Atlas of Tumor and Tumor Microenvironment Cells of Lymphovascular Space Invasion (LVSI) in High-Grade Serous Endometrial Adenocarcinoma: A Case Study.

Lymphovascular invasion (LVSI) is defined as the presence of tumor cells within a definite endothelial-lined space (lymphatics or blood vessels) in the organ surrounding invasive carcinoma. The presence of LVI is associated with an increased risk of lymph nodes and distant metastases. Lymphovascular invasion is described as cancer within blood or lymph vessels and is an independent risk factor for metastasis, recurrence, and mortality. This study aims to present the marker-based immunohistological characterization of cells around LVSI in a high-grade adenocarcinoma of the endometrium to build a cellular atlas of cells of LVSI. A cellular characterization of the cells around lymphovascular space invasion in a 67-year-old female patient with invasive high-grade serous endometrial adenocarcinomas is presented. Resected tumor tissue from a consented patient with invasive high-grade serous endometrial adenocarcinoma was obtained within an hour of surgery. The expressions of the epithelial markers (CK8, 18, and EpCAM), LCA (leukocyte common antigen) marker (CD45), proliferation marker (Ki67), apoptosis markers (cleaved PARP and cleaved caspase3), immune cell markers (CD3, CD4, CD8, CD56, CD68, CD163, FoxP3, PD-1, PD-L1), pro-inflammatory marker (IL-12-RB2), and fibroblast/mesenchyme markers (S100A7, SMA, and TE-7) of the resected tissue on the IHC stains were evaluated and scored by a pathologist. Acknowledging the deterministic role of LVSI in a high-grade adenocarcinoma of the endometrium, our study presents the first marker-based immunohistological atlas of the tumor and TME compartments in the context of epithelial cell markers, proliferation markers, apoptosis markers, macrophage markers, and fibroblast markers. Our study demonstrates that an aggressive disease like a high-grade adenocarcinoma of the endometrium inflicts the pro-metastatic event of LVSI by involving the immune landscape of both tumor and TME. This study demonstrates, for the first time, that the tumor cells within LVSI are positive for IL-12R-B2 and S100A4.

A landscape of patient-derived cancer-associated fibroblast signals in endometrial cancers.

In conversation with endometrial tumor cells, the endometrial cancer-associated fibroblasts (CAFs) are the "partners in crime" of uterine neoplasm's highly heterogeneous tumor microenvironment (TME). We designed a laboratory-friendly method to culture endometrial CAFs on a patient-to-patient basis for studying the CAF-TME and CAF-tumor cell interaction(s). Here, we present a comprehensive characterization of endometrial CAFs derived from patients' tumor tissues (T) and tumor-adjacent normal tissues (N). We used more than 80 T and N from 53 consecutive consented patients with endometrial cancers at the Avera Cancer Institute. We derived TCAF and NCAF in a non-enzymatic feeder-layer culture and characterized their expression of markers by qRT-PCR, flow cytometry, immunocytochemistry, immunofluorescence, and Western blot. Although similar in the expression pattern of EpCAM-/CK18-/vimentin+ as in ovarian CAFs, endometrial NCAFs, and TCAFs characteristically presented dual morphology in culture. Endometrial CAFs were EpCAM-/CK18-/CD45-/CD31-/SMA+/TE-7+/PDGFRA+/CXCL12+/Meflin+/CD155+/CD90+ with patient-specific positivity for S100A4/FAP/PD-L1/CD44. Endometrial CAFs expressed mRNAs for signaling proteins of several pathways and receptor-ligands, including (1) cell cycle pathway, (2) TGF pathway, (3) FGF pathway, (4) Wnt-beta-catenin pathway, (5) HER pathway, (6) tyrosine kinase receptor ligands, and (7) steroid receptors. We tested the hypoxic response of CAFs to show that endometrial CAFs upregulate MMP1 in a HIF-1a-independent manner. In trying to delineate the relationship between expressions of CAF markers and T-cells in the tumor tissue, we observed that FAP-positive CAFs that are derived from CD4/CD8 positive tumor tissue expressed CXCL12 mRNA. The data indicate the role of the CXCL12-CXCR4 pathway of the CAF-rich stroma in the lymphocytic infiltration of the tumor. We demonstrate that endometrial CAFs can be cultured in an enzymatic-digestion-independent manner, and their signaling landscape can be mapped toward understanding CAF-TME dialogue. Our data will help unearth the functional relevance of endometrial CAFs in the context of clinical outcomes and designing CAF-inclusive therapy in the future.

