Non-invasive surrogate markers of pulmonary hypertension are associated with poor survival in patients with cancer.

BACKGROUND: Cancer is one of the leading causes of death worldwide, and cardiopulmonary comorbidities may further adversely affect cancer prognosis. We recently described lung cancer-associated pulmonary hypertension (PH) as a new form of PH and comorbidity of lung cancer. While patients with lung cancer with PH had significantly reduced overall survival compared with patients without PH, the prevalence and impact of PH in other cancers remain unclear. METHODS: In this retrospective, observational cohort study, we analysed the prevalence and impact of PH on clinical outcomes in 1184 patients with solid tumours other than lung cancer, that is, colorectal, head and neck, urological, breast or central nervous system tumours, using surrogate markers for PH determined by CT. RESULTS: PH prevalence in this cohort was 10.98%. A Cox proportional hazard model revealed a significant reduction in the median survival time of patients with cancer with PH (837 vs 2074 days; p<0.001). However, there was no correlation between pulmonary metastases and PH. A subgroup analysis showed that PH was linked to decreased lung and cardiac function. Additionally, PH was associated with systemic arterial hypertension (p<0.001) and coronary artery disease (p=0.014), but not emphysema. CONCLUSIONS: In this study, fewer patients with cancer had surrogate parameters for PH compared with previously published results among patients with lung cancer. Consequently, the prevalence of PH in other cancers might be lower compared with lung cancer; however, PH still has a negative impact on prognosis. Furthermore, our data does not provide evidence that lung metastases cause PH. Thus, our results support the idea that lung cancer-associated PH represents a new category of PH. Our results also highlight the importance of further studies in the field of cardio-oncology.

Surgical Treatment of Patients with Endometriosis in the Certified Endometriosis Centers of the DACH Region - A Subanalysis of the Quality Assurance Study QS ENDO pilot.

Introduction After puberty, at least 10% of all women and girls suffer from endometriosis. Surgery is useful for both the diagnosis and therapy. To date, quality indicators for the surgical treatment of endometriosis are lacking. QS ENDO aims to record the quality of care provided in the DACH region and to introduce quality indicators for the diagnosis and treatment of endometriosis. In the first phase of the study, QS ENDO real, the reality of care was recorded using a questionnaire. The second phase, QS ENDO pilot, investigated the treatment of patients who underwent surgery in certified endometriosis centers in a defined time-period. Material and Methods The surgical data of 10 patients from each of the 44 endometriosis centers in the DACH region was recorded using an online tool. Collected data included the approach used, the endometriosis phenotype, a description of the surgical site, resection status, histological confirmation, the use of a classification, and any complications. All operations were carried out in October 2016 as the defined time-period. The surgical approaches used were compared with the recommendations in the current guidelines. Results The data of 435 patients with a median age of 34 years were evaluated. 315 (72.4%) were nulliparous. 120 patients had given birth to at least one child and 42.5% (51) of them had delivered their child by caesarean section. About 50% of all patients also had deep infiltrating endometriosis in addition to ovarian endometriosis, and the median NAS score was 7.5. With regards to the surgical treatment, endometriomas were completely resected in 81% (94) of patients. 87.3% of patients underwent resection of peritoneal endometriosis. Forty-one patients had a hysterectomy, with a total hysterectomy carried out in 26 (63.4%) and a supracervical hysterectomy in 15 (36.6%) patients. Of the 59 patients with bowel endometriosis, half had segmental resection and half had shaving of the anterior rectal wall. Complications requiring revision occurred in 0.9% of cases. Conclusion The surgical procedures carried out in the certified endometriosis centers of the DACH region are largely in line with the recommendations for appropriate surgical approaches in the current standard guidelines.

The Different Gene Expression Profile in the Eutopic and Ectopic Endometrium Sheds New Light on the Endometrial Seed in Endometriosis.

