Predicting Response to Immunotargeted Therapy in Endometrial Cancer via Tumor Immune Microenvironment: A Multicenter, Observational Study.

Only one-third of patients with advanced MSS/pMMR endometrial cancer exhibit a lasting response to the combination treatment of Pembrolizumab and Lenvatinib. The combined administration of these two drugs is based on Lenvatinib's ability to modulate the tumor microenvironment, enabling Pembrolizumab to exert its effect. These findings underscore the importance of exploring tumor microenvironment parameters to identify markers that can accurately select candidates for this type of therapy. An open non-randomized observational association study was conducted at six clinical centers, involving a total of 28 patients with advanced MSS/pMMR endometrial cancer who received Pembrolizumab and Lenvatinib therapy. Using TSA-associated multiplex immunofluorescence, we analyzed the proportion of CD8+ T lymphocytes, CD20+ B lymphocytes, FoxP3+ T regulatory lymphocytes, and CD163+ macrophages in tumor samples prior to immunotargeted therapy. The percentage of CD20+ B lymphocytes and the CD8-to-CD20 lymphocytes ratio was significantly higher in patients who responded to treatment compared to non-responders (responders vs. non-responders: 0.24 (0.1-1.24)% vs. 0.08 (0.00-0.15)%, p = 0.0114; 1.44 (0.58-2.70) arb. unit vs. 19.00 (3.80-34.78) arb. unit, p = 0.0031). The sensitivity and specificity of these biomarkers were 85.71% and 70.59%, and 85.71% and 85.71%, respectively. The proportion of CD20+ B lymphocytes and the CD8-to-CD20 lymphocytes ratio in the stroma of endometrial cancer serves as both a prognostic marker of response to immunotargeted therapy and a prognostic factor for progression-free survival in patients.

Integrin-associated transcriptional characteristics of circulating tumor cells in breast cancer patients.

Background: Integrins enable cell communication with the basal membrane and extracellular matrix, activating signaling pathways and facilitating intracellular changes. Integrins in circulating tumor cells (CTCs) play a significant role in apoptosis evasion and anchor-independent survival. However, the link between CTCs expressing different integrin subunits, their transcriptional profile and, therefore, their functional activity with respect to metastatic potential remains unclear. Methods: Single-cell RNA sequencing of CD45-negative cell fraction of breast cancer patients was performed. All CTCs were divided into nine groups according to their integrin profile. Results: СTCs without the gene expression of integrins or with the expression of non-complementary α and ß subunits that cannot form heterodimers prevailed. Only about 15% of CTCs expressed integrin subunits which can form heterodimers. The transcriptional profile of CTCs appeared to be associated with the spectrum of expressed integrins. The lowest potential activity was observed in CTCs without integrin expression, while the highest frequency of expression of tumor progression-related genes, namely genes of stemness, epithelial-mesenchymal transition (EMT), invasion, proinflammatory chemokines and cytokines as well as laminin subunits, were observed in CTCs co-expressing ITGA6 and ITGB4. Validation on the protein level revealed that the median of integrin ß4+ CTCs was higher in patients with more aggressive molecular subtypes as well as in metastatic breast cancer patients. One can expect that CTCs with ITGA6 and ITGB4 expression will have pronounced metastatic potencies manifesting in expression of EMT and stemness-related genes, as well as potential ability to produce chemokine/proinflammatory cytokines and laminins.

Spatial Heterogeneity of Integrins and Their Ligands in Primary Breast Tumors.

