Non-invasive analysis of hormonal variations and effect of postmenopausal Vagifem treatment on women using in vivo high wavenumber confocal Raman spectroscopy.

This study aims to evaluate the feasibility of applying high wavenumber (HW) confocal Raman spectroscopy for non-invasive assessment of menopause-related hormonal changes in the cervix as well as for determining the effect of Vagifem(®) treatment on postmenopausal women with atrophic cervix. A rapid HW confocal Raman spectroscopy system coupled with a ball lens fiber-optic Raman probe was utilized for in vivo cervical tissue Raman measurements at 785 nm excitation. A total of 164 in vivo HW Raman spectra (premenopausal (n = 104), postmenopausal-prevagifem (n = 34), postmenopausal-postvagifem (n = 26)) were measured from the normal cervix of 26 patients undergoing colposcopy. We established the biochemical basis of premenopausal, postmenopausal-prevagifem and postmenopausal-postvagifem cervix using semiquantitative biomolecular modeling derived from Raman-active biochemicals (i.e., lipids, proteins and water) that play a critical role in HW Raman spectral changes associated with the menopausal process. The diagnostic algorithms developed based on partial least squares-discriminant analysis (PLS-DA) together with leave-one patient-out, cross-validation yielded the diagnostic sensitivities of 88.5%, 91.2% and 88.5%, and specificities of 91.7%, 90.8% and 99.3%, respectively, for non-invasive in vivo discrimination among premenopausal, postmenopausal-prevagifem and postmenopausal-postvagifem cervix. This work demonstrates for the first time that HW confocal Raman spectroscopy in conjunction with biomolecular modeling can be a powerful diagnostic tool for identifying hormone/menopause-related variations in the native squamous epithelium of normal cervix, as well as for assessing the effect of Vagifem treatment on postmenopausal atrophic cervix in vivo during clinical colposcopic inspections.

Simultaneous fingerprint and high-wavenumber confocal Raman spectroscopy enhances early detection of cervical precancer in vivo.

Raman spectroscopy is a vibrational spectroscopic technique capable of nondestructively probing endogenous biomolecules and their changes associated with dysplastic transformation in the tissue. The main objectives of this study are (i) to develop a simultaneous fingerprint (FP) and high-wavenumber (HW) confocal Raman spectroscopy and (ii) to investigate its diagnostic utility for improving in vivo diagnosis of cervical precancer (dysplasia). We have successfully developed an integrated FP/HW confocal Raman diagnostic system with a ball-lens Raman probe for simultaneous acquistion of FP/HW Raman signals of the cervix in vivo within 1 s. A total of 476 in vivo FP/HW Raman spectra (356 normal and 120 precancer) are acquired from 44 patients at clinical colposcopy. The distinctive Raman spectral differences between normal and dysplastic cervical tissue are observed at ~854, 937, 1001, 1095, 1253, 1313, 1445, 1654, 2946, and 3400 cm(-1) mainly related to proteins, lipids, glycogen, nucleic acids and water content in tissue. Multivariate diagnostic algorithms developed based on partial least-squares-discriminant analysis (PLS-DA) together with the leave-one-patient-out, cross-validation yield the diagnostic sensitivities of 84.2%, 76.7%, and 85.0%, respectively; specificities of 78.9%, 73.3%, and 81.7%, respectively; and overall diagnostic accuracies of 80.3%, 74.2%, and 82.6%, respectively, using FP, HW, and integrated FP/HW Raman spectroscopic techniques for in vivo diagnosis of cervical precancer. Receiver operating characteristic (ROC) analysis further confirms the best performance of the integrated FP/HW confocal Raman technique, compared to FP or HW Raman spectroscopy alone. This work demonstrates, for the first time, that the simultaneous FP/HW confocal Raman spectroscopy has the potential to be a clinically powerful tool for improving early diagnosis and detection of cervical precancer in vivo during clinical colposcopic examination.

In vivo diagnosis of cervical precancer using Raman spectroscopy and genetic algorithm techniques.