The changes in endometrial cells, both in the eutopic endometrium of patients with and without endometriosis and in lesions at ectopic sites, are frequently described and often compared to tumorigenesis. In tumorigenesis, the concept of "seed and soil" is well established. The seed refers to tumor cells with metastatic potential, and the soil is any organ or tissue that provides a suitable environment for the seed to grow. In this systematic review (PRISMA-S), we specifically compared the development of endometriosis with the "seed and soil" hypothesis. To determine changes in the endometrial seed, we re-analyzed the mRNA expression data of the eutopic and ectopic endometrium, paying special attention to the epithelial-mesenchymal transition (EMT). We found that the similarity between eutopic endometrium without and with endometriosis is extremely high (~99.1%). In contrast, the eutopic endometrium of patients with endometriosis has a similarity of only 95.3% with the ectopic endometrium. An analysis of EMT-associated genes revealed only minor differences in the mRNA expression levels of claudin family members without the loss of other cell-cell junctions that are critical for the epithelial phenotype. The array data suggest that the changes in the eutopic endometrium (=seed) are quite subtle at the beginning of the disease and that most of the differences occur after implantation into ectopic locations (=soil).

Long-Term Maintenance of Viable Human Endometrial Epithelial Cells to Analyze Estrogen and Progestin Effects.

There are fewer investigations conducted on human primary endometrial epithelial cells (HPEECs) compared to human primary endometrial stromal cells (HPESCs). One of the main reasons is the scarcity of protocols enabling prolonged epithelial cell culture. Even though it is possible to culture HPEECs in 3D over a longer period of time, it is technically demanding. In this study, we successfully established a highly pure, stable, and long-term viable human conditionally reprogrammed endometrial epithelial cell line, designated as eCRC560. These cells stained positive for epithelial markers, estrogen and progesterone receptors, and epithelial cell-cell contacts but negative for stromal and endothelial cell markers. Estradiol (ES) reduced the abundance of ZO-1 in a time- and dose-dependent manner, in contrast to the dose-dependent increase with the progestin dienogest (DNG) when co-cultured with HPESCs. Moreover, ES significantly increased cell viability, cell migration, and invasion of the eCRC560 cells; all these effects were inhibited by pretreatment with DNG. DNG withdrawal led to a significantly disrupted monolayer of eCRC560 cells in co-culture with HPESCs, yet it markedly increased the adhesion of eCRC560 to the human mesothelial MeT-5A cells. The long-term viable eCRC560 cells are suitable for in vitro analysis of HPEECs to study the epithelial compartment of the human endometrium and endometrial pathologies.

Predictive Model for the Non-Invasive Diagnosis of Endometriosis Based on Clinical Parameters.

OBJECTIVES: Are other pain symptoms in addition to dysmenorrhea, dyspareunia, dyschezia, dysuria, and chronic pelvic pain correlated to endometriosis and suitable for a clinical prediction model? METHODS: We conducted a prospective study from 2016 to 2022, including a total of 269 women with numerous pain symptoms and other parameters. All women filled out two questionnaires and were examined by palpation and transvaginal ultrasound (TVUS). In cases of suspected deep endometriosis, magnetic resonance imaging (MRI) was performed. After the operation, endometriosis was diagnosed by histological examination. RESULTS: All in all, 30 significant parameters and 6 significant numeric rating scale (NRS) scores associated with endometriosis could be identified: 7 pain adjectives, 8 endometriosis-associated pain symptoms, 5 pain localizations, 6 parameters from the PainDETECT, consumption of analgesics, and allergies. Furthermore, longer pain duration (before, during, and after menstruation) was observed in women with endometriosis compared to women without endometriosis (34.0% vs. 12.3%, respectively). Although no specific pain for endometriosis could be identified for all women, a subgroup with endometriosis reported radiating pain to the thighs/legs in contrast to a lower number of women without endometriosis (33.9% vs. 15.2%, respectively). Furthermore, a subgroup of women with endometriosis suffered from dysuria compared to patients without endometriosis (32.2% vs. 4.3%, respectively). Remarkably, the numbers of significant parameters were significantly higher in women with endometriosis compared to women without endometriosis (14.10 ± 4.2 vs. 7.75 ± 5.8, respectively). A decision tree was developed, resulting in 0.904 sensitivity, 0.750 specificity, 0.874 positive predictive values (PPV), 0.802 negative predictive values (NPV), 28.235 odds ratio (OR), and 4.423 relative risks (RR). The PPV of 0.874 is comparable to the positive prediction of endometriosis by the clinicians of 0.86 (177/205). CONCLUSIONS: The presented predictive model will enable a non-invasive diagnosis of endometriosis and can also be used by both patients and clinicians for surveillance of the disease before and after surgery. In cases of positivety, as evaluated by the questionnaire, patients can then seek advice again. Similarly, patients without an operation but with medical therapy can be monitored with the questionnaire.

