1. STUDY OF MULTIMODAL HYPERSPECTROSCOPY IN A POPULATION OF WOMEN AT RISK FOR CERVICAL CANCER Olutosin Awolude α, Brenda Shultz β, Babatunde Akinwunmi α, Isaac Adewole α, Mark Faupel β α = College of Medicine, University of Ibadan/University College Hospital, Nigeria β = Guided Therapeutic Inc, USA

2. Abstract INTRODUCTION Early detection and treatment of its pre-cancerous lesions is an important secondary requisite for prevention of cervical cancer. However, the currently available screening tests are associated with limitations like low sensitivity, high false positive results, prohibitive costs, logistics and technicality. Hence, the need for a point of care tests to overcome these challenges. OBJECTIVE: To evaluate multimodal hyperspectroscopy (MHS) for the early detection of cervical cancer in a population of women previously screened for cervical cancer. METHODOLOGY: One hundred women enrolled in the study. MHS of the cervix using the LuViva Advanced Cervical Scan (Guided Therapeutics, Norcross, GA USA) was performed in a 1 minute procedure. All women then had an HPV sample and biopsies for histology taken. RESULTS: Of the 24 women with abnormal Pap tests (excluding HSIL), three had CIN2+, five had CIN1 and 16 were free of dysplasia at histopathology. All three of the CIN2+ recorded high likelihood of CIN2+ by MHS (100% sensitivity). HPV was negative for all three women. MHS classified one of five CIN1's and seven of the 15 women without dysplasia as having low or moderate risk for CIN2+, for a specificity of 40%. MHS identified as high risk 29 of 36 women (81%) that were VIA+. In contrast, 23 of 35 women (66%) with normal Pap tests and biopsy results were either moderate or low risk by MHS. CONCLUSIONS: MHS would have reduced the percentage of unnecessary colposcopy and biopsy by 40% without any false negatives and, also, was able to differentiate between VIA+ and Pap negative women. These suggest that MHS may have potential of a point of care primary and immediate objective screening procedure. 2

3. Introduction Early detection and treatment of its pre-cancerous lesions is an important secondary requisite for prevention of cervical cancer. However, the currently available screening tests are associated with limitations like: – low sensitivity –high false positive results –prohibitive costs –logistics and high technicality. 3

4. Introduction There remains, therefore, a need for a point of care test to improve the screening and treatment of cervical disease, especially with regard to improving sensitivity and lowering the false positive rate. 4

5. Introduction Such point of care tests include devices based on the principle of fluorescence and reflectance spectroscopy spectroscopic techniques have the capability to detect and to quantitatively assess changes in the cellular chemistry and tissue architecture associated with the progression of disease. One of such devices is luviva® –A multimodal Hyperspectoscopic device with a unique approach that has the potential to reduce morbidity from over treatment due to unnecessary referrals and significantly improve patient care while simultaneously reducing the enormous costs currently associated with cervical disease management. 5

6. LuViva The LuViva is a noninvasively collects and analyzes fluorescence and reflectance spectra from the cervix without contrast agents, such as acetic acid Light from the arc lamp is band pass filtered to limit exposure of the cervix to three distinct regions centered at 340nm, 400nm and 460nm which excite fluorophores associated with neoplastic processes. Each of three fluorescence wavelengths is applied automatically under software control one at a time in a predetermined order and scan pattern. 6

7. LuViva The resultant fluorescent spectral output of the cervical tissue is imaged onto a charge coupled device (CCD) camera and stored for processing and analysis. In addition to the fluorescence and reflectance spectroscopy channel, the LuViva also contains a separate colposcopy-quality imaging channel. The purpose of this imaging channel is two-fold: –1) to allow real-time imaging of the cervix for centration guidance while the cervical guide is being placed inside the vagina –2) to allow images to be taken in order to document placement of the device and appropriate centering of the cervix within the one-inch diameter measurement area. 7

8. OBJECTIVE To evaluate multimodal hyperspectroscopy (MHS) – LuViva®- for the early detection of cervical cancer in a population of women previously screened for cervical cancer. 8

9. METHODOLOGY One hundred women previously screened for cervical cancer using either VIA or Conventional cytology were enrolled in the study. –Each participant had sample for HPV DNA from the endocervix –MHS of the cervix using the LuViva Advanced Cervical Scan (Guided Therapeutics, Norcross, GA USA) was performed in a 1 minute procedure. –Biopsies for histology taken from the cervix, as gold standard, after application of acetic acid, from observed abnormal area and from the quadrants if no obvious abnormality is observed. 9