This study aimed to evaluate the clinical utility of applying near-infrared (NIR) Raman spectroscopy and genetic algorithm-partial least squares-discriminant analysis (GA-PLS-DA) to identify biomolecular changes of cervical tissues associated with dysplastic transformation during colposcopic examination. A total of 105 in vivo Raman spectra were measured from 57 cervical sites (35 normal and 22 precancer sites) of 29 patients recruited, in which 65 spectra were from normal sites, while 40 spectra were from cervical precancerous lesions (i.e., 7 low-grade CIN and 33 high-grade CIN). The GA feature selection technique incorporated with PLS was utilized to study the significant biochemical Raman bands for differentiation between normal and precancer cervical tissues. The GA-PLS-DA algorithm with double cross-validation (dCV) identified seven diagnostically significant Raman bands in the ranges of 925-935, 979-999, 1080-1090, 1240-1260, 1320-1340, 1400-1420, and 1625-1645 cm(-1) related to proteins, nucleic acids and lipids in tissue, and yielded a diagnostic accuracy of 82.9% (sensitivity of 72.5% (29/40) and specificity of 89.2% (58/65)) for precancer detection. The results of this exploratory study suggest that Raman spectroscopy in conjunction with GA-PLS-DA and dCV methods has the potential to provide clinically significant discrimination between normal and precancer cervical tissues at the molecular level.

The association with age, human tissue kallikreins 6 and 10 and hemostatic markers for survival outcome from epithelial ovarian cancer.

PURPOSE: To assess known cancer biomarkers CA-125, human tissue kallikreins KLK6 and KLK10, hemostatic markers and age with 5-year survival outcome from epithelial ovarian cancer. METHODS: Forty-one benign cyst cohorts and 83 patients diagnosed with ovarian cancer were recruited. The following assays were performed: fibrinogen, vWF antigen, D: -dimer, ATIII activity, tPA, PAI-1, uPAR, KLK6, KLK10 and CA-125. Follow-up visits of cancer patients of more than 60 months were noted. Data between those who survived past 60 months and mortality from cancer were analyzed. RESULTS: Only 24 patients lived past 60 months, and 31 died (advanced stage n = 27). Those living past 60 months were significantly older and associated with similar pre-operative levels seen in benign cyst cohorts especially for KLK6, fibrinogen, vWF, AT levels despite upregulation of D: -dimer, CA-125 and KLK10. Ovarian cancer cohorts living past 60 months were younger than those who died within 12 months (n = 12). Mortality within 12 months was associated with older age, upregulation of KLK6, fibrinogen, D: -dimer, vWF, tPA antigen and reduced ATIII levels. Similarly, mortality within 36 months of disease showed older age with upregulation of CA-125, KLK6 D: -dimer vWF antigen and tPA antigen levels. Late stage cancer (III/IV) showed upregulated CA-125, KLK6, KLK10, D: -dimer and reduced AT compared to early stage cancer (I/II). The 5-year survival rate for early cancer was 80%, advanced 22.9% and overall 5-year survival rate was 43.6%. CONCLUSION: Older age together with the novel biomarkers studied and their association with adverse outcome from epithelial ovarian cancer was seen especially within 12 and 36 months of disease. Those who lived past 60 months of disease showed similar pre-operative levels seen in benign cyst cohorts despite elevated D: -dimer, CA125 and KLK 10. An enlarged study is needed to confirm these findings.

Cervical dysplasia: assessing methylation status (Methylight) of CCNA1, DAPK1, HS3ST2, PAX1 and TFPI2 to improve diagnostic accuracy.

PURPOSE: Diagnosis of cervical neoplasia hinges upon microscopic inspection of cervical samples. This has inherent operator-dependent variability. Testing for high-risk human papilloma virus (HPV) may help to triage patients with pre-invasive disease in determining clinical intervention and follow-up. However, HPV presence/absence does not reflect the cervical epithelial cell's molecular status. Epigenetic modifications, e.g. DNA methylation, have been observed in the early stages of neoplastic change, preceding gene mutations. Here, we assess the correlation between cytologic/histologic results and combined DNA methylation data of 5 genes in different grades of cervical dysplasia. EXPERIMENTAL DESIGN: Cervical specimens collected via the liquid-based cytology system were each microscopically examined. Residual cells were subjected to DNA methylation analysis (Methylight) of gene loci CCNA1, PAX1, HS3ST2, DAPK1 and TFPI2. Methylation data were compared with cytologic/histologic reports. Statistical methods were applied to assess the ability of DNA methylation status to subtype the cervical neoplastic lesions according to their corresponding cytologic/histologic reports. RESULTS: A total of 165 subjects provided cytologically proven 63 HSIL, 49 LSIL and 53 normal samples. All patients with HSIL and LSIL underwent colposcopic examination. Patients with LSIL were all found to be CIN1; patients with HSIL were subsequently subdivided into 10 squamous cell carcinoma (SCC), 31 CIN3, 10 CIN2 and 12 CIN1. For each gene, there was increasing frequency of methylation from normal and LSIL (CIN1), through HSIL (CIN2 and CIN3), to SCC. Methylation of ≥1 of genes investigated was observed in 88% of combined HSIL (CIN2 and CIN3) and SCC cases. All genes showed significant increase in methylation level (PMR value) with increasing disease grade (p<0.005). CCNA1 was the only gene that was able to distinguish CIN2 from CIN3 specimens (p=0.016). Based on receiver operating characteristic (ROC) analysis, HS3ST2 was the most significant candidate in segregating HSIL/SCC from normal/LSIL cases (p<0.0001); at an optimal cutoff value, sensitivity and specificity between 70% and 80% were obtained. CONCLUSIONS: Development of DNA methylation status of a gene panel to improve diagnostic accuracy in cervical neoplasia is warranted.