QS ENDO Pilot - A Study by the Stiftung Endometrioseforschung (SEF) on the Quality of Care Provided to Patients with Endometriosis in Certified Endometriosis Centers in the DACH Region.

Introduction Endometriosis significantly reduces patients' quality of life and is additionally a burden on healthcare and social security systems. There are currently no quality indicators for the treatment of endometriosis. The care of patients with endometriosis must be considered inadequate. QS ENDO aims to record the quality of care available in the DACH region and to introduce quality indicators for the diagnosis and treatment of endometriosis as part of providing quality assurance in endometriosis care. The first phase, QS ENDO Real, recorded the reality of current care using a questionnaire. The second phase, QS ENDO Pilot, investigated the treatment of 435 patients who underwent surgical treatment within a defined one month period in certified endometriosis centers. Material and Methods An online tool was used to gather information about 9 points which covered both prior patient history and the process of clinical diagnosis. Surgery reports were reviewed to obtain information about the surgical approach, the investigated sites, findings of any histological examinations, the use of classification systems, and information about resection status. Results 85.3% of patients were asked all 4 questions about their prior medical history. All 5 diagnostic steps were carried out in 34.5% of patients. The 3 areas needed to describe potential sites of disease were recorded in 67.1% of patients. Samples for histological examination were taken in 84.1% of patients. The endometriosis stage was classified in 94.7% of surgeries. A combination of the rASRM and the ENZIAN classifications, which is needed for complex cases, was used in 46.1% of patients. Complete resection was achieved in 81.6% of surgical procedures. Conclusion For the first time, the quality of care in certified endometriosis centers has been recorded using QS ENDO Pilot. Despite the high certification standards, a substantial number of required indicators were omitted.

Near Infrared Photoimmunotherapy: A Review of Recent Progress and Their Target Molecules for Cancer Therapy.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed molecular targeted cancer treatment, which selectively kills cancer cells or immune-regulatory cells and induces therapeutic host immune responses by administrating a cancer targeting moiety conjugated with IRdye700. The local exposure to near-infrared (NIR) light causes a photo-induced ligand release reaction, which causes damage to the target cell, resulting in immunogenic cell death (ICD) with little or no side effect to the surrounding normal cells. Moreover, NIR-PIT can generate an immune response in distant metastases and inhibit further cancer attack by combing cancer cells targeting NIR-PIT and immune regulatory cells targeting NIR-PIT or other cancer treatment modalities. Several recent improvements in NIR-PIT have been explored such as catheter-driven NIR light delivery, real-time monitoring of cancer, and the development of new target molecule, leading to NIR-PIT being considered as a promising cancer therapy. In this review, we discuss the progress of NIR-PIT, their mechanism and design strategies for cancer treatment. Furthermore, the overall possible targeting molecules for NIR-PIT with their application for cancer treatment are briefly summarised.

Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy.

Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is limited to detecting a single marker per tissue section. Since immunotherapy has revolutionized the antineoplastic therapy, developing new immunohistochemistry strategies to detect multiple markers simultaneously to better understand tumor environment and predict or assess response to immunotherapy is necessary and urgent. Multiplex immunohistochemistry (mIHC)/multiplex immunofluorescence (mIF), such as multiplex chromogenic IHC and multiplex fluorescent immunohistochemistry (mfIHC), is a new and emerging technology to label multiple biomarkers in a single pathological section. The mfIHC shows a higher performance in cancer immunotherapy. This review summarizes the technologies, which are applied for mfIHC, and discusses how they are employed for immunotherapy research.

Unusual phenotypes in patients with a pathogenic germline variant in DICER1.