BACKGROUND: The diversity of cell-cell interactions in different regions of a tumor reflects the functional heterogeneity of cancer, which poses challenges in early diagnosis, selection of treatment strategies, and prognosis of breast cancer. Cancer cells interact with each other to form different morphological structures in the tumor and stromal host cells via integrins. The objective of this study was to characterize the morphological and spatial heterogeneity of primary breast tumors in the context of expression profiles of integrins and their ligands. METHODS: We studied spatial transcriptomics using the 10X Visium approach and the Niche Interactions and Communication Heterogeneity in Extracellular Signaling (NICHES) algorithm to map ligand-receptor signaling pathways and visualize the heterogeneity of signaling archetypes in tumor clusters. RESULTS: Cluster analysis of the expression profiles of tumor spots from the samples indicated pronounced inter-tumoral heterogeneity. Integrin-ligand functional clusters were associated with intratumoral heterogeneity, which was manifested by the presence of several morphological loci as observed in histological tumor samples. Inter-tumoral heterogeneity was manifested by a different number of functional clusters, ranging from 2 to 9 for each tumor sample. The main characteristic of these clusters was the significant predominance of non-complementary integrin subunits. Of the 42 functional integrin-ligand pairs in 21 clusters of five samples, 41 pairs occurred only once. The exception was the laminin subunit alpha-5 (LAMA5)-integrin beta 4 (ITGB4) pair, which was detected in two clusters of different samples. CONCLUSIONS: The spatial heterogeneity of integrin-ligand expression clusters in breast cancer contributes significantly to the functional heterogeneity of the tumor, which sets the stage for many scenarios of parenchymatous-stromal relationships, some of which may be effective in the emergence of metastasizing tumor seed cells. The intra- and inter-tumoral spatio-functional heterogeneity of the tumor tissue that we discovered may largely explain why it is difficult to achieve success in most patients with breast cancer using any therapeutic strategy targeting one molecule of the vast array, regardless of the importance of its pathogenetic significance.

Immune gene signatures as prognostic criteria for cancer patients.

Recently, the possibility of using immune gene signatures (IGSs) has been considered as a novel prognostic tool for numerous cancer types. State-of-the-art methods of genomic, transcriptomic, and protein analysis have allowed the identification of a number of immune signatures correlated to disease outcome. The major adaptive and innate immune components are the T lymphocytes and macrophages, respectively. Herein, we collected essential data on IGSs consisting of subsets of T cells and tumor-associated macrophages and indicating cancer patient outcomes. We discuss factors that can introduce errors in the recognition of immune cell types and explain why the significance of immune signatures can be interpreted with uncertainty. The unidirectional functions of cell types should be entirely addressed in the signatures constructed by the combination of innate and adaptive immune cells. The state of the antitumor immune response is the key basis for IGSs and should be considered in gene signature construction. We also analyzed immune signatures for the prediction of immunotherapy response. Finally, we attempted to explain the present-day limitations in the use of immune signatures as robust criteria for prognosis.

Phase I Trial of [99mTc]Tc-maSSS-PEG2-RM26, a Bombesin Analogue Antagonistic to Gastrin-Releasing Peptide Receptors (GRPRs), for SPECT Imaging of GRPR Expression in Malignant Tumors.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in prostate cancer (PCa) and in hormone-driven breast cancer (BCa). The aim of this phase I clinical trial was to evaluate safety, biodistribution, and dosimetry after the administration of the recently developed GRPR-targeting antagonistic bombesin analogue [99mTc]Tc-maSSS-PEG2-RM26 in PCa and BCa patients. Planar and whole-body SPECT/CT imaging was performed in six PCa patients and seven BCa patients 2, 4, 6, and 24 h post the intravenous administration of 40 µg of [99mTc]Tc-maSSS-PEG2-RM26 (600-700 MBq). No adverse events or pathological changes were observed. The rapid blood clearance of [99mTc]Tc-maSSS-PEG2-RM26 was observed with predominantly hepatobiliary excretion. The effective doses were 0.0053 ± 0.0007 for male patients and 0.008 ± 0.003 mSv/MBq for female patients. The accumulation of [99mTc]Tc-maSSS-PEG2-RM26 in tumors was observed in four out of six PCa and in seven out of seven BCa patients. In four BCa patients, a high uptake of the agent into the axillary lymph nodes was detected. Immunohistochemistry revealed positive GRPR expression in 60% of primary PCa, 71.4% of BCa tumors, and 50% of examined BCa lymph nodes. In conclusion, a single administration of [99mTc]Tc-maSSS-PEG2-RM26 was safe and well tolerated. [99mTc]Tc-maSSS-PEG2-RM26 SPECT may be useful for tumor detection in PCa and BCa patients, pending further studies.

Angiogenesis regulators S100A4, SPARC and SPP1 correlate with macrophage infiltration and are prognostic biomarkers in colon and rectal cancers.