10. RESULTS Sixty-five of the 100 women in the study were referred to colposcopy on the basis of either an abnormal Pap result or a positive VIA. – VIA+ was able to identify seven CIN1 and five CIN2, but only one CIN3, which is the goal of screening. – Referral Pap at the LSIL threshold identified 5 of 10 CIN1 as positive, missed one CIN2 and identified 3 of 5 CIN3 as positive for a sensitivity of 60%. –At the CIN3 threshold, the yield of positive biopsies for VIA was 3% (1/36) and was 12% (3/25) for Pap at the LSIL threshold. 10

11. Table 1: Cytology and VIA+ results as a function of histopathology Histology Cytology NormalCIN 1CIN 2CIN 3 Negative2941135 ASC-US10012 AGC20002 LSIL1350220 ASC-H00000 HSIL31015 VIA +2375136 Total711766100 11

12. Results Positive HPV results were lower in number than expected based on the population of referred women. –Overall, 11% (11/98) of all women in the study were positive for high risk HPV. This included only one of six with CIN2 and three of six with CIN3 (sensitivity = 50%). –The reasons for this are not completely clear but could include: pre-, intra-, or post-analytical errors and/or degradation due to environmental conditions. Another possible explanation is that the average age of women in the study was 45 years. It is known that HPV infection rates can be much lower in women above the age of 40 and that the sensitivity of HPV is also lower in this age group. I –In support of this, we found that HPV false negatives were more likely to occur in older Nigerian women (median age 51.5 years) compared with women having true positive HPV results (median age 43.0 years), although sample sizes are small. 12

13. Table 2: HPV results as a function of histopathology HistopathologyHistology NormalCIN 1CIN 2CIN 3 HPV+5213 HPV-641553 HPV (No Result)2000 Total711766 13

14. Triage Performance Of the 100 women enrolled in the study, –24 qualified as LuViva triage patients based on referral Pap results of ASC-US/AGUS (n = 4) or LSIL (n = 20). –An additional five patients with HSIL Pap smear were tested, but HSIL is contra-indicated for LuViva due to the high likelihood of significant cervical disease. Of the 24 women with abnormal Pap smear of at least LSIL, – three were found to have CIN2+ at histopathology. All three recorded RED (high likelihood of CIN2+) by LuViva for a sensitivity of 100%. It is of interest that HPV was negative for all three of these patients (sensitivity = 0%). 14

15. Triage Performance Of the remaining 21 women without CIN2+ – five were found to have CIN1. –LuViva recorded four of these as RED and one as YELLOW (moderate likelihood of CIN2+). –Fifteen referred patients were found to have cervicitis (n = 9) or normal histology (n = 6). Of these 15 patients, five were recorded as GREEN (low likelihood of CIN2+) and two were recorded as YELLOW by LuViva. –The remaining eight were recorded as RED by LuViva. Therefore, the sensitivity and specificity of the triage patients were as follows: 15

16. Table 3: Sensitivity and Specificity of LuViva ® at the Green/Yellow Threshold Sensitivity CIN 2+ Sensitivity CIN 1 Specificity No dysplasia 100% (3/3)0% (0/5)33% (5/15) 16

17. Table 3: Sensitivity and Specificity of LuViva ® at the Yellow/Red Threshold Sensitivity CIN 2+ Sensitivity CIN 1 Specificity No dysplasia 100% (3/3)20% (1/5)40% (6/15) 17

18. CONCLUSIONS MHS would have reduced the percentage of unnecessary colposcopy and biopsy by 40% without any false negatives Also, MHS was able to differentiate between VIA+ and Pap negative women. These suggest that MHS may have potential of a point of care primary and immediate objective screening procedure especially in a setting with poor health seeking behaviour in the population. 18

19. References J. Ferlay, F. Bray, P. Pisani and D.M. Parkin., GLOBOCAN 2000: Cancer Incidence, Mortality and Prevalence Worldwide, Version 1.0, IARC CancerBase No. 5. Lyon, IARC Press, 2001. Cancer Facts & Figures 2002, American Cancer Society, p17. Fahey MT, Irwig L, and Macaskill P, Meta-analysis of Pap test accuracy. American Journal of Epidemiology 1995;141 (7): 680-689. Sherman et al, Effects of age and human Papilloma viral load on colposcopy triage: data from the randomized Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial lesion Triage Study (ALTS), J. Natl. Can. Inst., 2002. 94(2):102-7. Wright T, Cox T, Massad L, Twiggs L, Wilkinson E 2001 Consensus Guidelines for the Management of Women with Cervical Cytological Abnormalities, JAMA, April 2002, Vol 287, No. 16, 2120-2129. The ASCUS-LSIL Triage Study (ALTS Group). Results of a randomized trial on the management of cytology interpretations of atypical squamous cells of undetermined significance. Am. J. Obstet. Gyncol. 2003, 188; 6: 1383-1392. Massad, LS and Collins, YC. Strength of correlations between colposcopic impression and biopsy histology. Gynecol. Onc. 2003, 89: 424-428. 19