Rhodes H, Malpica A, MacKinnon N, et al. Automated image analysis of digital colposcopy for the detection of cervical neoplasia. J Biomed Opt. 2008 Feb; 13(1):014029. 
16. Sankaranarayanan R, Rajkumar R, Theresa R, Esmy PO, Mahe C, Bagyalakshmi KR, et al. Initial results from a randomized trial of cervical visual screening in rural south India. Int J Cancer. 2004 Apr 10; 109(3):461–7. 
17. Bomfim-Hyppólito S, Franco ES, Franco RG de MM, de Albuquerque CM, Nunes GC. Cervicography as an adjunctive test to visual inspection with acetic acid in cervical cancer detection screening. Int J Gynaecol Obstet Off Organ Int Fed Gynaecol Obstet. 2006 Jan; 92(1):58–63. 
18. Li W, Van Raad V, Gu J, Hansson U, Hakansson J, Lange H, et al. Computer-Aided Diagnosis (CAD) for Cervical Cancer Screening and Diagnosis: A New System Design in Medical Image Processing. In: Computer Vision for Biomedical Image Applications - First International Workshop, CVBIA 2005, Proceedings. 2005; p. 240–50. 
19. Li W, Gu J, Ferris D, Poirson A. Automated image analysis of uterine cervical images - art. no. 65142P. Proc SPIE. 2007 Mar 1; 6514. 
20. Louwers JA, Zaal A, Kocken M, Berkhof J, Papagiannakis E, Snijders PJF, et al. The performance of Dynamic Spectral Imaging colposcopy depends on indication for referrals. Gynecol Oncol. 2015 Dec 1; 139(3):452–7.