Neuregulin-1 and ALS19 (ERBB4): at the crossroads of amyotrophic lateral sclerosis and cancer.

BACKGROUND: Neuregulin-1 (NRG1) is implicated in both cancer and neurologic diseases such as amyotrophic lateral sclerosis (ALS); however, to date, there has been little cross-field discussion between neurology and oncology in regard to these genes and their functions. MAIN BODY: Approximately 0.15-0.5% of cancers harbor NRG1 fusions that upregulate NRG1 activity and hence that of the cognate ERBB3/ERBB4 (HER3/HER4) receptors; abrogating this activity with small molecule inhibitors/antibodies shows preliminary tissue-agnostic anti-cancer activity. Notably, ERBB/HER pharmacologic suppression is devoid of neurologic toxicity. Even so, in ALS, attenuated ERBB4/HER4 receptor activity (due to loss-of-function germline mutations or other mechanisms in sporadic disease) is implicated; indeed, ERBB4/HER4 is designated ALS19. Further, secreted-type NRG1 isoforms may be upregulated (perhaps via a feedback loop) and could contribute to ALS pathogenesis through aberrant glial cell stimulation via enhanced activity of other (e.g., ERBB1-3/HER1-3) receptors and downstream pathways. Hence, pan-ERBB inhibitors, already in use for cancer, may be agents worthy of testing in ALS. CONCLUSION: Common signaling cascades between cancer and ALS may represent novel therapeutic targets for both diseases.

Tumor-TME Bipartite Landscape of PD-1/PD-L1 in Endometrial Cancers.

The bipartite landscape of tumor cells and stromal cells determines a tumor's response to treatment during disease management. In endometrial cancers (ECs), the mechanistic contribution of PD-L1/L2 and PD-1 signaling of the host's tumor microenvironment (TME) (CAF and immune cells) in the context of the tumor cells is elusive. To understand the tumor-stroma-immune crosstalk, we studied the compartmental pattern of PD-L1/L2 and PD-1 expression in EC tissues and their matched CAFs. Over 116 surgically resected tumors (T) and the tumor-adjacent normal tissues (N) were obtained from consented unselected consecutive patients. IHC was performed in T, N-epi-thelium, and the stromal mesenchymal environment (SME; mesenchyme) in the T and N tissues. The staining intensity and distribution patterns of PD-L1/L2 and PD-1 in the FFPE sections of T and N were evaluated by a pathologist using a standard scoring system of TPS and CPS. We tested the PD-L1/L2 and PD-1 immune landscape of tumor-TME pair and normal epithelial-stromal mesenchyme pairs from patients with different grades of disease vis-à-vis their CAF PD-L1 levels. We used qRT-PCR to determine the expressions of mRNAs, while the flow cytometry and ICC determined the level of expression of proteins. We observed higher levels of PD-L1 mRNA and protein expression in primary CAFs from the resected tumor tissue compared to the tumor-adjacent normal tissues. We also determined the expression of patients' soluble PD-L1/L2 as peripheral readouts of PD-L1/L2 and PD-1. As we evaluated the results in the context of their pathological parameters, such as grades, stages, lymphovascular invasion, percentage of myometrial invasion, and dMMR in patients, the dominance of PD-L1 expression in TME was positively correlated to the higher pathological grades of tumors, and its relationship with the dMMR. Since the neutralization of CD8-positive cytotoxic T-cells is PD-L1-dependent, our data indicate that irrespective of the PD-L1 positivity of tumor cells, the PD-L1-positive CAFs can play a critical role in bringing out an additional load of PD-L1 for an effective engagement of PD-1 within a tumor mass.

A CAF-Based Two-Cell Hybrid Co-Culture Model to Test Drug Resistance in Endometrial Cancers.

