Clinical use of an opto-nuclear probe for hybrid sentinel node biopsy guidance: first results.

INTRODUCTION: Guidelines advocate the use of combined detection techniques to achieve optimal results for sentinel node (SN) biopsy. The fluorescent and radioactive (dual-) tracer ICG-99mTc-nanocolloid has been shown to facilitate SN biopsy in several indications. It was reported that an opto-nuclear probe permitted the detection of near-infrared fluorescence and gamma-rays. The aim of the current study was to evaluate this device in a large patient group and to test it in both open and laparoscopic surgery implications. METHODS: Thirty-three patients scheduled for SN biopsy with the dual-tracer were retrospectively analyzed. Pre-operative lymphoscintigraphy was performed in all patients; in 18 patients (55%), a SPECT/CT scan was also performed. Radioactive and fluorescent signatures in the SNs were assessed in vivo and ex vivo using the opto-nuclear probe. RESULTS: One or more SNs were identified in all patients (identification rate 100%). Planar lymphoscintigraphic images revealed 95 hot spots that were considered as SNs. This number increased to 103 SNs when SPECT/CT was used. During surgery, 106 SNs were excised. In vivo, the fluorescence mode of the opto-nuclear probe was able to locate 79 SNs (74.5%). When the gamma-ray detection option of the same probe was used, this number increased to 99 SNs (93.3%). Ex vivo analysis revealed fluorescence in 93.3% of the excised nodes and radioactivity in 95.2%. CONCLUSIONS: This study underlines the feasibility of using the dual-tracer/opto-nuclear probe combination for SN resections. The use of the opto-nuclear technology has been extended to laparoscopic surgery. This study also underlines the fluorescence tracing can complement traditional radio-tracing approaches.

MicroRNA-200, associated with metastatic breast cancer, promotes traits of mammary luminal progenitor cells.

MicroRNAs are critical regulators of gene networks in normal and abnormal biological processes. Focusing on invasive ductal breast cancer (IDC), we have found dysregulated expression in tumor samples of several microRNAs, including the miR-200 family, along progression from primary tumors to distant metastases, further reflected in higher blood levels of miR-200b and miR-7 in IDC patients with regional or distant metastases relative to patients with primary node-negative tumors. Forced expression of miR-200s in MCF10CA1h mammary cells induced an enhanced epithelial program, aldehyde dehydrogenase (ALDH) activity, mammosphere growth and ability to form branched tubuloalveolar structures while promoting orthotopic tumor growth and lung colonization in vivo. MiR-200s also induced the constitutive activation of the PI3K-Akt signaling through downregulation of PTEN, and the enhanced mammosphere growth and ALDH activity induced in MCF10CA1h cells by miR-200s required the activation of this signaling pathway. Interestingly, the morphology of tumors formed in vivo by cells expressing miR-200s was reminiscent of metaplastic breast cancer (MBC). Indeed, the epithelial components of MBC samples expressed significantly higher levels of miR-200s than their mesenchymal components and displayed a marker profile compatible with luminal progenitor cells. We propose that microRNAs of the miR-200 family promote traits of highly proliferative breast luminal progenitor cells, thereby exacerbating the growth and metastatic properties of transformed mammary epithelial cells.

Limitations in predicting PAM50 intrinsic subtype and risk of relapse score with Ki67 in estrogen receptor-positive HER2-negative breast cancer.

PAM50/Prosigna gene expression-based assay identifies three categorical risk of relapse groups (ROR-low, ROR-intermediate and ROR-high) in post-menopausal patients with estrogen receptor estrogen receptor-positive (ER+)/ HER2-negative (HER2-) early breast cancer. Low risk patients might not need adjuvant chemotherapy since their risk of distant relapse at 10-years is below 10% with endocrine therapy only. In this study, 517 consecutive patients with ER+/HER2- and node-negative disease were evaluated for Ki67 and Prosigna. Most of Luminal A tumors (65.6%) and ROR-low tumors (70.9%) had low Ki67 values (0-10%); however, the percentage of patients with ROR-medium or ROR-high disease within the Ki67 0-10% group was 42.7% (with tumor sizes ≤2 cm) and 33.9% (with tumor sizes > 2 cm). Finally, we found that the optimal Ki67 cutoff for identifying Luminal A or ROR-low tumors was 14%. Ki67 as a surrogate biomarker in identifying Prosigna low-risk outcome patients or Luminal A disease in the clinical setting is unreliable. In the absence of a well-validated prognostic gene expression-based assay, the optimal Ki67 cutoff for identifying low-risk outcome patients or Luminal A disease remains at 14%.

