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We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities., (© 2024. The Author(s).)

The impact of multiple infections on the risk of cervical lesions is a subject of ongoing debate. This study aims to explore whether the richness of HPV genotype infections and the biodiversity of squamous and glandular cervical dysplasias could influence the progression of precancerous lesions. We conducted a cross-sectional analysis involving 469 women who attended the Colposcopy Unit at the European Institute of Oncology in Milan, Italy, from December 2006 to December 2014. HPV type richness was measured as the number of different genotypes per patient. We calculated the associations between richness and age, as well as histologic grade, along with Simpson's biodiversity index for cervical dysplasias. We observed significant inverse relationships between the richness of high-risk (HR) genotypes and both age ( p = 0.007) and histologic grade ( p < 0.001). Furthermore, as the histologic grade increased, the mean biodiversity index of cervical dysplasias decreased, with exceptions noted in cases of normal histology and adenocarcinoma in situ. Different histologic grades formed five clusters with distinct mean ages and mean biodiversity indices. These findings suggest that HPV genotype richness and the biodiversity of cervical dysplasias may play a crucial role in predicting the risk of high-grade cervical lesions, enabling personalized management of precancers.

Cervico-vaginal (CV) localization of extra-mammary Paget's disease (EMPD) of the vulva is extremely rare. In order to investigate the incidence risk and the pathognomonic clinical and pathological features of this condition, a retrospective analysis was conducted including 94 women treated for vulvar EMPD at the European Institute of Oncology, Milan, Italy, from October 1997 to May 2020. Overall nine patients developed CV involvement from EMPD, with a cumulative incidence of 2.5% (95% CI: 0.5-8.0%) at 5 years, 6.5% (95% CI: 1.9-15.1%) at 10 years and 14.0% (95% CI: 4.8-27.8%) at 15 years, respectively. All cases except one were firstly detected by abnormal glandular cytology. None reported vaginal bleeding or other suspicious symptoms. The colposcopic findings were heterogeneous and could sometimes be misdiagnosed. Cervical and/or vaginal biopsies were always performed for histopathological diagnosis by identification of Paget cells in the epithelium or stroma. Most patients developed invasive EMPD (5/9) of the cervix and/or vagina and underwent hysterectomy with partial or total colpectomy. CV involvement from EMPD should not be underestimated in women with a long-standing history of vulvar Paget's disease. Liquid-based cytology with immunocytochemistry represents a valuable tool for early diagnosis and should be routinely performed during the required lifelong follow-up.