The management of advanced or recurrent endometrial cancers presents a challenge due to the development of resistance to treatments. The knowledge regarding the role of the tumor microenvironment (TME) in determining the disease's progression and treatment outcome has evolved in recent years. As a TME component, cancer-associated fibroblasts (CAFs) are essential in developing drug-induced resistance in various solid tumors, including endometrial cancers. Hence, an unmet need exists to test the role of endometrial CAF in overcoming the roadblock of resistance in endometrial cancers. We present a novel tumor-TME two-cell ex vivo model to test CAF's role in resisting the anti-tumor drug, paclitaxel. Endometrial CAFs, both NCAFs (tumor-adjacent normal-tissue-derived CAFs) and TCAFs (tumor-tissue-derived CAFs) were validated by their expression markers. Both TCAFs and NCAFs expressed positive markers of CAF, including SMA, FAP, and S100A4, in varying degrees depending on the patients, while they consistently lacked the negative marker of CAF, EpCAM, as tested via flow cytometry and ICC. CAFs expressed TE-7 and immune marker, PD-L1, via ICC. CAFs better resisted the growth inhibitory effect of paclitaxel on endometrial tumor cells in 2D and 3D formats compared to the resistance of the tumoricidal effect of paclitaxel in the absence of CAFs. TCAF resisted the growth inhibitory effect of paclitaxel on endometrial AN3CA and RL-95-2 cells in an HyCC 3D format. Since NCAF similarly resisted the growth inhibitor action of paclitaxel, we tested NCAF and TCAF from the same patient to demonstrate the protective action of NCAF and TCAF in resisting the tumoricidal effect of paclitaxel in AN3CA in both 2D and 3D matrigel formats. Using this hybrid co-culture CAF and tumor cells, we established a patient-specific, laboratory-friendly, cost-effective, and time-sensitive model system to test drug resistance. The model will help test the role of CAFs in developing drug resistance and contribute to understanding tumor cell-CAF dialogue in gynecological cancers and beyond.

LAG-3 transcriptomic expression patterns across malignancies: Implications for precision immunotherapeutics.

BACKGROUND: Lymphocyte activation gene 3 (LAG-3) or CD223 is a transmembrane protein that serves as an immune checkpoint which attenuates T-cell activation. Many clinical trials of LAG-3 inhibitors have had modest effects, but recent data indicate that the LAG-3 antibody relatlimab, together with nivolumab (anti-PD-1), provided greater benefit than nivolumab alone in patients with melanoma. METHODS: In this study, the RNA expression levels of 397 genes were assessed in 514 diverse cancers at a clinical-grade laboratory (OmniSeq: https://www.omniseq.com/). Transcript abundance was normalized to internal housekeeping gene profiles and ranked (0-100 percentile) using a reference population (735 tumors; 35 histologies). RESULTS: A total of 116 of 514 tumors (22.6%) had high LAG-3 transcript expression (≥75 percentile rank). Cancers with the greatest proportion of high LAG-3 transcripts were neuroendocrine (47% of patients) and uterine (42%); colorectal had among the lowest proportion of high LAG-3 expression (15% of patients) (all p < 0.05 multivariate); 50% of melanomas were high LAG-3 expressors. There was significant independent association between high LAG-3 expression and high expression of other checkpoints, including programmed death-ligand 1 (PD-L1), PD-1, and CTLA-4, as well as high tumor mutational burden (TMB) ≥10 mutations/megabase, a marker for immunotherapy response (all p < 0.05 multivariate). However, within all tumor types, there was inter-patient variability in LAG-3 expression level. CONCLUSIONS: Prospective studies are therefore needed to determine if high levels of the LAG-3 checkpoint are responsible for resistance to anti-PD-1/PD-L1 or anti-CTLA-4 antibodies. Furthermore, a precision/personalized immunotherapy approach may require interrogating individual tumor immunograms to match patients to the right combination of immunotherapeutic agents for their malignancy.

Characterization and Clinical Relevance of Endometrial CAFs: Correlation between Post-Surgery Event and Resistance to Drugs.

Cancer-associated fibroblasts (CAFs) within a solid tumor can support the progression of cancer. We studied the identification and characterization of patient-derived endometrial CAFs in the context of their clinical relevance in endometrial cancers. We established patient-derived primary cultures of CAFs from surgically resected tumors (TCAF) and tumor-adjacent normal (NCAF) tissues in 53 consented patients with success rates of 97.7% and 75%, respectively. A passage of CAF was qualified by the (1) absence of CK 8,18,19, EpCAM, CD45, and CD31, and (2) presence of SMAalpha, S100A4, CD90, FAP, TE-7, CD155, PD-L1, TGFB, PDGFRA (qRT-PCR, flow cytometry, Western blot, ICC). Out of the 44 established CAFs, 31 were aggressive (having an early, i.e., 4-7 week, establishment time and/or >3 passages) compared to 13 which were non-aggressive. A post-surgery-event (PSE) was observed in 7 out of 31 patients bearing aggressive CAFs, 2 of whom were also positive for CTCs, while none of the 13 patients bearing non-aggressive CAFs had events. A positive correlation was found between patients with grade 3 (p = 0.025) as well as stage 3/4 diseases (p = 0.0106) bearing aggressive CAFs and the PSE. Finally, aggressive TCAFs from patients with PSE resisted the effects of paclitaxel and lenvatinib on the growth of HUVEC and endometrial tumor cells. Our study is the first to report a correlation between the PSE and the aggressive nature of CAFs in endometrial cancers and provides an undeniable reason to study the in-depth mechanism of CAF function towards the development of treatment resistance in endometrial cancers.