High wavenumber Raman spectroscopy for in vivo detection of cervical dysplasia.

Raman spectroscopy is a vibrational spectroscopic technique capable of optically probing the biomolecular changes associated with neoplastic transformation. The purpose of this study was to apply near-infrared (NIR) Raman spectroscopy in the high wavenumber (HW) region (2800-3700 cm(-1)) for in vivo detection of cervical dysplasia. A rapid-acquisition NIR Raman spectroscopy system associated with a ball-lens fiber-optic Raman probe was developed for in vivo spectroscopic measurements at 785 nm excitation. A total of 92 in vivo HW Raman spectra (46 normal, 46 dysplasia) were acquired from 46 patients with Pap smear abnormalities of the cervix. Significant difference in Raman intensities of prominent Raman bands at 2850 and 2885 cm(-1) (CH(2) stretching of lipids), 2940 cm(-1) (CH(3) stretching of proteins), and the broad Raman band of water (peaking at 3400 cm(-1) in the 3100-3700 cm(-1) range) were observed in normal and dysplasia cervical tissue. The diagnostic algorithms based on principal components analysis and linear discriminant analysis together with the leave-one-patient-out cross-validation method on in vivo HW Raman spectra yielded a diagnostic sensitivity of 93.5% and specificity of 97.8% for dysplasia tissue identification. This study demonstrates for the first time that HW Raman spectroscopy has the potential for the noninvasive, in vivo diagnosis and detection of precancer of the cervix.

Tumor molecular profiling of responders and non-responders following pembrolizumab monotherapy in chemotherapy resistant advanced cervical cancer.

Optimal treatment for advanced cervical cancer after first line chemotherapy remains undefined. Immune checkpoint inhibition with pembrolizumab, a programmed cell death protein 1(PD-1) inhibitor, is under investigation. We analyzed the micro-environmental and molecular genetic profile of tumors from 4 patients with metastatic cervical cancer treated with off-label second-line pembrolizumab in an effort to identify predictive biomarkers. All patients received 2 mg/kg of pembrolizumab, 3-weekly until disease progression. Immunohistochemistry(IHC) for PD-1, PD-L1, CD3 and CD8, as well as next generation sequencing (NGS) for 50 cancer-related genes were performed on tumor samples. All patients tolerated treatment well with no discontinuation of treatment due to toxicity. One patient experienced dramatic and prolonged partial response, and remains stable on pembrolizumab with a progression free survival (PFS) of 21 months at the time of reporting of this series. Three patients experienced disease progression as best response. In the exceptional responder, there was no tumoral expression of PD-L1, however, combined positive score (CPS) for PD-L1 was 1 and we identified somatic mutations in ERBB4(R612W), PIK3CA(E542K) and RB1(E365K). In 2 patients, despite progressive disease defined by RECIST v1.1, symptom stabilization on pembrolizumab was observed. The tumors of both patients had PD-1 expression in ≥1% of stromal lymphocytes. All patients with response or clinical benefit had CPS for PD-L1 ≥ 1. NGS revealed PIK3CA mutations in 3 tumors. Pembrolizumab is a promising therapeutic option in advanced cervical cancer. Further evaluation of biomarkers may guide optimal patient selection.

The association between fibrinogen, von Willebrand Factor, antithrombin III, and D-dimer levels and survival outcome by 36 months from ovarian cancer.