Laparoscopic lymphadenectomy in advanced cervical cancer: prognostic and therapeutic value.

OBJECTIVE: Clinical benefit of surgical staging in locally advanced cervical cancer has not yet been proved. The goal of this study was to analyze the prognostic and therapeutic value of laparoscopic para-aortic lymphadenectomy with selective excision of suspicious pelvic nodes in patients with locally advanced cervical cancer. METHODS: This is a retrospective study including 109 women treated in a single institution from 2000 to 2009. The International Federation of Gynecology and Obstetrics stage was IB2 in 12 women, IIB in 58 women, and IIIB in 39 women. None had suspicious para-aortic nodes by presurgical imaging evaluation. All patients underwent extraperitoneal para-aortic laparoscopic lymphadenectomy with selective excision of enlarged pelvic nodes and received pelvic radiotherapy with concomitant chemotherapy. Extended lumboaortic radiation therapy was added to patients with metastatic para-aortic nodes. The mean ± SD follow-up time was 43.1 ± 33.7 months. RESULTS: Metastatic lymph nodes were identified in 23 (21.1%) of 109 patients in the para-aortic area and in 24 (53.3%) of 45 patients who underwent selective excision of pelvic nodes. Patients with nodal metastases had increased risk of mortality than those with negative nodes independently of the location (pelvic and/or para-aortic) of the metastases (hazard ratio, 4.07; 95% confidence interval, 1.36-12.16 for patients with pelvic metastases [P = 0.012]; and 3.73; 95% confidence interval, 1.38-10.09 for patients with para-aortic metastases [P = 0.010]). In the subset of women with para-aortic metastases treated by extended lumboaortic radiation therapy, neither the number of lymph nodes removed nor the number of positive nodes were associated with survival (P = 0.556 and P = 0.195, respectively). CONCLUSION: Para-aortic and pelvic lymphadenectomy provides valuable information about mortality risk in patients with locally advanced cervical cancer.

Predictors of absence of cervical intraepithelial neoplasia in the conization specimen.

OBJECTIVE: Over 10% of women who undergo conization for cervical intraepithelial neoplasia (CIN) show no lesion in the surgical specimen. We aimed to determine whether these patients can be identified before conization using clinical, virological and/or cyto-histological characteristics, to avoid unnecessary treatment. METHODS: Of 687 women with CIN treated by conization in the Hospital Clinic of Barcelona between 2008 and 2011, all patients (n=110, 16%) showing no lesion in the surgical specimen were included as the study group. The control group included a series of randomly selected women with CIN in the cone specimen (n=220). Pre-conization clinical characteristics as well as high-risk human papillomavirus (hr-HPV) status determined by Hybrid Capture 2 were analyzed as possible predictors of absence of lesion. RESULTS: A negative pre-conization hr-HPV test or a low viral load (<10 relative light units) significantly increased the probability of absence of CIN in the conization specimen (75.0%, and 52% respectively) compared with patients with a high viral load (26.7%, p<0.001). This association was confirmed in the multivariate analysis (p<0.001). The risk of developing persistent/recurrent disease after treatment was significantly lower in patients with negative hr-HPV test or a low viral load (16.1% CIN1, 0% CIN2-3), than in patients with a high viral load (27.6% CIN1, 4.1% CIN2-3, p=0.031). CONCLUSION: Women with negative pre-conization hr-HPV test results or a low viral load have a high probability of having no lesion in the conization specimen. These patients should be excluded from immediate surgical excision and considered for follow-up.

Human papillomavirus as a favorable prognostic biomarker in squamous cell carcinomas of the vagina.