Pathogenic germline DICER1 variants are associated with pleuropulmonary blastoma, multinodular goiter, embryonal rhabdomyosarcoma and other tumour types, while mosaic missense DICER1 variants in the RNase IIIb domain are linked to cause GLOW (global developmental delay, lung cysts, overgrowth, and Wilms' tumor) syndrome. Here, we report four families with germline DICER1 pathogenic variants in which one member in each family had a more complex phenotype, including skeletal findings, facial dysmorphism and developmental abnormalities. The developmental features occur with a variable expressivity and incomplete penetrance as also described for the neoplastic and dysplastic lesions associated with DICER1 variants. Whole exome sequencing (WES) was performed on all four cases and revealed no further pathogenic or likely pathogenic dominant, homozygous or compound heterozygous variants in three of them. Notably, a frameshift variant in ARID1B was detected in one patient explaining part of her phenotype. This series of patients shows that pathogenic DICER1 variants may be associated with a broader phenotypic spectrum than initially assumed, including predisposition to different tumours, skeletal findings, dysmorphism and developmental abnormalities, but genetic work up in syndromic patients should be comprehensive in order not to miss additional underlying /modifying causes.

Claudin-10 Expression Is Increased in Endometriosis and Adenomyosis and Mislocalized in Ectopic Endometriosis.

Claudins, as the major components of tight junctions, are crucial for epithelial cell-to-cell contacts. Recently, we showed that in endometriosis, the endometrial epithelial phenotype is highly conserved, with only minor alterations. For example, claudin-11 is strongly expressed; however, its localization in the endometriotic epithelial cells was impaired. In order to better understand the role of claudins in endometrial cell-to-cell contacts, we analyzed the tissue expression and localization of claudin-10 by immunohistochemistry analysis and two scoring systems. We used human tissue samples (n = 151) from the endometrium, endometriosis, and adenomyosis. We found a high abundance of claudin-10 in nearly all the endometrial (98%), endometriotic (98−99%), and adenomyotic (90−97%) glands, but no cycle-specific differences and no differences in the claudin-10 positive endometrial glands between cases with and without endometriosis. A significantly higher expression of claudin-10 was evident in the ectopic endometrium of deep-infiltrating (p < 0.01) and ovarian endometriosis (p < 0.001) and in adenomyosis in the cases with endometriosis (p ≤ 0.05). Interestingly, we observed a shift in claudin-10 from a predominant apical localization in the eutopic endometrium to a more pronounced basal/cytoplasmic localization in the ectopic endometria of all three endometriotic entities but not in adenomyosis. Significantly, despite the impaired endometriotic localization of claudin-10, the epithelial phenotype was retained. The significant differences in claudin-10 localization between the three endometriotic entities and adenomyosis, in conjunction with endometriosis, suggest that most of the aberrations occur after implantation and not before. The high similarity between the claudin-10 patterns in the eutopic endometrial and adenomyotic glands supports our recent conclusions that the endometrium is the main source of endometriosis and adenomyosis.

EpCAM- and EGFR-Specific Antibody Drug Conjugates for Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new era of targeting therapy. Since the epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) are over expressed on triple-negative breast cancer, we developed novel ADCs by conjugating benzylguanine (BG)-modified monomethyl auristatin E (MMAE) to EpCAM- and EGFR-specific SNAP-tagged single chain antibody fragments (scFvs). Rapid and efficient conjugation was achieved by SNAP-tag technology. The binding and internalization properties of scFv-SNAP fusion proteins were confirmed by flow cytometry and fluorescence microscopy. The dose-dependent cytotoxicity was evaluated in cell lines expressing different levels of EGFR and EpCAM. Both ADCs showed specific cytotoxicity to EGFR or EpCAM positive cell lines via inducing apoptosis at a nanomolar concentration. Our study demonstrated that EGFR specific scFv-425-SNAP-BG-MMAE and EpCAM-specific scFv-EpCAM-SNAP-BG-MMAE could be promising ADCs for the treatment of TNBC.

The effect of warm and humidified gas insufflation in gynecological laparoscopy on maintenance of body temperature: a prospective randomized controlled multi-arm trial.