Embryonal rhabdomyosarcoma of the uterine cervix (cERMS) is rare and frequently associated with DICER1 mutations. We report 94 tumors that arose in patients aged 7 to 59 (median=23) years and presented with vaginal bleeding (52), protruding vaginal mass (17), cervical polyp (8), or expelled tumor fragments per vagina (5). Nine had DICER1 syndrome, 8 of whom had other syndromic manifestations including ovarian Sertoli-Leydig cell tumor (7), multinodular goiter (3), pleuropulmonary blastoma (2), pineoblastoma (1), and osteosarcoma (1). Syndromic patients were younger than nonsyndromic patients (16 vs. 24 y). Tumor size ranged from 2 to 24 (median=4.5) cm. Ninety-two tumors were polypoid, most being grape-like (77 of 92). They were characterized by aggregates of primitive cells, almost always exhibiting a cambium layer, within a variably myxoedematous stroma and were hypocellular (63), moderately cellular (22), or hypercellular (9). Entrapped glands, typically scant, were present in 84 tumors. Primitive hyperchromatic ovoid to spindled cells with minimal cytoplasm predominated but differentiated rhabdomyoblasts with abundant eosinophilic cytoplasm (having cross-striations in 30) were seen in 83 tumors; they were often sparse but predominated in three. Nine tumors showed areas of intersecting fascicles and 4 zones with densely cellular (solid) growth. Cartilage was present in 38. Anaplasia was seen in 15 tumors, as was necrosis. Mitotic activity ranged from 1 to 58/10 high-power fields (median=8). The varied microscopic features resulted in a spectrum of differential diagnostic considerations, mainly typical and cellular forms of fibroepithelial polyps, Mullerian adenosarcoma, and other sarcomas. Follow-up was available for 79 patients ranging from 6 to 492 (median=90) months. Treatment information was available in 62 and included polypectomy in 6 patients (2 also received chemotherapy), limited resection in 26 (14 also received chemotherapy), hysterectomy in 29 (15 with adjuvant chemotherapy), and biopsies only in 1 (with chemotherapy). Staging was possible in 56 tumors; according to the "uterine sarcoma" system (tumor size and extent) they were: stage I (10/56; could not be further subclassified as size not available), IA (22/56), IB (18/56), IIA (2/56), IIB 3/56), IIIC (1/56). According to the "adenosarcoma" system (depth of invasion and extent) they were: stage IA (26/56), IB (14/56), IC (10/56), IIA (2/56), IIB (3/56), IIIC (1/56). Eight patients had local recurrence following incomplete excision (10%). Eleven of 79 patients had extrauterine recurrences (14%) and 9 died of disease (11%). Older age was associated with extrauterine recurrence (median 44 vs. 22; P =0.002) and decreased disease-specific survival (median 44 vs. 22; P =0.02). For patients with tumors initially confined to the cervix, the adenosarcoma staging system was superior to the uterine sarcoma staging system for predicting survival ( P =0.02). Three patients with DICER1 syndrome who underwent fertility-preserving surgery developed a second primary cERMS 7, 7, and 12 years after their primary tumor. All 9 patients with DICER1 syndrome had tumors confined to the cervix and none died of disease. This study highlights the intriguing clinical aspects of cERMS including its long-known tendency to occur in the young but also more recently appreciated association with DICER1 syndrome. Establishing the diagnosis may still be difficult because of the hazard of sampling a neoplasm which in areas may appear remarkably bland and also because of its potential confusion with other neoplasms. This study indicates that this tumor has a good prognosis at this site and in selected cases a conservative surgical approach is a realistic consideration., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Avalanche photodiodes, which operate above the breakdown voltage in Geiger mode connected with avalanche-quenching circuits, can be used to detect single photons and are therefore called single-photon avalanche diodes SPAD's. Circuit configurations suitable for this operation mode are critically analyzed and their relative merits in photon counting and timing applications are assessed. Simple passive-quenching circuits (PQC's), which are useful for SPAD device testing and selection, have fairly limited application. Suitably designed active-quenching circuits (AQC's) make it possible to exploit the best performance of SPAD's. Thick silicon SPAD's that operate at high voltages (250-450 V) have photon detection efficiency higher than 50% from 540- to 850-nm wavelength and still [approximately]3% at 1064 nm. Thin silicon SPAD's that operate at low voltages (10-50 V) have 45% efficiency at 500 nm, declining to 10% at 830 nm and to as little as 0.1% at 1064 nm. The time resolution achieved in photon timing is 20 ps FWHM with thin SPAD's; it ranges from 350 to 150 ps FWHM with thick SPAD's. The achieved minimum counting dead time and maximum counting rate are 40 ns and 10 Mcps with thick silicon SPAD's, 10 ns and 40 Mcps with thin SPAD's. Germanium and III-V compound semiconductor SPAD's extend the range of photon-counting techniques in the near-infrared region to at least 1600-nm wavelength. Key words: Photon counting, photon timing, avalanche photodiodes, quenching circuits.

The feasibiltiy of using compounds avalanche photodiodes (APD) for measuring the pulse waveform of long-wavelength laser diodes is studied through experimental procedures. A gain-switched laser diode emitting at 1.3 micrometer is treated with a high concentration of dopants to reduce the structure's bandgap. The measurements are obtained by means of an ultrafast III-IV photodiode. Results show that APDscan be used for measuring the pulse shape of laser diodes under various light intensity.

At the present time, Single Photon Avalanche Diodes (SPADs) are the enabling devices in many applications, ranging from medical imaging to laser ranging and from remote sensing to quantum key distribution. Even though they belong to different scientific domains, these applications share the need for a detector capable of attaining high count rates possibly without trading it off with other key detector's features, such as afterpulsing probability, photon detection efficiency, and dark counts. In this work, we present the characterization of a fast integrated active quenching circuit capable of driving high-performance external custom-technology SPADs for single photon detection in the visible wavelength range. Combining the prompt intervention of the electronic circuitry and the performance of a custom-technology SPAD, we attained count rates up to 250 MCps while keeping the afterpulsing probability within 2%.

Objectives: To investigate the prevalence of high-risk human papillomavirus (HPV)-negative cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma (ICC) and to analyze the distribution of other genotypes in this subset., Methods: In total, 431 women who underwent excisional surgical treatment for CIN or ICC at the European Institute of Oncology, Milan, Italy, from January 2016 to December 2017 were retrospectively analyzed. The Linear Array HPV genotyping test (Roche Diagnostics) was performed on a postaliquot from high-risk-HPV-negative liquid-based cervical specimens, when available. Patient characteristics and the prevalence of high-risk-HPV-negative CIN grade 2 or worse (CIN2+) were tabulated. We used t tests to compare age between high-risk-HPV-positive and high-risk-HPV-negative patients., Results: Overall, 8.9% of CIN2+ and 7.5% of ICC cases were high-risk HPV negative. There was no age difference between high-risk-HPV-negative CIN2+ women (mean [SD], 41.3 [8.7] years) and high-risk-HPV-positive women (mean [SD], 39.5 [9.0] years) (P = .28). The Linear Array result was available in 22 cases. Most high-risk-HPV-negative patients were positive for a single other genotype infection (32.6%). HPV 73 was the most prevalent genotype, followed by HPV 53 and HPV 84. HPV 26 was detected in 1 case of ICC., Conclusions: Our results showed a not-negligible proportion of high-risk-HPV-negative CIN2+, suggesting that cotesting would not miss these cases., (© American Society for Clinical Pathology, 2021. All rights reserved.For permissions, please e-mail: journals.permissions@oup.com.)