OBJECTIVE: Recent evidence has confirmed two independent pathways in the development of vaginal squamous cell carcinoma (VaSCC): one related to and the other independent of human papillomavirus (HPV). The aim of our study was to evaluate whether HPV status has prognostic significance in this neoplasm. METHODS: All confirmed primary VaSCCs diagnosed and treated from 1995 to 2009 in two institutions were retrospectively evaluated (n=57). HPV infection was detected by PCR using SPF-10 primers and typed with the INNO-LIPA HPV assay and p16(INK4a) expression by immunohistochemistry. Disease-free and overall survival (DFS and OS) were analyzed by Kaplan-Meier analysis with the log-rank test and a multivariate Cox proportional hazard's model. RESULTS: HR-HPV DNA was detected in 70.2% patients. HPV16 was the most prevalent genotype (67.5% of cases). p16(INK4a) was positive in 97.5% HPV-positive and 17.6% HPV-negative tumors (p<.001). FIGO stage was associated with DFS (p=.042) and OS (p=.008). HPV-positive tumors showed better DFS (p=.042) and OS (p=.035) than HPV-negative tumors. Multivariate analysis confirmed better DFS and OS of HPV-positive patients independent of age and stage. This reduced risk of progression and mortality in HPV-positive patients was limited to women with FIGO stages I and II tumors (HR=0.26; 95% CI 0.10-0.69; p=0.006). CONCLUSIONS: HPV-positive early stage (FIGO I and II) VaSCCs have a better prognosis than early HPV-negative tumors. HPV detection and/or p16(INK4a) immunostaining can be easily implemented in routine pathology and should be considered as valuable prognostic biomarkers in the study of patients with VaSCC.

Clinical, colposcopic and pathological characteristics of cervical and vaginal high-grade lesions negative for HPV by Hybrid Capture 2.

OBJECTIVE: Less than 5% of women with cervical or vaginal biopsy proven high-grade squamous intraepithelial lesions (HG-SIL) show a negative Hybrid Capture 2 (HC2) result. We analyzed 1) human papillomavirus (HPV) genotypes by PCR in order to determine whether these cases represent infections by common or unusual types, and 2) the clinical, colposcopic and pathological differential characteristics of these patients. METHODS: 646 women with a histological diagnosis of HG-SIL and a HC2 test collected within 6 months prior to the diagnosis were identified. Patients with a negative HC2 result were selected. HPV was typed in the biopsy specimen in all by PCR using SPF10 and GP5+/6+ primers, and p16(INK4a) immunostaining was performed. The clinical and colposcopy findings of these women were compared with a control group of HG-SIL with positive HC2 result. RESULTS: 20 women (3.1%) with HG-SIL had a negative HC2. All biopsies were positive for p16(INK4). PCR analysis detected HPV types included in HC2 test in 55% of the cases, with an identical percentage of common viruses between women with relative light unit values above or below 0.40 (p=.361). False negative HC2 tests increased with age (p=.002) and were more frequent in patients with non satisfactory colposcopy or small sized lesions (p<.001). CONCLUSION: A negative HC2 test is an infrequent event in women with HG-SIL. Common HPV types are identified in over half of the cases. Older women and patients with small lesions or non satisfactory colposcopy have a higher rate of HC2 negative results.

Does human papillomavirus infection imply a different prognosis in vulvar squamous cell carcinoma?

BACKGROUND: Two independent pathways in the development of vulvar squamous cell carcinoma (VSCC) have been described, one related to and the other independent of high-risk human papillomavirus (HR-HPV). The aim of our study was to evaluate whether the HPV status has a prognostic significance or can predict response to radiotherapy. METHODS: All VSCC diagnosed from 1995 to 2009 were retrospectively evaluated (n=98). HPV infection was detected by amplification of HPV DNA by PCR using SPF-10 primers and typed by the INNO-LIPA HPV research assay. p16(INK4a) expression was determined by immunohistochemistry. Disease-free and overall survival (DFS and OS) were estimated by Kaplan-Meier analysis with the log-rank test and a multivariate Cox proportional hazard's model. RESULTS: HR-HPV DNA was detected in 19.4% of patients. HPV16 was the most prevalent genotype (73.7% of cases). p16(INK4a) stained 100% HPV-positive and 1.3% HPV-negative tumors (p<.001). No differences were found between HPV-positive and -negative tumors in terms of either DFS (39.8% vs. 49.8% at 5 years; p=.831), or OS (67.2% vs. 71.4% at 5 years; p=.791). No differences in survival were observed between HPV-positive and -negative patients requiring radiotherapy (hazard ratio [HR] 1.04, 95% confidence interval [CI] .45 to 2.41). FIGO stages III-IV (p=.002), lymph node metastasis (p=.030), size ≥ 20 mm (p=.023), invasion depth (p=.020) and ulceration (p=.032) were associated with increased mortality but in multivariated only lymph node metastasis retained the association (HR 13.28, 95% CI 1.19 to 148.61). CONCLUSIONS: HPV-positive and -negative VSCCs have a similar prognosis. Radiotherapy does not increase survival in HPV-positive women.