Thirty-five patients diagnosed to have ovarian cancer (early FIGO stage I/II n = 11, advanced FIGO stage III/IV, n = 24) were evaluated for hemostatic parameters relating to survival outcome by 36 months of disease. Systemic plasminogen activators and inhibitors were evaluated and we found no significant association with survival outcome and eventually only fibrinogen, von Willebrand Factor (vWF), antithrombin III (ATIII), and D-dimer levels were determined for their association with disease outcome by 12 months, 24 months, and 36 months. Twenty-four patients succumbed to the disease by 36 months (early n = 2, advanced n = 22). The 11 surviving patients (advanced n = 3, including one deceased at 52 months) is still living past 36 months and 82 months at the time of analysis. Elevated fibrinogen, vWF, and D-dimer together with reduced ATIII levels were found to be associated with poor survival outcome by 12 months of disease. Moreover, elevated vWF and D-dimer with reduced ATIII levels was strongly implicated with poor survival outcome by 36 months from ovarian cancer. The overall survival rate at 36 months from ovarian cancer was 31.4%. It is therefore suggested that fibrinogen, vWF, ATIII, and D-dimer levels be used together as prognostic markers for disease outcome especially in patients with advanced ovarian cancer within 36 months of disease. An expanded study is required to confirm these findings.

Termination of early pregnancy (menstrual induction) with 16-phenoxy-omega-tetranor PGE2 methylsulfonylamide.

PIP: The efficacy and side effects of a new synthetic compound, 16-phenoxy-W-17, 18, 19, 20-tetranor prostaglandin E2 methylsulfonylamide, for menstrual induction. The study was conducted in an outpatient clinic in 240 patients with a delay in menstruation of 6-14 days. In 206 patients (86%) the immunologic pregnancy test was positive before prostaglandin administration. Most of the remaining 34 (14%) patients with a negative pregnancy test had symptoms suggestive of pregnancy. Whenever there was doubt that amenorrhea was due to reasons other than pregnancy, the patients were not treated with prostaglandins. The patients' age range was 16-32 years; the parity range was 0-6. The treatment was successful in 228 patients (95%), i.e., there was uterine bleeding after prostaglandin administration followed by a negative pregnancy test within 14 days. In 6 patients with failed treatment, there was uterine bleeding after prostaglandin administration lasting for 2-13 days but the pregnancy remained positive. Prostaglandin failed to induce bleeding in 4 patients. In 3 of the patients the pregnancy test was positive before and 14 days after prostaglandin administration. The uterus was evacuated by vacuum aspiration in 9 patients. In 1 patient who had no uterine bleeding, pregnancy test was negative both before and 2 weeks after prostaglandin administration. 2 patients were admitted to the hospital the day after prostaglandin treatment for excessive bleeding. Curettage was carried out in both these patients and the bleeding stopped. 1 patient was readmitted to the hospital 8 hours after prostaglandin administration with severe pain in the lower abdominal region. On further examination a tubal pregnancy was diagnosed and confirmed by laparotomy. 2 patients were successfully treated for pelvic infection with antibiotics 7-10 days after prostaglandin administration. All patients experienced mild uterine pain a few minutes after prostaglandin administration. 16 patients required analgesics. The majority of the patients described the bleeding as heavier and longer in duration than their normal menstrual period. The average length of bleeding was 7.3 days with a range of 2-13 days. Side effects included 1 or 2 episodes of vomiting in 13 patients and headache in 9 patients.^ieng

The role of surgery in the treatment of trophoblastic disease.

Over 600 of the 850 cases of gestational trophoblastic disease that have been treated in our department during the last 20 years were reviewed to determine the role of surgery in the management of the disease. The surgical procedures employed included hysterectomy, both as a prophylactic and definitive procedure, thoracotomy, excision of vaginal nodules and neurosurgical removal of cerebral secondaries. It was found that removal of a large tumor mass improved the response to subsequent chemotherapy. Removal of vaginal nodules usually was not necessary unless there was hemorrhage. Chemotherapy with precise hormonal monitoring remains the most important means of treating choriocarcinoma.

Maternal death associated with hydatidiform molar pregnancy.

Three maternal deaths with hydatidiform molar pregnancies associated with severe hemorrhage and coagulopathy are described. Symptoms of coagulation abnormalities are few in mild degrees of intravascular coagulation. This paper addresses the importance of anticipating a coagulopathy in hydatidiform molar pregnancy even before evacuation of the abnormal conceptus since evacuation aggravates fibrinolytic activity and therefore increases hemorrhage in these conditions.

The 'Pap' or cervical smear and the role of colposcopy in screening for carcinoma of the cervix.

