Small Number Of Tumor Cells Research Articles

Detection of comprehensive oncogenic driver alteration of non-small cell lung cancer from cytology specimens by multi-gene PCR panel.

8579 Background: The analysis of the next-generation sequencing (NGS) panel test requires a sufficient amount of tissue specimens (TS), but the collection of tissue specimens can be sometimes challenging in clinical practice. Despite being easier and safer to collect than TS, cytology specimens (CS) are considered unsuitable for NGS due to the small number of tumor cells. The AmoyDx Pan Lung Cancer PCR Panel (AmoyDx), a multi-gene PCR panel that amplified mutation or fusion variants of 11 genes (EGFR, ALK, ROS1, RET, MET HER2, BRAF, KRAS, and NTRK1-3) by real-time PCR, can be performed with smaller amounts of nucleic acid than NGS, and CS may be suitable for multi-gene analysis using this panel. In this study, we evaluated the feasibility of multi-gene analysis using CS, and the concordance rate of gene alteration between CS and TS in non–small cell lung cancer (NSCLC). Methods: We enrolled consecutive NSCLC patients obtaining CS through bronchoscopy, including transbronchial brushing (TBB) and transbronchial needle aspiration (TBNA) from September 2020 to March 2023. These CS were preserved as frozen pellets. The difference in the amount of nucleic acid, the success rate and the concordance rate of gene alteration with AmoyDx were evaluated between CS and pared TS collected simultaneously with CS. Results: A total of 131 (Ad/Sq/NSCLC-NOS: 113/2/16) cases were enrolled in this study. Fifty-nine specimens were obtained through TBB and another 72 were obtained through TBNB. The median nucleic acid concentration (NanoDrop [ng/μL]) isolated from CS was comparable to that isolated from TS (DNA 114/99, p=0.96; RNA 36/27, p=0.63). However, the median DNA concentration in CS was significantly lower (80/102, p=0.01) in TBB samples, while in TBNA samples, it was higher (134/90, p=0.03). Almost all CS (99.2%,130/131) and TS (99.2%,130/131) were analyzed successfully with AmoyDx, with only one RNA analysis failure in each group. Among CS, 78 (60%) samples exhibit gene alteration, including EGFR/KRAS/BRAF/HER2/ALK/ROS1/MET/RET: 44/9/2/5/8/1/8/1, while 77 (59%); 44/9/2/6/6/1/8/1 in TS. The positive percent agreement, negative percent agreement, and overall percent agreement of CS using analysis compared with the results of TS using analysis were 99% (76/77), 96% (52/54), and 98% (128/131), respectively. Conclusions: CS exhibit an extremely high success rate for multi-gene analysis, with nucleic acid yield comparable to TS. Moreover, the concordance rate of gene alteration between CS and TS is also remarkably high. CS emerge as a viable alternative to TS for analyzing driver gene alteration in multi-gene PCR analysis.

Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer

BackgroundEnumeration of circulating tumor cells (CTCs) has proven clinical significance for monitoring patients with metastatic cancers. Multiplexed gene expression profiling of CTCs is a potential tool for assessing disease status and monitoring treatment response. The Parsortix® technology enables the capture and harvest of CTCs from blood based on cell size and deformability. The HyCEAD™ (Hybrid Capture Enrichment Amplification and Detection) assay enables simultaneous amplification of short amplicons for up to 100 mRNA targets, and the Ziplex™ instrument quantifies the amplicons for highly sensitive gene expression profiling down to single cell levels. The aim of the study was to functionally assess this system.MethodsThe HyCEAD/Ziplex platform was used to quantify the expression levels for 72 genes using as little as 20 pg of total RNA or a single cultured tumor cell. Assay performance was evaluated using cells or total RNA spiked into Parsortix harvests of healthy donor blood. The assay was also evaluated using total RNA obtained from Parsortix harvests of blood from metastatic breast cancer (MBC) patients or healthy volunteers (HVs).ResultsUsing genes with low expression in WBC RNA and/or in unspiked Parsortix harvests from HVs, the assay distinguished between the different breast cancer and ovarian cancer cell lines with as little as 20 pg of total RNA (equivalent to a single cell) in the presence of 1 ng of WBC RNA. Single cultured cells spiked into Parsortix harvests from 10 mL of HV blood were also detected and distinguished from each other. CVs from repeatability experiments were less than 20%. Hierarchical clustering of clinical samples differentiated most MBC patients from HVs.ConclusionHyCEAD/Ziplex provided sensitive quantification of expression of 72 genes from 20 pg of total RNA from cultured tumor cell lines or from single cultured tumor cells spiked into lysates from Parsortix harvests of HV blood. The HyCEAD/Ziplex platform enables the quantification of selected genes in the presence of residual nucleated blood cells in Parsortix harvests. The HyCEAD/Ziplex platform is an effective tool for multiplexed molecular characterization of mRNA in small numbers of tumor cells harvested from blood.