Introduction: Increasing evidence suggests that it is necessary to find effective and robust clinically validated prognostic biomarkers that can identify "high-risk" colorectal cancer (CRC) patients. Currently, available prognostic factors largely include clinical-pathological parameters and focus on the cancer stage at the time of diagnosis. Among cells of tumor microenvironment (TME) only Immunoscore classifier based on T lymphocytes showed high predictive value. Methods: In the present study, we performed the complex analysis of mRNA and protein expression of crucial regulators of tumor angiogenesis and tumor progression, expressed by tumor-associated macrophages (TAMs): S100A4, SPP1 and SPARC. Colon and rectal cancer patients were investigated independently and in a combined cohort (CRC). For mRNA expression, we analyzed RNA sequencing data obtained from TCGA (N=417) and GEO (N=92) cohorts of colorectal cancer patients. For protein expression, we performed IHC digital quantification of tumor tissues obtained from 197 patients with CRC treated in the Department of abdominal oncology in Clinics of Tomsk NRMC. Results: High S100A4 mRNA expression accurately predicted poor survival for patients with CRC independently of cancer type. SPARC mRNA level was independent prognostic factors for survival in colon but not in rectal cancer. SPP1 mRNA level had significant predictive value for survival in both rectal and colon cancers. Analysis of human CRC tissues revealed that S100A4, SPP1 and SPARC are expressed by stromal compartments, in particular by TAMs, and have a strong correlation with macrophage infiltration. Finally, our results indicate that chemotherapy-based treatment can change the predictive direction of S100A4 for rectal cancer patients. We found that S100A4 stromal levels were higher in patients with better response to neoadjuvant chemotherapy/chemoradiotherapy, and S100A4 mRNA levels predicted better DFS among non-responders. Discussion: These findings can help improve the prognosis of patients with CRC based on S100A4, SPP1 and SPARC expression levels.

Spatial Profile of Tumor Microenvironment in PD-L1-Negative and PD-L1-Positive Triple-Negative Breast Cancer.

The problem of finding more precise stratification criteria for identifying the cohort of patients who would obtain the maximum benefit from immunotherapy is acute in modern times. In our study were enrolled 18 triple-negative breast cancer patients. The Ventana SP142 test was used for PD-L1 detection. Spatial transcriptomic analysis by 10x Genomics was used to compare PD-L1-positive and PD-L1-negative tumors. The seven-color multiplex immunofluorescence (by Akoya) was used for the detection of the type of cells that carried the PD1 receptor and the PD-L1 ligand. Using pathway analysis, we showed that PD-L1-positive tumors demonstrate signatures of a cell response to cytokines, among others, and PD-L1-negative tumors demonstrate signatures of antigen presentation. PD-L1-positive and PD-L1-negative tumors have different tumor microenvironment (TME) compositions according to CIBERSORT analysis. Multiplex immunohistochemistry (IHC) confirmed the prevalence of PD1-negative M2 macrophages and PD1-negative T lymphocytes in PD-L1-positive tumors. PD-L1-positive tumors are not characterized by direct contact between cells carrying the PD1 receptor and the PD-L1 ligand. So, the absence of specific immune reactions against the tumor, predominance of pro-tumor microenvironment, and rare contact between PDL1 and PD1-positive cells may be the potential reasons for the lack of an immune checkpoint inhibitor (ICI) effect in triple-negative breast cancer patients.

Heterogeneity of Circulating Epithelial Cells in Breast Cancer at Single-Cell Resolution: Identifying Tumor and Hybrid Cells.

Circulating tumor cells and hybrid cells formed by the fusion of tumor cells with normal cells are leading players in metastasis and have prognostic relevance. This study applies single-cell RNA sequencing to profile CD45-negative and CD45-positive circulating epithelial cells (CECs) in nonmetastatic breast cancer patients. CECs are represented by transcriptionally-distinct populations that include both aneuploid and diploid cells. CD45- CECs are predominantly aneuploid, but one population contained more diploid than aneuploid cells. CD45+ CECs mostly diploid: only two populations have aneuploid cells. Diploid CD45+ CECs annotated as different immune cells, surprisingly harbored many copy number aberrations, and positively correlated to tumor grade. It is noteworthy that cancer-associated signaling pathways areabundant only in one aneuploid CD45- CEC population, which may represent an aggressive subset of circulating tumor cells. Thus, CD45- and CD45+ CECs are highly heterogeneous in breast cancer patients and include aneuploid cells, which are most likely circulating tumor and hybrid cells, respectively, and diploid cells. DNA ploidy analysis can be an effective instrument for identifying tumor and hybrid cells among CECs. Further follow-up study is needed to determine which subsets of circulating tumor and hybrid cells contribute to breast cancer metastasis.