BACKGROUND: Hypothermia is defined as a decrease in body core temperature to below 36 °C. If intraoperative heat-preserving measures are omitted, a patient's temperature will fall by 1 - 2 °C. Even mild forms of intraoperative hypothermia can lead to a marked increase in morbidity and mortality. Using warm and humidified gas insufflation in laparoscopy may help in the maintenance of intraoperative body temperature. METHODS: In this prospective randomized controlled study, we investigated effects of temperature and humidity of the insufflation gas on intra- and postoperative temperature management. 150 patients undergoing gynecologic laparoscopic surgery were randomly assigned to either insufflation with non-warmed, non-humidified CO2 with forced air warming blanket (AIR), humidified warm gas without forced air warming blanket (HUMI) or humidified warm gas combined with forced air warming blanket (HUMI+). We hypothesized that the use of warmed laparoscopic gas would have benefits in the maintenance of body temperature and reduce the occurrence of hypothermia. RESULTS: The use of warm and humidified gas insufflation alone led to more hypothermia episodes with longer duration and longer recovery times as well as significantly lower core body temperature compared to the other two groups. In the comparison of the AIR group and HUMI + group, HUMI + patients had a significantly higher body temperature at arrival at the PACU (Post Anaesthesia Care Unit), had the least occurrence of hypothermia and suffered from less shivering. CONCLUSION: The use of warm and humidified gas insufflation alone does not sufficiently warm the patients. The optimal temperature management is achieved in the combination of external forced air warming and insufflation of warm and humidified laparoscopy gas.

Warm and humidified insufflation gas during gynecologic laparoscopic surgery reduces postoperative pain in predisposed patients-a randomized, controlled multi-arm trial.

BACKGROUND: Postoperative pain remains a common problem in gynecologic laparoscopy, especially in head zone-related regions, triggered by intra-abdominal pressure during capnoperitoneum. Humidified and prewarmed insufflation gas may ameliorate pain and be beneficial. METHODS: This prospective randomized controlled parallel group multi-arm single-center study investigated the effects of temperature and humidity of insufflation gas on postoperative pain during gynecologic laparoscopy with a duration ≥ 60 min. Female participants (18-70 years) were blinded and randomly assigned-computer generated-to either insufflation with dry cold CO2 with forced air warming blanket ("AIR"), humidified warm gas without forced air warming blanket ("HUMI"), or humidified warm gas with forced air warming blanket ("HUMI +"). We hypothesized that using humidified warm gas resulted in lower pain scores and less analgesic consumption. The primary endpoint postoperative pain was assessed for different pain localizations every 12 h during 7 days after surgery. Secondary endpoints were demand for painkillers and epidural anesthetics, length of stay in recovery room, and hospital stay. (Registration: ClinicalTrials.gov NCT02781194-completed). RESULTS: 150 participants were randomized. Compared to group "AIR" (n = 48), there was significantly less pain in group "HUMI +" (n = 48) in the recovery room (- 1.068; 95% CI - 2.08 to - 0.061), as well as significantly less ibuprofen use at day two (- 0.5871 g ± 0.258; p-value = 0.0471). Other variables did not change significantly. Stratification for presence of endometriosis or non-previous abdominal surgery in patient history revealed significantly less pain in both groups "HUMI" (n = 50) and "HUMI +" versus group "AIR." Related side effects were not noted. CONCLUSION: In the overall population, the use of warm, humidified insufflation gas did not yield clinically relevant effects; however, in predisposed patients with endometriosis and who could otherwise expect high pain levels, warm and humidified gas may be beneficial.

Toward Homogenous Antibody Drug Conjugates Using Enzyme-Based Conjugation Approaches.

In the last few decades, antibody-based diagnostic and therapeutic applications have been well established in medicine and have revolutionized cancer managements by improving tumor detection and treatment. Antibodies are unique medical elements due to their powerful properties of being able to recognize specific antigens and their therapeutic mechanisms such as blocking specific pathways, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Furthermore, modification techniques have paved the way for improving antibody properties and to develop new classes of antibody-conjugate-based diagnostic and therapeutic agents. These techniques allow arming antibodies with various effector molecules. However, these techniques are utilizing the most frequently used amino acid residues for bioconjugation, such as cysteine and lysine. These bioconjugation approaches generate heterogeneous products with different functional and safety profiles. This is mainly due to the abundance of lysine and cysteine side chains. To overcome these limitations, different site-direct conjugation methods have been applied to arm the antibodies with therapeutic or diagnostics molecules to generate unified antibody conjugates with tailored properties. This review summarizes some of the enzyme-based site-specific conjugation approaches.