Patient-Derived Primary Cancer-Associated Fibroblasts Mediate Resistance to Anti-Angiogenic Drug in Ovarian Cancers.

Ovarian cancers rank first in both aggressiveness and dismal prognosis among gynecological neoplasms. The poor outcome is explained by the fact that most patients present with late-stage disease and progress through the first line of treatment. Ovarian neoplasms, especially epithelial ovarian cancers, are diagnosed at advanced/metastatic stages, often with a high angiogenesis index, one of the hallmarks of ovarian cancers with rapid progression and poor outcome as resistance to anti-angiogenic therapy develops. Despite therapy, the metastatic progression of aggressive ovarian cancer is a spectacularly selective function of tumor cells aided and abetted by the immune, mesenchymal and angiogenic components of the tumor microenvironment (TME) that enforces several pro-metastatic event(s) via direct and indirect interactions with stromal immune cells, cancer-associated fibroblasts (CAFs), and vascular endothelial cells. Since transdifferentiation of tumor endothelium is one of the major sources of CAFs, we hypothesized that ovarian CAF plays a critical role in resisting anti-angiogenic effects via direct crosstalk with endothelium and hence plays a direct role in the development of resistance to anti-angiogenic drugs. To test the hypothesis, we set up a hybrid ex vivo model for co-culture comprising Patient-Derived ex vivo primary CAFs from ovarian tumor samples and human umbilical vein endothelial cells (HUVEC). Patient-Derived CAFs were characterized by the mRNA and protein expression of positive (SMA, S100A4, TE-7, FAP-A, CD90/THY1), negative (EpCAM, CK 8,18, CD31, CD44, CD45), functional (PDGFRA, TGFB1, TGFB2, TGFRA) and immunological markers (PD-L1, PD-L2, PD-1) associated with CAFs by qRT-PCR, flow cytometry, Western blot, and ICC. Data from our HUVEC-on-CAF ex vivo Hybrid Co-Culture (HyCC) study demonstrate the pro-angiogenic effect of Patient-Derived ovarian CAFs by virtue of their ability to resist the effect of anti-angiogenic drugs, thereby aiding the development of resistance to anti-angiogenic drugs. Ascertaining direct experimental proof of the role of CAFs in developing resistance to specific anti-angiogenic drugs will provide an opportunity to investigate new drugs for counteracting CAF resistance and "normalizing/re-educating" TME in aggressive ovarian cancers. Our data provide a unique experimental tool for the personalized testing of anti-angiogenic drugs, positively predicting the development of future resistance to anti-angiogenic drugs well before it is clinically encountered in patients.

Identification and Morphological Characterization of Features of Circulating Cancer-Associated Macrophage-like Cells (CAMLs) in Endometrial Cancers.

The blood of patients with solid tumors contains circulating tumor-associated cells, including epithelial cells originating from the tumor mass, such as circulating tumor cells (CTCs), or phagocytic myeloid cells (differentiated monocytes), such as circulating cancer-associated macrophage-like cells (CAMLs). We report for the first time the identification and in-depth morphologic characterization of CAMLs in patients with endometrial cancers. We isolated CAMLs by size-based filtration on lithographically fabricated membranes followed by immunofluorescence, using a CD45+/CK 8,18,19+/EpCAM+/CD31+/macrophage-like nuclear morphology, from > 70 patients. Irrespective of the histological and pathological parameters, 98% of patients were positive for CAMLs. Two size-based subtypes of CAMLs, <20 µm (tiny) and >20 µm (giant) CAMLs, of distinctive polymorphic morphologies with mononuclear or fused polynuclear structures in several morphological states were observed, including apoptotic CAMLs, CAML−WBC doublets, conjoined CAMLs, CAML−WBC clusters, and CTC−CAML−WBC clusters. In contrast, CAMLs were absent in patients with non-neoplastic/benign tumors, healthy donors, and leucopaks. Enumerating CTCs simultaneously from the same patient, we observed that CTC-positive patients are positive for CAMLs, while 55% out of all CAML-positive patients were found positive for CTCs. Our study demonstrated for the first time the distinctive morphological characteristics of endometrial CAMLs in the context of the presence of CTCs in patients.