Colposcopy prediction of progression in human papillomavirus infections with minor cervical lesions.

OBJECTIVES: To evaluate the risk of progression to cervical intraepithelial neoplasia (CIN) grade 2 or 3 in women with positive human papillomavirus (HPV) testing and low-grade (low-grade squamous intraepithelial lesions), borderline (atypical squamous cells of undetermined significance), or no cervical lesions, and to determine the accuracy of initial colposcopy to predict progression. METHODS: Women with HPV infection and low-grade squamous intraepithelial lesions, atypical squamous cells, or normal cytology were recruited and grouped according to cytologic or histologic diagnosis. Exclusion criteria were histologic CIN 2 or 3, previous cervical cancer and HPV infection, cervical disease, or treatment for CIN 2 or 3 in the past 3 years. Four-hundred sixty-five women were included and monitored by cytology, Hybrid Capture-2 test, and colposcopy every 6 months. Colposcopy results were described as normal, with minor or major changes, and lesion size was recorded in quadrants. RESULTS: Forty-three women (9.3%) had progression to CIN 2 or 3. No significant differences were found in rate of progression between women with low-grade squamous intraepithelial lesions, atypical squamous cells, or negative results (8.2%, 13.4%, and 9.8%, respectively; P=.679). Neither colposcopy pattern (P=.284) nor lesion size (P=.170) at recruitment provided any information on the risk of progression. History of cervical lesion and worsening of the colposcopy pattern during follow-up were associated with progression (P<.001). CONCLUSION: Initial colposcopy findings do not provide relevant information on the risk of progression in HPV-positive women with minor or no cervical lesions. These women have a similar risk of progression and should benefit from the same follow-up strategies.

Tumor fibroso solitario de la vulva / Solitary fibrous tumours of the vulva

El tumor fibroso solitario (TFS) es una tumoración mesenquimal poco frecuente identificada inicialmente en la pleura, pero que puede presentarse en prácticamente todas las regiones. En los últimos años, se han descrito algunos casos aislados originados en el tracto genital femenino. Presentamos el caso de una mujer de 35 años de edad que consultó por una tumoración vulvar, a la que se le practicó exéresis quirúrgica. En el examen anatomopatológico la tumoración medía 20mm de diámetro, presentaba características típicas de TFS y estaba constituida por una proliferación fusocelular sin atipias, con áreas alternas hiper ehipocelulares, abundante colágena y un patrón vascular hemangiopericitomatoso. En la inmunohistoquímica era intensamente positiva paraCD34 y bcl-2 y negativa para S-100 y actina. El TFS presenta un comportamiento agresivo en alrededor de un 25% de los casos y debe considerarse siempre un tumor potencialmente maligno, por lo que es preciso un seguimiento clínico. Por tanto, su correcta identificación y diferenciación de otras lesiones del área genital femenina con características similares son de importancia crucial (AU)

Solitary fibrous tumour (SFT) is a rare mesenchymal tumour initially identified in the pleura, but that can be present in virtually all regions. Isolated cases arising in the female genital tract have been described in recent years. We report the case of a woman aged 35who presented with a vulvar tumour, which was resected. On pathological examination thet umour measured 20 mm in diameter and showed typical features of SFT, consisting of spindle cell proliferation without atypia, alternating with hyper- and hypo-cellular areas, presence of abundant collagen and with avascular hemangiopericy tomatous pattern. Immunohistochemistry was strongly positive forCD34 and bcl-2 and negative for S-100 and actin. SFT shows aggressive behaviour in approximately 25% of cases and should always be considered a potentially malignant tumour fusiforand requires a clinical follow-up. Therefore, the identification and differentiation from other lesions with similar features in the female genital area is essential (AU)

HPV-negative vulvar intraepithelial neoplasia (VIN) with basaloid histologic pattern: an unrecognized variant of simplex (differentiated) VIN.

