PD-L1 quantification across tumor types using the reverse phase protein microarray: implications for precision medicine.

BACKGROUND: Anti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely used to assess PD-L1 expression, their clinical utility remains controversial due to the partial predictive value and lack of standardized cut-offs across antibody clones. Using a high throughput immunoassay, the reverse phase protein microarray (RPPA), coupled with a fluorescence-based detection system, this study compared the performance of six anti-PD-L1 antibody clones on 666 tumor samples. METHODS: PD-L1 expression was measured using five antibody clones (22C3, 28-8, CAL10, E1L3N and SP142) and the therapeutic antibody atezolizumab on 222 lung, 71 ovarian, 52 prostate and 267 breast cancers, and 54 metastatic lesions. To capture clinically relevant variables, our cohort included frozen and formalin-fixed paraffin-embedded samples, surgical specimens and core needle biopsies. Pure tumor epithelia were isolated using laser capture microdissection from 602 samples. Correlation coefficients were calculated to assess concordance between antibody clones. For two independent cohorts of patients with lung cancer treated with nivolumab, RPPA-based PD-L1 measurements were examined along with response to treatment. RESULTS: Median-center PD-L1 dynamic ranged from 0.01 to 39.37 across antibody clones. Correlation coefficients between the six antibody clones were heterogeneous (range: -0.48 to 0.95) and below 0.50 in 61% of the comparisons. In nivolumab-treated patients, RPPA-based measurement identified a subgroup of tumors, where low PD-L1 expression equated to lack of response. CONCLUSIONS: Continuous RPPA-based measurements capture a broad dynamic range of PD-L1 expression in human specimens and heterogeneous concordance levels between antibody clones. This high throughput immunoassay can potentially identify subgroups of tumors in which low expression of PD-L1 equates to lack of response to treatment.

Kinase-driven metabolic signalling as a predictor of response to carboplatin-paclitaxel adjuvant treatment in advanced ovarian cancers.

BACKGROUND: The biological mechanisms underlying early- and advanced-stage epithelial ovarian cancers (EOCs) are still poorly understood. This study explored kinase-driven metabolic signalling in early and advanced EOCs, and its role in tumour progression and response to carboplatin-paclitaxel treatment. METHODS: Tumour epithelia were isolated from two independent sets of primary EOC (n=72 and 30 for the discovery and the validation sets, respectively) via laser capture microdissection. Reverse phase protein microarrays were used to broadly profile the kinase-driven metabolic signalling of EOC with particular emphasis on the LBK1-AMPK and AKT-mTOR axes. Signalling activation was compared between early and advanced lesions, and carboplatin-paclitaxel-sensitive and -resistant tumours. RESULTS: Advanced EOCs were characterised by a heterogeneous kinase-driven metabolic signature and decreased phosphorylation of the AMPK-AKT-mTOR axis compared to early EOC (P<0.05 for AMPKα T172, AMPKα1 S485, AMPKß1 S108, AKT S473 and T308, mTOR S2448, p70S6 S371, 4EBP1 S65, GSK-3 α/ß S21/9, FOXO1 T24/FOXO3 T32, and FOXO1 S256). Advanced tumours with low relative activation of the metabolic signature and increased FOXO1 T24/FOXO3 T32 phosphorylation (P=0.041) were associated with carboplatin-paclitaxel resistance. CONCLUSIONS: If validated in a larger cohort of patients, the decreased AMPK-AKT-mTOR activation and phosphorylation of FOXO1 T24/FOXO3 T32 may help identify carboplatin-paclitaxel-resistant EOC patients.

The challenge of preoperative identification of uterine myomas: Is ultrasound trustworthy? A prospective cohort study.

PURPOSE: To correlate preoperative ultrasound examination with intraoperative and anatomo-pathological findings, including estimation of number, localization and size of uterine myomas, uterine diameters and volume. METHODS: A prospective study on 126 women undergoing surgery for uterine myomatosis at Campus Bio-medico between May 2013 and April 2014. The patients were divided into two groups: one submitted to hysterectomy and the other submitted to open myomectomy. Ultrasound scans were performed 1 day before surgery by the same expert sonographer. The number of myomas at ultrasound was compared to intraoperative visualization and anatomo-pathological findings. Wilcoxon Test was applied to compare data registered with each technique. RESULTS: There was no significant difference between the number of myomas recorded at visualization and at ultrasound, while there was a significant difference between visualization and anatomo-pathology (p = 0.0006). The analysis showed a non-significant difference between myoma number at ultrasound and at anatomo-pathology in the two groups, if the number of myomas was less than or equal to six. Contrarily, we observed a significant difference if the number of myomas was more than six (p = 0.003). CONCLUSIONS: Our data show that ultrasound has limits in identifying the exact number of uterine myomas. This mapping is particularly needed in a minority of patients with usually desiring fertility who need a debulking procedure due to the large size and/or number of myomas or myoma location causing symptomatology. In patients with more than six myomas, voluminous uterus, a second-level examination such as Magnetic Resonance may be helpful.

Functional characterization of epithelial ovarian cancer histotypes by drug target based protein signaling activation mapping: implications for personalized cancer therapy.