LIMCH1 as a New Potential Metastasis Predictor in Breast Cancer.

BACKGROUND AND OBJECTIVE: High LIMCH1 expression in lung and renal cancer is determined as a favorable prognostic factor. However, prognostic value of LIMCH1 expression in breast cancer has not been studied yet. Therefore, this study was performed to determine the prognostic value of LIMCH1 expression in breast cancer patients. METHODS: This retrospective study included 89 patients with invasive breast carcinoma of no special type. These patients referred to  Cancer Research Institute of Tomsk National Research Medical Center from 2007 to 2018. LIMCH1 protein expression in tumor cells was detected by immunohistochemical analysis in this study. Statistical analysis was done to investigate the possible relationship between LIMCH1 protein expression and clinicopathological parameters, risk of metastasis, distant metastasis free survival, and overall survival. RESULTS: IHC analysis of breast cancer tissue samples revealed that LIMHC1 protein expression was found in 29.2% (26/89) of the cases. Lymph node and distant metastases were more frequent in patients with LIMCH1 protein expression. LIMCH1 protein expression increased the risk of distant metastasis based on our findings. LIMCH1 protein affected metastatic-free survival regardless of the T, as well as other clinical and pathological parameters (p=0.0146, HR=3.2058 (1.26; 8.17)). Moreover, LIMCH1 protein expression was associated with worse overall survival (p=0.0071, HR=2.73 (1.28; 5.85)) in our breast cancer patients. CONCLUSION: LIMCH1 protein expression was associate with metastases development, providing prognostic stratification. In breast cancer, LIMCH1 protein expression was found as an unfavorable prognostic factor of distant metastasis-free survival based on our findings.

The Novel Association of Early Apoptotic Circulating Tumor Cells with Treatment Outcomes in Breast Cancer Patients.

Stemness and epithelial-mesenchymal plasticity are widely studied in the circulating tumor cells of breast cancer patients because the roles of both processes in tumor progression are well established. An important property that should be taken into account is the ability of CTCs to disseminate, particularly the viability and apoptotic states of circulating tumor cells (CTCs). Recent data demonstrate that apoptosis reversal promotes the formation of stem-like tumor cells with pronounced potential for dissemination. Our study focused on the association between different apoptotic states of CTCs with short- and long-term treatment outcomes. We evaluated the association of viable CTCs, CTCs with early features of apoptosis, and end-stage apoptosis/necrosis CTCs with clinicopathological parameters of breast cancer patients. We found that the proportion of circulating tumor cells with features of early apoptosis is a perspective prognosticator of metastasis-free survival, which also correlates with the neoadjuvant chemotherapy response in breast cancer patients. Moreover, we establish that apoptotic CTCs are associated with the poor response to neoadjuvant chemotherapy, and metastasis-free survival expressed at least two stemness markers, CD44 and CD133.

B Lymphocytes Are a Predictive Marker of Eribulin Response and Overall Survival in Locally Advanced or Metastatic Breast Cancer: A Multicenter, Two-Cohort, Non-Randomized, Open-Label, Retrospective Study.

Triple-negative breast cancer has no specific treatment and unfavorable prognosis. Eribulin is one of the drugs widely used in this cohort of patients. In addition to its antimitotic effect, eribulin has an immunomodulant effect on the tumor microenvironment. In this study, we discover immunological markers, such as tumor-infiltrating lymphocytes, CD8+, CD4+, FoxP3+, CD20+ lymphocytes, and their PD1 positivity or negativity, with the ability to predict benefits from eribulin within locally advanced or metastatic triple-negative breast cancer. The primary objective was to explore the association of composition of immune cells in the microenvironment with response to eribulin. The key secondary objective was overall survival. Seven-color multiplex immunofluorescence was used to phenotype lymphocytes in the primary tumor. It has been shown that the PD1-negative-to-PD1-positive B cells ratio in primary tumors more than 3 is an independent predictor of the short-term effectiveness of eribulin [OR (95%CI) 14.09 (1.29-153.35), p=0.0029] and worse overall survival [HR (95%CI) 11.25 (1.37-70.25), p=0.0009] in patients with locally advanced or metastatic triple-negative breast cancer.

Upregulated flotillins and sphingosine kinase 2 derail AXL vesicular traffic to promote epithelial-mesenchymal transition.

Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called upregulated flotillin-induced trafficking (UFIT). Here, we found that in non-tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane domains and endosomes. Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation.