Osteosarcoma with cell-cycle and fibroblast growth factor genomic alterations: case report of Molecular Tumor Board combination strategy resulting in long-term exceptional response.

There is a paucity of information about molecularly driven therapy in osteosarcomas. We report a 31-year-old woman with chemotherapy-refractory metastatic osteosarcoma who was successfully treated with the combination of palbociclib (CDK4/6 inhibitor) and lenvatinib (multikinase FGFR inhibitor), selected based on next generation sequencing that showed CDK4 and CCND2 amplifications (upregulates CDK4/6), and FGF6 (ligand for FGFR1,2 and 4), FGF23 (ligand for FGFR1,2,3, and 4) and FRS2 (adaptor protein for FGFR signaling) amplifications. The patient's tumor showed 68% reduction in positron emission tomography (PET) avidity, lasting 31 months after therapy initiation, when a solitary recurrence occurred, was resected, and treatment continued. The patient remains on matched targeted therapy at 51 + months from the start of the combination. Treatment was given at reduced dosing (lenvatinib 10 mg oral daily (approved dose = 24 mg daily)) and palbociclib 75 mg oral daily, one week on and one week off (approved dose = 125 mg oral daily, three weeks on/one week off) and is tolerated well. Therefore, co-targeting the aberrant cyclin and FGFR pathways resulted in long-term exceptional response in a patient with refractory advanced osteosarcoma.

Precision Medicine in Solid Tumors: How Far We Traveled So Far?

The future of disease management in solid tumors will rely heavily on how effectively we understand precision medicine and how successfully we can deliver personalized medicine [...].

A Laboratory-Friendly CTC Identification: Comparable Double-Immunocytochemistry with Triple-Immunofluorescence.

The source of circulating tumor cells (CTC) in the peripheral blood of patients with solid tumors are from primary cancer, metastatic sites, and a disseminated tumor cell pool. As 90% of cancer-related deaths are caused by metastatic progression and/or resistance-associated treatment failure, the above fact justifies the undeniable predictive and prognostic value of identifying CTC in the bloodstream at stages of the disease progression and resistance to treatment. Yet enumeration of CTC remains far from a standard routine procedure either for post-surgery follow-ups or ongoing adjuvant therapy. The most compelling explanation for this paradox is the absence of a convenient, laboratory-friendly, and cost-effective method to determine CTC. We presented a specific and sensitive laboratory-friendly parallel double-detection format method for the simultaneous isolation and identification of CTC from peripheral blood of 91 consented and enrolled patients with various malignant solid tumors of the lung, endometrium, ovary, esophagus, prostate, and liver. Using a pressure-guided method, we used the size-based isolation to capture CTC on a commercially available microfilter. CTC identification was carried out by two expression marker-based independent staining methods, double-immunocytochemistry parallel to standard triple-immunofluorescence. The choice of markers included specific markers for epithelial cells, EpCAM and CK8,18,19, and exclusion markers for WBC, CD45. We tested the method's specificity based on the validation of the staining method, which included positive and negative spiked samples, blood from the healthy age-matched donor, healthy age-matched leucopaks, and blood from metastatic patients. Our user-friendly cost-effective CTC detection technique may facilitate the regular use of CTC detection even in community-based cancer centers for prognosis, before and after surgery.

Targeting Cell Cycle Progression in HER2+ Breast Cancer: An Emerging Treatment Opportunity.