Vulvar intraepithelial neoplasia (VIN) is classified into 2 clinicopathologic subtypes, classic, related to human papillomavirus (HPV) infection and affecting relatively young women, and simplex (differentiated), negative for HPV and affecting elderly women. Histologically, classic VIN may be basaloid and characterized by a replacement of the whole epidermis by a homogeneous population of small, "undifferentiated" keratinocytes, which are diffusely positive for p16(INK4a) and negative for p53. Simplex VIN is characterized by atypia of the basal layer with high degree of cellular differentiation and shows negative staining for p16(INK4a) and frequent positivity for p53. Simplex VIN is frequently associated with squamous cell hyperplasia and lichen sclerosus. From a series of 110 invasive squamous cell carcinomas of the vulva negative for HPV by highly sensitive polymerase chain reaction, 51 had VIN lesions located at least 1 cm away from the tumor. In 4 (7.8%) cases, the VIN had basaloid histologic features. All cases showed obvious architectural disorganization with a homogeneous population of basaloid, undifferentiated keratinocytes with scanty cytoplasm replacing the whole epidermis. Immunohistochemically, all cases were negative for p16(INK4a) and strongly positive for p53 with suprabasilar extension of positive cells. All patients were postmenopausal (median age 61.0 y; range, 45-76). Squamous cell hyperplasia was identified in 1 case and lichen sclerosus in 1 case. The invasive squamous cell carcinoma was of keratinizing type in 3 cases and basaloid in 1 case. In conclusion, simplex, HPV-negative VIN may occasionally have basaloid morphology. Immunostaining for p16(INK4a) and p53 protein may be helpful in the identification of these lesions and the differential diagnosis with classic, HPV-positive basaloid VIN.

Value of p16(INK4a) as a marker of progression/regression in cervical intraepithelial neoplasia grade 1.

OBJECTIVE: The objective of this study was to evaluate the usefulness of p16(INK4a) staining to classify cervical intraepithelial neoplasia grade 1 according to its progression/regression risk. STUDY DESIGN: Patients with a histologic diagnosis of cervical intraepithelial neoplasia grade 1 were prospectively recruited (n = 138). Simultaneous detection of high-risk human papillomaviruses and p16(INK4a) evaluation were performed. Follow-up was conducted every 6 months by cytology and colposcopy and annually by high-risk human papillomavirus testing, for at least 12 months (mean, 29.0). Progression was defined as a histologic diagnosis of cervical intraepithelial neoplasia grades 2-3, regression as a negative cytology and high-risk human papillomaviruses, and persistence as a cytologic result of low-grade squamous intraepithelial lesions and/or a positive test for high-risk human papillomaviruses. RESULTS: Progression was observed in 14 women (10.1%), 66 (47.6%) regressed, and 58 (42.0%) had a persistent disease. p16(INK4a) was positive in 77 (55.8%) initial biopsy specimens. Progression to cervical intraepithelial neoplasia grades 2-3 was identified in 14 of 77 (18.2%) women with positive and none of 61 (0.00%) women with negative p16(INK4a) immunostaining (P < .001). CONCLUSION: p16(INK4a) negative cervical intraepithelial neoplasia grade 1 lesions rarely progress and may benefit from a less intensive follow-up.

p16 INK4a immunostaining identifies occult CIN lesions in HPV-positive women.

To evaluate whether p16 staining could help to recognize underestimated cervical intraepithelial neoplasia (CIN) in women positive for high-risk human papillomavirus (HR-HPV) with negative biopsy. Out of 1,259 women undergoing a histologic study and a simultaneous HR-HPV detection using the Hybrid Capture 2 test, we selected all patients testing positive for HR-HPV and having a negative biopsy (n=139), as well as all women testing negative for HR-HPV with a biopsy of either CIN 1 (26 cases) or CIN 2 to 3 (11 cases). Of the remaining 1,083 women, we randomly selected for the purpose of controls, 50 cases negative for HR-HPV with negative biopsy and 100 cases positive for HR-HPV and with biopsy of CIN (50 CIN 1, 50 CIN 2-3). In all cases, immunohistochemical staining for p16 and a second evaluation of the initial biopsy was carried out. Thirty-four out of 139 biopsies (24.5%) testing positive for HR-HPV but having a negative biopsy were positive for p16. Thirty of these cases (21.6%) were classified as harboring a CIN (11 CIN 1, 19 CIN 2/3) after reevaluation. Both the number of cases reclassified as CIN of any grade, or as CIN 2/3, were significantly higher for cases with HR-HPV load above 100 relative light unit (P<0.005). Particular attention should be paid to biopsies from patients having positive Hybrid Capture 2. The risk of harboring undetected CIN of any type or CIN 2/3 is significantly higher for patients with high HR-HPV load. Immunostaining with p16 should be considered as a highly desirable addition to the histologic evaluation of cervical biopsy specimens in HR-HPV-positive women.