Cervical cancer is the commonest female genital tract cancer in Singaporean women with an annual age-standardized rate of 17.4 per 100,000. High risk factors are early sexual intercourse, multiple sexual partners and cigarette smoking. Population screening with annual cervical (Pap) smears after beginning sexual activity until age 35 and at 5 year intervals after that can reduce both incidence and mortality rate from invasive cervical cancer. Benign, premalignant and malignant conditions may be identified in smears. The term cervical intra-epithelial neoplasia (CIN) reflects better the continuum of change in precursor lesions and is preferred over the older dual terminology of dysplasia/carcinoma-in-situ for precursors of cervical cancer. Colposcopy is essential for evaluation of all patients with abnormal cervical smears. Colposcopy is used to identify the site, severity and extent of abnormality as well as to aid directed biopsy, plan treatment and allow use of conservative methods to treat the precursor lesions. Colposcopy however, has no role as a primary screening procedure for cervical cancer but instead cervical smears are used for screening.

Update on molar pregnancy and choriocarcinoma.

The term gestational trophoblastic disease encompasses a wide spectrum of disorders ranging from the benign hydatidiform mole to the malignant choriocarcinoma and placental site trophoblastic tumor. Recent advances in cytogenetics and pathological criteria have brought to light the occurrence of the partial mole, which is probably more common than the complete mole. The World Health Organisation has proposed a classification to rectify the current confusion that exists with clinical and pathological terms. Evacuation of the uterus followed by meticulous followup with sensitive beta subunit Human Chorionic Gonadotropin assay remain the cornerstone of management of molar pregnancy. Prompt chemotherapy is the most important aspect in managing malignant sequelae. Newer chemotherapy regimes have helped to salvage failures from the usual drugs. Judicial use of surgery in metastatic trophoblastic disease can reduce the duration and toxicity of chemotherapy. All patients with gestational trophoblastic disease should be managed in a tertiary care centre with the expertise and facilities easily available to manage these cases.

Optimal treatment in gestational trophoblastic disease.

Gestational trophoblastic diseases are a heterogenous group of conditions ranging from the benign hydatidiform mole to the malignant choriocarcinoma. Optimal therapy in this group of diseases rest in the correct diagnosis, assessing their risk for malignant behavior using prognostic scoring systems and administering appropriate treatment. Their rarity makes it imperative that these patients are treated in special centres by experts. Benign moles are treated surgically with evacuation of the uterus or hysterectomy. In malignant gestational trophoblastic disease, chemotherapy is the treatment of choice; single agent for non-metastatic and low-risk metastatic disease and combination chemotherapy for high-risk metastatic disease. Judicious use of surgery and radiotherapy in these cases will improve the survival rate. With appropriate treatment, the cure rates approach 100% in the low-risk group and 80% to 85% in the high risk group.

Recent advances in the management of gestational trophoblastic disease.

What was once an almost uniformly fatal disease has now become highly curable, with survival rates of 100% being reported for the early stages of the disease. This is the direct result of improved techniques of diagnosis, recognition of high risk groups, effective and intensive chemotherapy, adjunctive surgery in selected cases and most important of all, a close follow-up of all patients with monitoring of the tumour marker, human chorionic gonadotrophin (hCG). This review will highlight the advances made in these areas and how these have helped in reducing morbidity and mortality from gestational trophoblastic disease (GTD).

Induction of labour with prostaglandin E2 and amniotomy--a multicentre, three dose schedule study in 1533 patients.

Prostaglandin E2 administered orally and combined with amniotomy was used for induction of labour at or near term in 1533 patients. The study was carried out as a collaborative project between the University Departments of Obstetrics and Gynaecology in Singapore, Medan (Indonesia) and Kuala Lumpur (Malaysia). The overall success rates for Medan, Kuala Lumpur and Singapore were 95.6%, 88.1% and 84.7% respectively.

Epithelial ovarian cancer.

The incidence of epithelial ovarian cancer in women aged 40 years and younger is 3-17%. The management of these women is challenging and requires balancing the need to treat epithelial ovarian cancer adequately and preserving reproductive potential. Fertility-sparing surgery, especially for early stage epithelial ovarian cancer, seems to be associated with equivalent clinical and cancer outcomes while preserving reproductive potential. A complete staging and cytoreductive procedure retaining the uterus, and at least one grossly normal ovary, is the minimum recommended procedure. Adjuvant chemotherapy with a platinum-taxane combination is recommended as clinically indicated, and is associated with better cancer and survival outcomes. Adjuvant treatment does not seem to increase the risk of congenital anomalies in subsequent pregnancies. Targeted therapy and ovarian cryopreservation are largely experimental and cannot be recommended as part of the clinical standard of care.