The development of HER2-targeted therapies has dramatically improved patient survival and patient management and increased the quality of life in the HER2+ breast cancer patient population. Due to the activation of compensatory pathways, patients eventually suffer from resistance to HER2-directed therapies and develop a more aggressive disease phenotype. One of these mechanisms is the crosstalk between ER and HER2 signaling, especially the CDK4/6-Cyclin D-Rb signaling axis that is commonly active and has received attention for its potential role in regulating tumor progression. CDK 4/6 inhibitors interfere with the binding of cell-cycle-dependent kinases (CDKs) with their cognate partner cyclins, and forestall the progression of the cell cycle by preventing Rb phosphorylation and E2F release that consequentially leads to cancer cell senescence. CDK 4/6 inhibitors, namely, palbociclib, ribociclib, and abemaciclib, in combination with anti-estrogen therapies, have shown impressive outcomes in hormonal receptor-positive (HR+) disease and have received approval for this disease context. As an extension of this concept, preclinical/clinical studies incorporating CDK 4/6 inhibitors with HER2-targeted drugs have been evaluated and have shown potency in limiting tumor progression, restoring therapeutic sensitivity, and may improving the management of the disease. Currently, several clinical trials are examining the synergistic effects of CDK 4/6 inhibitors with optimized HER2-directed therapies for the (ER+/-) HER2+ population in the metastatic setting. In this review, we aim to interrogate the burden of HER2+ disease in light of recent treatment progress in the field and examine the clinical benefit of CDK 4/6 inhibitors as a replacement for traditional chemotherapy to improve outcomes in HER2+ breast cancer.

Bête Noire of Chemotherapy and Targeted Therapy: CAF-Mediated Resistance.

In tumor cells' struggle for survival following therapy, they resist treatment. Resistance to therapy is the outcome of well-planned, highly efficient adaptive strategies initiated and utilized by these transformed tumor cells. Cancer cells undergo several reprogramming events towards adapting this opportunistic behavior, leading them to gain specific survival advantages. The strategy involves changes within the transformed tumors cells as well as in their neighboring non-transformed extra-tumoral support system, the tumor microenvironment (TME). Cancer-Associated Fibroblasts (CAFs) are one of the components of the TME that is used by tumor cells to achieve resistance to therapy. CAFs are diverse in origin and are the most abundant non-transformed element of the microenvironment in solid tumors. Cells of an established tumor initially play a direct role in the establishment of the CAF population for its own microenvironment. Like their origin, CAFs are also diverse in their functions in catering to the pro-tumor microenvironment. Once instituted, CAFs interact in unison with both tumor cells and all other components of the TME towards the progression of the disease and the worst outcome. One of the many functions of CAFs in influencing the outcome of the disease is their participation in the development of resistance to treatment. CAFs resist therapy in solid tumors. A tumor-CAF relationship is initiated by tumor cells to exploit host stroma in favor of tumor progression. CAFs in concert with tumor cells and other components of the TME are abettors of resistance to treatment. Thus, this liaison between CAFs and tumor cells is a bête noire of therapy. Here, we portray a comprehensive picture of the modes and functions of CAFs in conjunction with their role in orchestrating the development of resistance to different chemotherapies and targeted therapies in solid tumors. We investigate the various functions of CAFs in various solid tumors in light of their dialogue with tumor cells and the two components of the TME, the immune component, and the vascular component. Acknowledgment of the irrefutable role of CAFs in the development of treatment resistance will impact our future strategies and ability to design improved therapies inclusive of CAFs. Finally, we discuss the future implications of this understanding from a therapeutic standpoint and in light of currently ongoing and completed CAF-based NIH clinical trials.

Addressing activation of WNT beta-catenin pathway in diverse landscape of endometrial carcinogenesis.

The WNT-beta-catenin pathway (WP) is one of the major oncogenic pathways in solid tumors. Wnt beta-catenin pathway plays a unique role in a wide range of endometrial dysfunctions, from embryo implantation failure to severe pathogenic changes like endometriosis and endometrial cancer. Although abnormal activation of the pathway has long been known to be associated with endometrial tumorigenesis, the pathway's exact mode of involvement remains to be understood. As more evidence has been presented in favor of a crucial role of the WP in solid tumors, including endometrial cancer, anti-WP drugs are currently being tested to manage the disease. Aggressive tumor cells are nurtured by the tumor microenvironment (TME). The genetic alterations within tumor cells are the primary driving force to activate the extra-tumoral micro-environment. TME (a) provides metabolic support for the proliferation of tumor cells, (b) orchestrates immune-evasion, (c) initiates mechanistic signaling for several metastasis-associated phenotypes, and (d) supports cellular events for the development of drug resistance. To get metabolic as well as immune support from the tumor microenvironment, tumor cells cross-talk with components of the TME, most critically to the cancer-associated fibroblasts. Thus it is expected that the tumor-TME cross-talk throughout the process of tumorigenesis and metastasis is one of the characteristic features of an aggressive tumor. Here we review the WP's mechanistic involvement as a common culprit (Un Colpevole Comune) in endometrial tumor cells and endometrial cancer-associated fibroblast (CAF). In this review, we have attempted to discuss the activation of the WP in the genesis and progression of endometrial cancers, including endometrial tumor biology, tumor microenvironment, cancer-associated fibroblasts, and wnt-beta catenin genetic alteration. We interrogated the available literature on the various aspects of endometrial carcinogenesis leading to the pathway's activation. We examined how genetic alterations in WP directly influence tumor cell signaling to bring out different tumor cell phenotypes, and present palpable evidence to envision a role of WP inhibitors in the future management of the disease.