Identification of differentially methylated genes in first-trimester placentas with trisomy 16.

The presence of an extra chromosome in the embryo karyotype often dramatically affects the fate of pregnancy. Trisomy 16 is the most common aneuploidy in first-trimester miscarriages. The present study identified changes in DNA methylation in chorionic villi of miscarriages with trisomy 16. Ninety-seven differentially methylated sites in 91 genes were identified (false discovery rate (FDR) < 0.05 and Δß > 0.15) using DNA methylation arrays. Most of the differentially methylated genes encoded secreted proteins, signaling peptides, and receptors with disulfide bonds. Subsequent analysis using targeted bisulfite massive parallel sequencing showed hypermethylation of the promoters of specific genes in miscarriages with trisomy 16 but not miscarriages with other aneuploidies. Some of the genes were responsible for the development of the placenta and embryo (GATA3-AS1, TRPV6, SCL13A4, and CALCB) and the formation of the mitotic spindle (ANKRD53). Hypermethylation of GATA3-AS1 was associated with reduced expression of GATA3 protein in chorionic villi of miscarriages with trisomy 16. Aberrant hypermethylation of genes may lead to a decrease in expression, impaired trophoblast differentiation and invasion, mitotic disorders, chromosomal mosaicism and karyotype self-correction via trisomy rescue mechanisms.

Phase I Trial of 99mTc-(HE)3-G3, a DARPin-Based Probe for Imaging of HER2 Expression in Breast Cancer.

Radionuclide molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression may enable a noninvasive discrimination between HER2-positive and HER2-negative breast cancers for stratification of patients for HER2-targeted treatments. DARPin (designed ankyrin repeat proteins) G3 is a small (molecular weight, 14 kDa) scaffold protein with picomolar affinity to HER2. The aim of this first-in-humans study was to evaluate the safety, biodistribution, and dosimetry of 99mTc-(HE)3-G3. Methods: Three cohorts of patients with primary breast cancer (each including at least 4 patients with HER2-negative and 5 patients with HER2-positive tumors) were injected with 1,000, 2,000, or 3,000 µg of 99mTc-(HE)3-G3 (287 ± 170 MBq). Whole-body planar imaging followed by SPECT was performed at 2, 4, 6, and 24 h after injection. Vital signs and possible side effects were monitored during imaging and up to 7 d after injection. Results: All injections were well tolerated. No side effects were observed. The results of blood and urine analyses did not differ before and after studies. 99mTc-(HE)3-G3 cleared rapidly from the blood. The highest uptake was detected in the kidneys and liver followed by the lungs, breasts, and small intestinal content. The hepatic uptake after injection of 2,000 or 3,000 µg was significantly (P < 0.05) lower than the uptake after injection of 1,000 µg. Effective doses did not differ significantly between cohorts (average, 0.011 ± 0.004 mSv/MBq). Tumor-to-contralateral site ratios for HER-positive tumors were significantly (P < 0.05) higher than for HER2-negative at 2 and 4 h after injection. Conclusion: Imaging of HER2 expression using 99mTc-(HE)3-G3 is safe and well tolerated and provides a low absorbed dose burden on patients. This imaging enables discernment of HER2-positive and HER2-negative breast cancer. Phase I study data justify further clinical development of 99mTc-(HE)3-G3.

Parameters of Tumor Microenvironment Determine Effectiveness of Anti-PD-1/PD-L1 Therapy.

Undoubtedly, one of the most promising approaches to the treatment of cancer is creation of the pathogenetically based therapeutic drugs. Researchers from all over the world are trying to answer the question on how to select a target that would be effective and, in general, they are quite successful at that. The Nobel Prize-winning discovery of mechanisms for regulating activity of the immune system cells through checkpoint molecules, as well as discovery of the ability of tumor cells to use these mechanisms to suppress immune responses was an impetus for the development of modern immunotherapy, and now such inhibitors of the immune checkpoints as PD-1/PD-L1 are included in the routine chemotherapy. Use of such drugs can prolong the patient's life, but, unfortunately, not cure the disease. This is partially due to heterogeneity of tumor cells and microenvironment, but the main reasons may be in the complex relationships between the tumor and microenvironment, which, at times, are so plastic that they can change, adjusting to newly emerging conditions. Main characteristic of the tumor microenvironment is the type of the ongoing immune-inflammatory response (IIR), and since inhibitors of the immune checkpoints act on the cells involved in IIR, it is obvious that the outcomes of cancer therapy, including outcomes of hyperprogressive disease, can be associated with this parameter. The presented review reveals the essence of interactions between the tumor and its microenvironment during therapy with PD-L1 inhibitors.