Current techniques in screening for cervical cancer in Spain: updated recommendations.

A population-based survey (AFRODITA Study) was conducted in Spain in order to estimate the coverage and factors associated with cervical cancer cytological screening. The results of this survey indicate that the rate of screening for cervical cancer in Spain is 75.6% in women between 18 and 65 years. This high rate of opportunistic cervical cancer screening possibly has increased in the last 5 years. However, screening participation still needs to be improved in older women, women living in rural areas, women at a low socioeconomic level, and women living in certain autonomous regions. Conversely, an overuse of cytology has been observed in Spain, as a result of opportunistic screening. A survey in 2005, carried out in 14 public and private Spanish cytological laboratories, showed that among 409,443 women, the mean rate of abnormal cytology (a diagnosis of at least atypical cells of undetermined significance on a Pap smear) was 3.5% with a range of 0.5%-7.0% in Spain. We believe that this low rate of abnormal Pap smears is the result of repeated annual opportunistic screening in a low-risk population of women. A new Spanish consensus protocol for screening for cervical carcinoma was developed in 2006 by the Spanish Society of Gynaecology and Obstetrics, the Spanish Association of Cervical Pathology and Colposcopy, the Spanish Society of Cytology, and the Spanish Society of Anatomic Pathology. In order to rationalize the use of cervical cancer screening in Spain, the recommendations of the new Spanish consensus screening protocol must be followed.

Clinical role of cervical cancer vaccination: when and whom to vaccinate?

The recent development of two highly effective vaccines against persistent infection by the 2 most important types of human papillomavirus (HPV) (16 and 18) and against high grade premalignant lesions (CIN2+) has opened a new scenario for the primary prevention of cervical cancer. The optimum target population for vaccination should be individually defined taking the following into account: 1) the efficacy of the vaccine, 2) the epidemiological context and 3) the vaccination programs available in each country. To achieve the maximum preventive benefits, the vaccine should be administered before the initiation of sexual relations. So, the HPV vaccine should be integrated in the school vaccination programs of adolescents together with other vaccines. The vaccination of sexually active women may considerably increase the speed with which results in the fight against this disease will be achieved. Developed countries will probably consider the vaccination of these women, although vaccination strategies and the efforts to reach this population will be conditioned by the resources of each country and by the estimations of the cost-efficacy relationship in each situation. Women with a previous history of premalignant cervical disease or with an abnormal screening test should not be excluded from the potential benefits which the vaccine may provide. There is no contraindication for the administration of the vaccine in immunosupressed women. However, it is still unknown under what specific circumstances of immunosuppression the immunogenicity of the vaccine may be affected and there are currently ongoing studies for an answer to this question.

High-risk cervical epithelial neoplasia grade 1 treated by loop electrosurgical excision: follow-up and value of HPV testing.