Clear cell carcinoma of the ovary is a rare and distinct histotype of epithelial ovarian carcinomas. Women diagnosed with clear cell carcinomas are usually younger and diagnosed at earlier stages than those with the most common high-grade serous histology. Endometriosis is considered a main risk factor for the development of clear cell carcinoma of the ovary, and it can be considered a precursor of of this tumor, as it is identified in more than 50% of patients with clear cell carcinoma. Different molecular pathways and alterations heve been identified in ovarian clear cell carcinoma, including the most common mutations of AT-rich interaction domain 1A [ARID1A] and phosphatidylinositol-4,5-bisphosphate 3-kinase [PIK3] catalytic subunit alpha [PIK3CA]. The prognosis of patients at early stage is favorable, while patients with advanced or recurrent disease experience a poor oncologic outcomes. Despite a lower rate of responses due to an intrinsic chemoresistance, the treatment strategy for advanced disease resembles the treatment of high-grade serous carcinoma, which includes aggressive cytoreductive surgery and platinum-based chemotherapy. For this reason, the role of adjuvant chemotherapy in patients with stage I disease undergoing complete surgical staging is still under debate. Alternative treatments, including biological agents that target different pathways constitute the most promising treatment strategies, and well-designed, collaborative international trials should be designed in order to improve the oncologic outcomes and the quality of life of patients with this aggressive disease., (Copyright © 2021. Published by Elsevier Inc.)

In this paper we present a new technology for the fabrication of Single Photon Avalanche Diodes (SPADs) aimed at combining the advantages of thin and thick SPADs. The new detector is manufactured in a thick epitaxial layer designed to improve the Photon Detection Efficiency (PDE) in the red and near-infrared range while maintaining a good timing resolution. Experimental characterization of the new red-enhanced SPAD (RE-SPAD) confirmed a significant improvement of the PDE compared with thin SPADs; for example the PDE at a wavelength of 800 nm has increased from 15% to about 40%. Nevertheless the temporal resolution is still good, with a timing jitter of about 90 ps FWHM. In the same operating conditions the dark count rate is comparable with the one attainable with a thin SPAD (e.g. less than 25 cps for a 50 µm diameter device cooled down to −5°C). Moreover, being planar, the new technology is compatible with the fabrication of arrays of detectors. [ABSTRACT FROM PUBLISHER]

Single-photon detection is an invaluable tool for many applications ranging from basic research to consumer electronics. In this respect, the Single Photon Avalanche Diode (SPAD) plays a key role in enabling a broad diffusion of these techniques thanks to its remarkable performance, room-temperature operation, and scalability. In this paper we present a silicon technology that allows the fabrication of SPAD-arrays with an unprecedented combination of low timing jitter (95 ps FWHM) and high detection efficiency at red and near infrared wavelengths (peak of 70% at 650 nm, 45% at 800 nm). We discuss the device structure, the fabrication process, and we present a thorough experimental characterization of the fabricated detectors. We think that these results can pave the way to new exciting developments in many fields, ranging from quantum optics to single molecule spectroscopy.

Objectives: Tumor-associated antigens (TAAs) are frequently overexpressed in several cancer types. The aim of this study was to investigate the expression of TAAs in breast cancer., Material and Methods: A total of 250 selected invasive breast cancers including 50 estrogen receptor (ER)-positive (Luminal B like), 50 triple-negative (TN), 50 ER-positive lobular type, 50 ER- and progesterone receptor (PgR)-positive (Luminal A like) and 50 cerbB2-positive breast cancers, were assessed for New York esophageal squamous cell carcinoma-1 (NY-ESO-1), Wilms tumor antigen (WT-1) and PReferentially expressed Antigen of MElanoma (PRAME) antigen expression by immunohistochemistry (IHC)., Results: A significantly higher expression of cancer testis (CT)-antigens NY-ESO-1 and WT-1 antigen was detected in TN breast cancers compared with ER-positive tumors. NY-ESO-1 overexpression (score 2 + and 3+) assessed by monoclonal and polyclonal antibodies was detected in 9 (18%) TN cancers as compared to 2 (4%) ER-positive tumors (p = 0.002). WT1 over-expression (score 2 + and 3+) was confirmed in 27 (54%) TN tumor samples as compared to 6 (12%) ER-positive (p < 0.0001). PRAME over-expression (score 2 + and 3+) was detected in 8 (16%) HER2 positive tumor samples as compared to no TN and ER-positive cancers (p = 0.0021)., Conclusions: NY-ESO-1 and WT1 antigens are overexpressed in TN breast cancers. Because of the limited therapeutic options for this patient subgroup, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)