Premalignant Changes in the Bronchial Epithelium Are Prognostic Factors of Distant Metastasis in Non-Small Cell Lung Cancer Patients.

BACKGROUND: The study assessed the possibility of dividing patients into groups based on the assessment of morphological changes in the epithelium of small-caliber bronchi located near the primary tumor in order to predict high and low risks of distant metastasis of non-small cell lung cancer. METHODS: In 171 patients with non-small cell lung cancer (T1-4N0-3M0) in small-caliber bronchi taken at a distance of 3-5 cm from the tumor, various variants of morphological changes in the bronchial epithelium (basal cell hyperplasia (BCH), squamous cell metaplasia (SM), and dysplasia (D)) were assessed. Long-term results of treatment, namely, distant metastasis, were assessed after 2 and 5 years. RESULTS: During the follow-up period, distant metastases were found in 35.1% (60/171) of patients. Most often, they were observed in patients of the high-risk group: BCH+SM-D- (51.6%, 40/95) and BCH-SM+D+ (54.4%, 6/11). Less often, distant metastases were observed in low-risk group patients: BCH+SM+D- (6.7%, 3/45) and BCH-SM-D- (10.0%, 2/20). Tumor size, grade, and stage were significant predictors of metastasis only in the high-risk group. The 5-year metastasis-free survival was better in the low-risk group of distant metastases. CONCLUSIONS: Isolated BCH or dysplasia in small bronchi distant from foci of tumor is associated with a high-risk distant metastasis and less 5-year metastasis-free survival.

Imaging-Guided Therapy Simultaneously Targeting HER2 and EpCAM with Trastuzumab and EpCAM-Directed Toxin Provides Additive Effect in Ovarian Cancer Model.

Efficient treatment of disseminated ovarian cancer (OC) is challenging due to its heterogeneity and chemoresistance. Overexpression of human epidermal growth factor receptor 2 (HER2) and epithelial cell adhesion molecule (EpCAM) in approx. 30% and 70% of ovarian cancers, respectively, allows for co-targeted treatment. The clinical efficacy of the monoclonal antibody trastuzumab in patients with HER2-positive breast, gastric and gastroesophageal cancers makes it readily available as the HER2-targeting component. As the EpCAM-targeting component, we investigated the designed ankyrin repeat protein (DARPin) Ec1 fused to a truncated variant of Pseudomonas exotoxin A with reduced immunogenicity and low general toxicity (LoPE). Ec1-LoPE was radiolabeled, evaluated in ovarian cancer cells in vitro and its biodistribution and tumor-targeting properties were studied in vivo. The therapeutic efficacy of Ec1-LoPE alone and in combination with trastuzumab was studied in mice bearing EpCAM- and HER2-expressing SKOV3 xenografts. SPECT/CT imaging enabled visualization of EpCAM and HER2 expression in the tumors. Co-treatment using Ec1-LoPE and trastuzumab was more effective at reducing tumor growth and prolonged the median survival of mice compared with mice in the control and monotherapy groups. Repeated administration of Ec1-LoPE was well tolerated without signs of hepatic or kidney toxicity. Co-treatment with trastuzumab and Ec1-LoPE might be a potential therapeutic strategy for HER2- and EpCAM-positive OC.

Heterogeneous Manifestations of Epithelial-Mesenchymal Plasticity of Circulating Tumor Cells in Breast Cancer Patients.

To date, there is indisputable evidence of significant CTC heterogeneity in carcinomas, in particular breast cancer. The heterogeneity of CTCs is manifested in the key characteristics of tumor cells related to metastatic progression - stemness and epithelial-mesenchymal (EMT) plasticity. It is still not clear what markers can characterize the phenomenon of EMT plasticity in the range from epithelial to mesenchymal phenotypes. In this article we examine the manifestations of EMT plasticity in the CTCs in breast cancer. The prospective study included 39 patients with invasive carcinoma of no special type. CTC phenotypes were determined by flow cytometry before any type of treatment. EMT features of CTC were assessed using antibodies against CD45, CD326 (EpCam), CD325 (N-cadherin), CK7, Snail, and Vimentin. Circulating tumor cells in breast cancer are characterized by pronounced heterogeneity of EMT manifestations. The results of the study indicate that the majority of heterogeneous CTC phenotypes (22 out of 24 detectable) exhibit epithelial-mesenchymal plasticity. The variability of EMT manifestations does not prevent intravasation. Co-expression of EpCAM and CK7, regardless of the variant of co-expression of Snail, N-cadherin, and Vimentin, are associated with a low number of CTCs. Intrapersonal heterogeneity is manifested by the detection of several CTC phenotypes in each patient. Interpersonal heterogeneity is manifested by various combinations of CTC phenotypes in patients (from 1 to 17 phenotypes).