OBJECTIVE: This study was undertaken to evaluate the value of high-risk human papillomavirus (HPV) testing in the follow-up of cervical intraepithelial neoplasia grade 1/low-grade squamous intraepithelial lesion treated by loop electrosurgical excision procedure because of the risk criteria established by the American Society for Colposcopy and Cervical Pathology (ie, unsatisfactory colposcopy or positive endocervical curettage, persistence of cervical intraepithelial neoplasia grade 1/low-grade squamous intraepithelial lesion, or high-risk HPV infection for longer than 2 years and older than 40 years). STUDY DESIGN: Seventy-seven women with cervical intraepithelial neoplasia grade 1/low-grade squamous intraepithelial lesion treated by loop electrosurgical excision procedure and followed-up with colposcopy, cytology, and high-risk HPV detection using Hybrid Capture II. RESULTS: More than 67% (67.6%) of women had cervical intraepithelial neoplasia grade 1 in the specimen; 22% a cervical intraepithelial neoplasia grade 2-3; and 10.4% had no lesion. Pretreatment HPV testing was positive in 100% of cervical intraepithelial neoplasia grade 2-3, in 93.5% of cervical intraepithelial neoplasia 1, and in 14.3% of cases with no lesion (P < .01). Pretreatment high-risk HPV testing was positive in all cases eventually developing residual/recurrent disease. Fifty percent of women with pretreatment viral load more than 100 relative light units had residual/recurrent disease develop. Posttreatment high-risk HPV testing during the follow-up reached a sensitivity and negative predictive value of 100% for detecting residual/recurrent disease. CONCLUSION: Patients with low-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia grade 1 and risk factors have a significant risk of harboring a cervical intraepithelial neoplasia grade 2-3 lesion. A conservative approach should be considered when basal high-risk HPV test is negative. High pretreatment high-risk HPV loads should be considered a risk factor for developing residual/recurrent disease. Posttreatment Hybrid Capture II has an extremely high sensitivity for detecting recurrences.

Pre- and post-conization high-risk HPV testing predicts residual/recurrent disease in patients treated for CIN 2-3.

OBJECTIVE: To evaluate whether high-risk human papillomavirus (HR-HPV) detection and viral load prior to treatment and status of cone margins can predict residual/recurrent disease as well as the ability of current diagnostic tools to identify residual/recurrent disease during follow-up of high-grade cervical intraepithelial neoplasia (CIN) treated by conization using loop electrosurgical procedure (LEEP). METHODS: Two hundred and three women (mean age 38.6 +/- 9.7; range 22-83) with CIN2-3 treated by LEEP conization and confirmed in the surgical specimen, attending follow-up visits were included. Age, HR-HPV detection and viral load determined by HybridCapture 2, and cone margins were evaluated as possible predictors of residual/recurrent disease. Value of single and repeated cytology as well as HR-HPV detection and viral load during follow-up were analyzed as screening tools of recurrence. RESULTS: Residual/recurrent disease was demonstrated by colposcopy guided biopsy in 36 patients (17.7%). High HR-HPV load (>1000 RLU) prior to LEEP and positive cone margins were significantly associated with higher risk of recurrence (31.8% vs. 9.4%, P = 0.005; and 36.4% vs. 11.9%, P < 0.001 respectively). HR-HPV detection at 6-12 m after LEEP showed higher sensitivity than a single or repeated cytology (97.2% vs. 83.3% and 94.4% respectively) although it showed less specificity (81.4% vs. 92.2% and 82.6%). The combination of HR-HPV detection and the first cytology during follow-up detected all patients with residual/recurrent disease (sensitivity 100%, negative predictive value 100%) with an acceptable specificity (76.6%). CONCLUSION: The inclusion of HR-HPV testing with cytology in follow-up of patients treated for CIN2-3 would allow for fewer post-treatment visits and avoid unnecessary cytologies. High HR-HPV load prior to LEEP or positive margins should be considered as risk factors for developing residual/recurrent disease.

Pre- and postsynaptic maturation of the neuromuscular junction during neonatal synapse elimination depends on protein kinase C.

The distribution of acetylcholine receptors (AChRs) within and around the neuromuscular junction changes dramatically during the first postnatal weeks, a period during which polyneuronal innervation is eliminated. We reported previously that protein kinase C (PKC) activation accelerates postnatal synapse loss. Because of the close relationship between axonal retraction and AChR cluster dispersal, we hypothesize that PKC can modulate morphological maturation changes of the AChR clusters in the postsynaptic membrane during neonatal axonal reduction. We applied substances affecting PKC activity to the neonatal rat levator auris longus muscle in vivo. Muscles were then stained immunohistochemically to detect both AChRs and axons. We found that, during the first postnatal days of normal development, substantial axonal loss preceded the formation of areas in synaptic sites that were free of AChRs, implying that axonal loss could occur independently of changes in AChR cluster organization. Nevertheless, there was a close relationship between axonal loss and AChR organization; PKC modulates both, although differently. Block of PKC activity with calphostin C prevented both AChR loss and axonal loss between postnatal days 4 and 6. PKC may act primarily to influence AChR clusters and not axons, insofar as phorbol ester activation of PKC accelerated changes in receptor aggregates but produced relatively little axon loss.