Molecular profiling of advanced malignancies guides first-line N-of-1 treatments in the I-PREDICT treatment-naïve study.

BACKGROUND: Malignancies are molecularly complex and become more resistant with each line of therapy. We hypothesized that offering matched, individualized combination therapies to patients with treatment-naïve, advanced cancers would be feasible and efficacious. Patients with newly diagnosed unresectable/metastatic, poor-prognosis cancers were enrolled in a cross-institutional prospective study. METHODS: A total of 145 patients were included in the study. Genomic profiling (tissue and/or circulating tumor DNA) was performed in all patients, and PD-L1 immunohistochemistry, tumor mutational burden, and microsatellite status assessment were performed in a subset of patients. We evaluated safety and outcomes: disease-control rate (stable disease for ≥ 6 months or partial or complete response), progression-free survival (PFS), and overall survival (OS). RESULTS: Seventy-six of 145 patients (52%) were treated, most commonly for non-colorectal gastrointestinal cancers, carcinomas of unknown primary, and hepatobiliary malignancies (53% women; median age, 63 years). The median number of deleterious genomic alterations per patient was 5 (range, 0-15). Fifty-four treated patients (71%) received ≥ 1 molecularly matched therapy, demonstrating the feasibility of administering molecularly matched therapy. The Matching Score, which reflects the percentage of targeted alterations, correlated linearly with progression-free survival (R2 = 0.92; P = 0.01), and high (≥ 60%) Matching Score was an independent predictor of improved disease control rate [OR 3.31 (95% CI 1.01-10.83), P = 0.048], PFS [HR 0.55 (0.28-1.07), P = 0.08], and OS [HR 0.42 (0.21-0.85), P = 0.02]. Serious adverse event rates were similar in the unmatched and matched groups. CONCLUSIONS: Personalized combination therapies targeting a majority of a patient's molecular alterations have antitumor activity as first-line treatment. These findings underscore the feasibility and importance of using tailored N-of-1 combination therapies early in the course of lethal malignancies. TRIAL REGISTRATION: I-PREDICT ( NCT02534675 ) was registered on August 25, 2015.

Cancer-Associated Fibroblast Functions as a Road-Block in Cancer Therapy.

The journey of a normal resident fibroblast belonging to the tumor microenvironment (TME) from being a tumor pacifier to a tumor patron is fascinating. We introduce cancer-associated fibroblast (CAF) as a crucial component of the TME. Activated-CAF partners with tumor cells and all components of TME in an established solid tumor. We briefly overview the origin, activation, markers, and overall functions of CAF with a particular reference to how different functions of CAF in an established tumor are functionally connected to the development of resistance to cancer therapy in solid tumors. We interrogate the role of CAF in mediating resistance to different modes of therapies. Functional diversity of CAF in orchestrating treatment resistance in solid tumors portrays CAF as a common orchestrator of treatment resistance; a roadblock in cancer therapy.

A tipping-point for apoptosis following dual inhibition of HER2 signaling network by T-DM1 plus GDC-0980 maximizes anti-tumor efficacy.