Neoadjuvant chemotherapy combined with intraoperative radiotherapy is effective to prevent recurrence in high-risk non-small cell lung cancer (NSCLC) patients.

BACKGROUND: Basal cell hyperplasia (BCH) and squamous metaplasia (SM) in the small bronchi distant from the tumor is associated with a high risk of non-small cell lung cancer (NSCLC) recurrence. Here, we assessed whether neoadjuvant chemotherapy (NAC), intraoperative radiotherapy (IORT), or adjuvant chemotherapy (AC) is effective to prevent recurrence in NSCLC patients (n=171) with different premalignant lesions in the small bronchi. METHODS: BCH, SM, and dysplasia (D) were identified in the samples of lung tissue distant from the tumor. NSCLC patients were treated by surgery, different combinations of NAC and IORT, and AC. RESULTS: Based on the type of bronchial lesions, NSCLC patients were classified into four groups: BCH+SM-D- (55.6%, 95/171), BCH+SM+D- (26.3%; 45/171), BCH-SM+D+ (6.4%, 11/171), and BCH-SM-D- (11.7%, 20/171). During 5 years, recurrent carcinoma was found in 13.4% (23/171) of patients and represented by metachronous metastases in the thoracic lymph nodes (82.6%, 19/23) and by a relapse in the bronchial stump (17.4%, 4/23). Recurrence was frequent in BCH+SM+D- patients (87.0%, 20/23), rare in BCH+SM-D- and BCH-SM-D- patients (13.0%, 3/23), and absent in BCH-SM+D+ patients (0/23). The 5-year recurrence-free survival was also shorter in BCH+SM+D- patients (HR 27.35; 95% CI: 6.31-118.48; P<0.0001). In the high-risk (BCH+SM+D-) group, recurrence occurred mainly in cases without NAC and IORT (88.2%, 15/17) and was absent (0/15) when these therapies were combined. NAC- and IORT-negative patients also showed poor overall survival (HR 4.35; 95% CI: 1.96-9.66; P<0.0001) and tended to have decreased recurrence-free survival (P=0.075). Importantly, the recurrence rate was not different between AC-treated and AC-naïve BCH+SM+D- patients. CONCLUSIONS: The combination of NAC and IORT is an effective strategy to prevent recurrence in high-risk NSCLC patients.

The Activity of KIF14, Mieap, and EZR in a New Type of the Invasive Component, Torpedo-Like Structures, Predetermines the Metastatic Potential of Breast Cancer.

Intratumor morphological heterogeneity reflects patterns of invasive growth and is an indicator of the metastatic potential of breast cancer. In this study, we used this heterogeneity to identify molecules associated with breast cancer invasion and metastasis. The gene expression microarray data were used to identify genes differentially expressed between solid, trabecular, and other morphological arrangements of tumor cells. Immunohistochemistry was applied to evaluate the association of the selected proteins with metastasis. RNA-sequencing was performed to analyze the molecular makeup of metastatic tumor cells. High frequency of metastases and decreased metastasis-free survival were detected in patients either with positive expression of KIF14 or Mieap or negative expression of EZR at the tips of the torpedo-like structures in breast cancers. KIF14- and Mieap-positive and EZR-negative cells were mainly detected in the torpedo-like structures of the same breast tumors; however, their transcriptomic features differed. KIF14-positive cells showed a significant upregulation of genes involved in ether lipid metabolism. Mieap-positive cells were enriched in genes involved in mitophagy. EZR-negative cells displayed upregulated genes associated with phagocytosis and the chemokine-mediated signaling pathway. In conclusion, the positive expression of KIF14 and Mieap and negative expression of EZR at the tips of the torpedo-like structures are associated with breast cancer metastasis.