Molecular Targeting Therapy against EGFR Family in Breast Cancer: Progress and Future Potentials.

：The epidermal growth factor receptor (EGFR) family contains four transmembrane tyrosine kinases (EGFR1/ErbB1, Her2/ErbB2, Her3/ErbB3 and Her4/ErbB4) and 13 secreted polypeptide ligands. EGFRs are overexpressed in many solid tumors, including breast, pancreas, head-and-neck, prostate, ovarian, renal, colon, and non-small-cell lung cancer. Such overexpression produces strong stimulation of downstream signaling pathways, which induce cell growth, cell differentiation, cell cycle progression, angiogenesis, cell motility and blocking of apoptosis.The high expression and/or functional activation of EGFRs correlates with the pathogenesis and progression of several cancers, which make them attractive targets for both diagnosis and therapy. Several approaches have been developed to target these receptors and/or the EGFR modulated effects in cancer cells. Most approaches include the development of anti-EGFRs antibodies and/or small-molecule EGFR inhibitors. This review presents the state-of-the-art and future prospects of targeting EGFRs to treat breast cancer.

Prevention of Intraoperative Hypothermia in Laparoscopy by the Use of Body-Temperature and Humidified CO 2 : a Pilot Study.

Introduction Hypothermia is defined as a decrease in body core temperature to below 36 °C. If intraoperative heat-preserving measures are omitted, a patient's temperature will fall by 1 - 2 °C. Even mild forms of intraoperative hypothermia can lead to a marked increase in morbidity and mortality. The temperature of the insufflation gas is usually disregarded in the treatment and prevention of hypothermia. This study was conducted to investigate the effect of body-temperature and humidified CO 2 on the intraoperative temperature profile and avoidance of hypothermia in laparoscopic surgery. Material and Methods In this retrospective, non-randomised case control study, 110 patients whose planned operation lasted at least 60 minutes were identified from 376 patients by means of an algorithm. Dry (20% humidity) CO 2 at room temperature was insufflated in 51 patients (control group). 59 patients were insufflated with humidified (98% humidity) CO 2 at body temperature (37 °C) (study group). These conditions were achieved with the HumiGard MR860 Surgical Humidification System (Fisher & Paykel Healthcare Limited, Auckland, New Zealand). The intraoperative temperature profile was evaluated by measurements every 10 minutes. Statistical analysis was performed with IBM ® SPSS ® Statistics 23.0.0. Results The intraoperative temperature in the control group fell steadily, while a continuous rise in temperature was observed in the study group. Warming was demonstrated in the study group with a start-end temperature difference of 0.09 °C, which differed significantly from the control group, in which it was - 0.09 °C (p = 0.011). The middle-end difference of 0.11 °C showed even higher significance in favour of the warmed gas (p = 0.003). The rate of hypothermia at the start of the operation fell from 50 to 36% in the study group and increased from 36 to 42% in the control group. Conclusion These results show that the use of body-temperature and humidified insufflation gas for laparoscopy can help to prevent intraoperative hypothermia.

Clinicopathologic and Molecular Features of Paired Cases of Metachronous Ovarian Serous Borderline Tumor and Subsequent Serous Carcinoma.