Epithelial ovarian carcinoma (EOC) is a deadly disease, with a 5-year survival of 30%. The aim of the study was to perform broad-scale protein signaling activation mapping to evaluate if EOC can be redefined based on activated protein signaling network architecture rather than histology. Tumor cells were isolated using laser capture microdissection (LCM) from 72 EOCs. Tumors were classified as serous (n = 38), endometrioid (n = 13), mixed (n = 8), clear cell (CCC; n = 7), and others (n = 6). LCM tumor cells were lysed and subjected to reverse-phase protein microarray to measure the expression/activation level of 117 protein drug targets. Unsupervised hierarchical clustering analysis was utilized to explore the overall signaling network. ANOVA was used to detect significant differences among the groups (p < 0.05). Regardless of histology, unsupervised analysis revealed five pathway-driven clusters. When the EOC histotypes were compared by ANOVA, only CCC showed a distinct signaling network, with activation of EGFR, Syk, HER2/ErbB2, and SHP2 (p = 0.0007, p = 0.0021, p < 0.0001, and p = 0.0410, respectively). The histological classification of EOC fails to adequately describe the underpinning protein signaling network. Nevertheless, CCC presents unique signaling characteristics compared to the other histotypes. EOC may need to be characterized by functional signaling activation mapping rather than pure histology.

Reverse phase protein microarrays and their utility in drug development.

The majority of human diseases, including cancer, are characterized by abnormal protein function. Proteins regulate virtually every cellular process and exhibit multiple kinds of post-translational modification that modulate expression levels and activation states, such as phosphorylation by protein kinases. Additionally proteins interact with each other in complex regulatory networks and signal transduction pathways modulated by feedback mechanisms. These pathways are disrupted in disease and altered by therapeutic drugs. Reverse phase protein microarray (RPMA) technology allows simultaneous measurement of numerous phosphorylated, glycosylated, cleaved, or total cellular proteins from complex mixtures in many samples at once. Therefore, RPMAs can provide a portrait of a cell's signaling pathways in diseased states, before and after treatment with drugs, and allows comparison of changes in drug-resistant and sensitive cells. Furthermore, the technology offers a means of connecting genomic abnormalities in cancer to targetable alterations in protein signaling pathways, even for genetic events that seem otherwise undruggable. Consequently, the RPMA platform has great utility in many steps of drug development including target identification, validation of a pharmaceutical agent's efficacy, understanding mechanisms of action, and discovery of biomarkers that predict or guide therapeutic response. RPMAs have become a powerful tool for drug development and are now being integrated into human clinical cancer trials, where they are being used to personalize therapy.

Intraoperative contact ultrasonography during open myomectomy for uterine fibroids.

OBJECTIVE: To evaluate the benefit of intraoperative ultrasound applied directly to the uterine serosa during surgery for uterine fibroids. DESIGN: Prospective study. SETTING: University hospital, tertiary care. PATIENT(S): Women admitted for open myomectomy due to uterine fibroids. INTERVENTION(S): Intraoperative ultrasound (IUS) and intraoperative palpation were performed to detect the number of residual fibroids at the end of surgery, then the number of fibroids was recorded at anatomopathology examination. MAIN OUTCOME MEASURE(S): Residual fibroids detected at IUS and intraoperative palpation at the end of open myomectomy. RESULT(S): The comparison between the number of residual fibroids at IUS and at intraoperative palpation was statistically significant. CONCLUSION(S): Intraoperative ultrasound is more efficient than palpation in detecting residual leiomyomata at the end of open myomectomy.

A gastrointestinal stromal tumor presenting as a pelvic mass: A case report.

Gastrointestinal stromal tumors (GISTs) represent 0.1-1% of gastrointestinal malignancies. They are commonly asymptomatic and found incidentally during laparoscopy, surgical procedures or radiological studies. Diagnosis is based on histology and immunohistochemistry, while the role of imaging studies is not diagnosis-specific. We present the case of a 38-year-old patient complaining of an increase in her abdominal circumference. Consequently, a vaginal examination, a transvaginal ultrasound and an MRI of the abdomen and pelvis were carried out. It should be noted that a preoperative diagnosis of GISTs is uncommon, due to the rarity and many presentations of the disease. Ultrasound and MRI are not able to differentiate a GIST from ovarian cancer. However, if a pelvic mass is detected, the possibility of a non-gynaecological tumor has to be considered.

Update on lymphadenectomy in early and advanced ovarian cancer.

PURPOSE OF REVIEW: Pelvic and para-aortic lymphnode sampling is an integral part of the staging system of ovarian cancer. The issue concerning lymphadenectomy in the management of the disease is still debated, however. The purpose of this paper is to review the role of systematic lymphadenectomy in patients affected by early and advanced-stage ovarian cancer. RECENT FINDINGS: Some retrospective studies have revealed an increased survival rate in early-stage ovarian cancer patients after lymphadenectomy. Recently, the first randomized prospective trial, on lymphadenectomy in advanced-stage disease, was published. It evidenced an improvement in progression-free survival in patients who had undergone lymphadenectomy. SUMMARY: Systematic lymphadenectomy has a diagnostic value in early-stage ovarian cancer, thanks to the possibility of accurate clinical staging. As up to 22% of women, who were presumed to have early-stage ovarian cancers, are upstaged during the lymphadenectomy procedure, accurate staging can help to avoid unnecessary postoperative chemotherapy. In patients affected by advanced ovarian cancer, systematic lymphadenectomy statistically significantly improves progression-free survival and reduces recurrence rates despite a higher incidence of postoperative complications. As improvement of overall survival is not statistically significant, further studies are needed to balance risks and benefits of systematic lymphadenectomy in advanced-stage disease.