HER2 signaling network and its complex relationship with the PI3K-AKT-mTOR pathway explain the acquired resistance to anti-HER2 therapy observed in clinics. Such complexity has been clinically evident from the limited efficacy of data in the BOLERO-1 and BOLERO-3 trials, which tested combinations of trastuzumab (T), everolimus, and chemotherapy in women with HER2+ advanced BC. In the following MARIANNE trial also, a combination of T-DM1 plus pertuzumab delivered a non-inferior but yet not superior PFS compared to trastuzumab plus a taxane. Algorithmic inhibition of PI3K/mTOR along with T or T-DM1 is, therefore, an attractive drug combination, and we tested the combination(s) in HER2+ BC, especially in T-resistant and PIK3CA mutated conditions. GDC-0980, a dual pan-PI3K/mTOR inhibitor alone or in combination with T or T-DM1, was examined in a panel of HER2+ T-sensitive (BT474, SKBR3), HER2+ T-resistant (BT474HerR), HER2+/PIK3CA mutant (HCC1954, MDA-MB453), and HER2+/PTEN mutant (HCC1569) BC cell lines. GDC-0980 re-sensitized trastuzumab-resistant, PIK3CA mutant, or PTEN mutant cells to T and acted additively with T. Importantly, this activity was more when GDC-0980 is combined with T-DM1. The combination (with T or with T-DM1) was then tested in the HER2+/T-sensitive, HER2+/T-resistant, and HER2+/PIK3CA mutated BC xenograft models for the anti-tumor effect. Along with its anti-tumor effect, GDC-0980 effectively decreased tumor angiogenesis (CD31 staining). Maximum anti-tumor (from tumor growth inhibition to tumor regression) efficiency was observed in all three xenograft models when T-DM1 was combined with GDC-0980. The anti-proliferative effects of GDC-0980 as evidenced by a decreased p-AKT (Ser473, The308), p-P70S6K, p-S6RP, and p-4EBP1, along with blockade of clonogenic 3D growth was accompanied by the initiation of apoptotic activity (annexin V, CASPASE3, cleaved PARP1 and mitochondrial depolarization); and was significantly superior when GDC-0980 combined with T-DM1. Interestingly, both trastuzumab and T-DM1 induce PD-L1 expression in HER2 amplified BC cells. Our data provide evidence that an oncogenic mutation of PIK3CA and HER2-amplification may represent biomarkers to identify patients who may benefit most from the use of GDC-0980 and an opportunity to include immunotherapy in the combination of anti-HER2 therapy.

A Triple-Parameter-Based Laboratory-Friendly Fluorescence Imaging to Identify Apoptosis in Live Cells.

Cellular signals to resist apoptosis have been attributed as one of the mechanisms of tumorigenesis. Hence, apoptosis is a cardinal target for drug development in cancers, and several antitumor drugs have been designed to induce apoptosis in tumor cells. Recently, venetoclax, a Bcl2 inhibitor that induces apoptosis, has been approved by the FDA for the treatment of CLL and SLL patients. Proapoptotic antitumor drugs have been traditionally developed and tested, targeting apoptosis in tumor cells. The mechanism of such drug actions has been functionally connected to the mechanism of apoptosis. The identification of apoptosis in a tumor cell takes into account different characteristics in several steps of apoptosis. Thus, it is understandable that modes of identification of apoptosis observed in tumor cells in a laboratory have also been tuned to different characteristics in several parameters of apoptosis. Here, we present a detailed methodology for a triple-parameter-based co-fluorescence imaging to identify apoptosis in live tumor cells. The procedure involves co-fluorescence staining specific for three cardinal features of apoptosis in live cells. The procedure is simple, time-sensitive, and can be performed successfully in a laboratory-friendly manner.

Targeted Neoadjuvant Therapies in HR+/HER2-Breast Cancers: Challenges for Improving pCR.

A strong association of pCR (pathological complete response) with disease-free survival or overall survival is clinically desirable. The association of pCR with disease-free survival or overall survival in ER+/HER2-breast cancers following neoadjuvant systemic therapy (NAT) or neoadjuvant endocrine therapy (NET) is relatively low as compared to the other two subtypes of breast cancers, namely triple-negative and HER2+ amplified. On the bright side, a neoadjuvant model offers a potential opportunity to explore the efficacy of novel therapies and the associated genomic alterations, thus providing a rare personalized insight into the tumor's biology and the tumor cells' response to the drug. Several decades of research have taught us that the disease's biology is a critical factor determining the tumor cells' response to any therapy and hence the final outcome of the disease. Here we propose two scenarios wherein apoptosis can be induced in ER+/HER2- breast cancers expressing wild type TP53 and RB genes following combinations of BCL2 inhibitor, MDM2 inhibitor, and cell-cycle inhibitor. The suggested combinations are contextual and based on the current understanding of the cell signaling in the ER+/HER2- breast cancers. The two combinations of drugs are (1) BCL2 inhibitor plus a cell-cycle inhibitor, which can prime the tumor cells for apoptosis, and (2) BCL2 inhibitor plus an MDM2 inhibitor.