Although risk factors have been established for the development of serous carcinoma after a diagnosis of serous borderline tumor (SBT), comprising atypical proliferative serous tumor (APST) (ie, conventional SBT) and noninvasive low-grade serous carcinoma (niLGSC) (ie, micropapillary SBT), subsequent invasive carcinoma still occurs in a subset of women who are not at increased risk. Whether subsequent serous carcinoma in women with a prior SBT represents malignant progression/recurrence or an independent primary tumor is unclear, and the combined clinicopathologic and molecular features of SBTs and their subsequent carcinomas have not been fully characterized. In this study, we analyzed a cohort of 42 women initially diagnosed with SBT who subsequently developed serous carcinoma of a total of 1025 cases of ovarian SBT from a nationwide population-based cohort. Review of the diagnostic slides was performed from this subset of SBTs and matched metachronous invasive serous carcinomas (39 low grade, 3 high grade). DNA was extracted from tissue blocks available for 41 cases (both SBT and carcinoma, n=36; SBT only, n=3; carcinoma only, n=2). Samples were subjected to digital droplet PCR to analyze mutation hotspots in KRAS (codon 12) and BRAF (V600E), which are frequently found in low-grade serous tumors. Eighty-one percent of SBTs (34/42) were APST, and 19% (8/42) were niLGSC. Forty percent of cases (17/42) were FIGO stage I, the majority of which were APST (14/17; 82%). The median time to development of carcinoma was 9 years (range, 0.6 to 25 y). Mutations in SBTs were distributed as follows: 5/39 (13%) BRAF mutant, 22/39 (56%) KRAS mutant, and 12/39 (31%) wild-type for both genes. There was a significant relationship between SBT gene mutation and histologic type, with BRAF mutations occurring exclusively in APST and a higher frequency of niLGSC among SBTs wild-type for BRAF and KRAS (P=0.01). The diffuse presence of tumor cells with abundant eosinophilic cytoplasm was significantly associated with the BRAF mutation (P=0.001). Mutational analyses of matched SBT/carcinoma pairs revealed concordant profiles in 33/36 (92%) cases, of which 19 (53%) were KRAS mutant, 4 (11%) were BRAF mutant, and 10 (28%) were wild type for both genes. The 3 discordant cases consisted of a wild-type niLGSC with a subsequent BRAF-mutant invasive LGSC, a KRAS-mutant APST with a KRAS-mutant LGSC, and a BRAF-mutant APST with subsequent development of a KRAS-mutant high-grade serous carcinoma. In conclusion, some women with SBTs can subsequently develop serous carcinoma, occasionally over 10 years later. Most subsequent carcinomas are low grade, but a small subset can be high grade. The type of gene mutation in SBT correlates with various histologic features. While most cases of serous carcinoma developing after a diagnosis of SBT probably represent tumor progression, a minority are independent primary tumors, presumably arising from endosalpingiosis.

Simultaneous and Independent Dual Site-Specific Self-Labeling of Recombinant Antibodies.

Antibody-based diagnostic and therapeutic reagents armed with effector molecules such as dyes and drugs offer hope in the battle against cancer. Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve multiple reaction steps. The conjugation of two different effector molecules to a single antibody also remains a major challenge. Here we describe a simple, controlled, and robust method for the dual site-specific conjugation of an antibody with two effector molecules in a single-pot reaction using the self-labeling SNAP and CLIP protein tags. We verified the principle of the method by labeling an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425) simultaneously with IRDye700 and Alexa-Fluor647. This dual-labeled antibody bound to EGFR+ ovarian cancer cell lines and tissue samples with high specificity, and its phototherapeutic efficacy was confirmed by the selective killing of EGFR+ cells in vitro.

Mutation of NRAS is a rare genetic event in ovarian low-grade serous carcinoma.

Activating mutations involving the members of the RAS signaling pathway, including KRAS, NRAS, and BRAF, have been reported in ovarian low-grade serous carcinoma and its precursor lesion, serous borderline tumor (SBT). Whether additional genetic alterations in the RAS oncogene family accumulate during the progression of SBT to invasive low-grade serous carcinoma (LGSC) remains largely unknown. Although mutations of KRAS and BRAF occur at a very early stage of progression, even preceding the development of SBT, additional driving events, such as NRAS mutations, have been postulated to facilitate progression. In this study, we analyzed NRAS exon 3 mutational status in 98 cases that were diagnosed with SBT/atypical proliferative serous tumor, noninvasive LGSC, or invasive LGSC. Of the latter, NRAS Q61R (CAA to CGA) mutations were detected in only 2 of 56 (3.6%) cases. The same mutation was not detected in any of the SBTs (atypical proliferative serous tumors) or noninvasive LGSCs. Mutational analysis for hotspots in KRAS and BRAF demonstrated a wild-type pattern of KRAS and BRAF in one of the NRAS-mutated cases. Interestingly, another LGSC case with NRAS mutation harbored a concurrent BRAF V600L mutation. These findings indicate that, although recurrent NRAS mutations are present, their low prevalence indicates that NRAS plays a limited role in the development of LGSC. Further studies to identify other oncogenic events that drive LGSC progression are warranted.