Targeting myeloid-derived suppressor cells in combination with tumor cell vaccination predicts anti-tumor immunity and breast cancer dormancy: an in silico experiment

Among the different breast cancer subsets, triple-negative breast cancer (TNBC) has the worst prognosis and limited options for targeted therapies. Immunotherapies are emerging as novel treatment opportunities for TNBC. However, the surging immune response elicited by immunotherapies to eradicate cancer cells can select resistant cancer cells, which may result in immune escape and tumor evolution and progression. Alternatively, maintaining the equilibrium phase of the immune response may be advantageous for keeping a long-term immune response in the presence of a small-size residual tumor. Myeloid-derived suppressor cells (MDSCs) are activated, expanded, and recruited to the tumor microenvironment by tumor-derived signals and can shape a pro-tumorigenic micro-environment by suppressing the innate and adaptive anti-tumor immune responses. We recently proposed a model describing immune-mediated breast cancer dormancy instigated by a vaccine consisting of dormant, immunogenic breast cancer cells derived from the murine 4T1 TNBC-like cell line. Strikingly, these 4T1-derived dormant cells recruited fewer MDSCs compared to aggressive 4T1 cells. Recent experimental studies demonstrated that inactivating MDSCs has a profound impact on reconstituting immune surveillance against the tumor. Here, we developed a deterministic mathematical model for simulating MDSCs depletion from mice bearing aggressive 4T1 tumors resulting in immunomodulation. Our computational simulations indicate that a vaccination strategy with a small number of tumor cells in combination with MDSC depletion can elicit an effective immune response suppressing the growth of a subsequent challenge with aggressive tumor cells, resulting in sustained tumor dormancy. The results predict a novel therapeutic opportunity based on the induction of effective anti-tumor immunity and tumor dormancy.

Study on the mechanism of low shear stress restoring the viability of damaged breast tumor cells

Blood vessel is one of the ‘pathways’ for cancer to metastasize, in which cells are exposed to the fluid shear stress. Although most cells are damaged by fluid shear stress, a small number of tumor cells survive and metastasize when they are exposed to low shear stress (LSS) of tiny capillary. It is important to study the survival state of damaged cells during LSS. In this study, high shear stress (HSS) was applied to simulate the blood circulation and damage cells. The viability and mitochondrial function of cells were detected after HSS and LSS loading, respectively. Further, the expression of mitochondrial related proteins and genes were detected by western blot and real-time quantitative polymerase chain reaction, respectively. The role of cytochrome c (Cyt C) was also verified in this process. The experimental results showed that the viability of HSS damaged cells was increased significantly when they were exposed to LSS subsequently. The function of mitochondria was improved via reducing the release of Cyt C by LSS during this process. This study is expected to provide potential target for suppressing hematogenous metastasis.

Abstract 5324: Isolation of small number of tumor cells for proliferation studies using DEPArray technology

Abstract Cancer is universally recognized as a heterogeneous disease. The different cell clones present in a tumor can display distinct molecular and phenotypic profiles, which may influence proliferation capabilities, metastatic potential and response to therapy. To better understand cancer progression and possibly design effective therapeutic strategies, it is fundamental to design cell-sorting technologies able to isolate rare, viable tumor cell clones from tissue or liquid biopsies. Fluorescence-activated cell sorting (FACS) is the most widely used technique to isolate cell populations from heterogeneous samples. However, FACS analysis presents technical limitations when applied to rare cell populations and/or samples with low cellularity and limitations associated with the low total number of target cells in the sample. DEParray™ PLUS (DA) is a highly automated image-based cell sorting technology capable of isolating pure, single and rare cells from heterogeneous samples. Here we evaluate the capabilities of the DA system to isolate a small number of viable tumor cells and assess the proliferation capacity of DA-sorted cells in comparison with FACS-sorted cells. Human Jurkat T lymphoblast cell line were cultured in RPMI medium and a double staining procedure was optimized to label cells with CellTraceFarRed (CTFR), division tracking dye and Live/Dead (LD) Fixable marker. CTFR/LD staining was performed for each experiment and, at days 0 and 7 of culture, CTFR and LD fluorescences were analyzed by means of BD CANTO II Flow cytometry. At day 0, FACS analysis discriminate dead from live cells, while at day 7 evaluate cell proliferation and viability after 7 days in culture. Comparison tests were carried out following two different recovery schemes: 1 recovery of 100 CTFR+/LD- cells pool and 10 recoveries of 10 CTFR+/LD- cells pool using DA Plus and BD FACSAria Fusion. For each experiment, the results were compared to cells seeded manually, which represent our unsorted control. When comparing the results obtained with a 100-cells pool, no significant difference in cell viability and proliferation were observed between DA-sorted and FACSAria-sorted cells after 7 days in culture. On the other hand, the 10 cells pool sorted with DA PLUS showed a significantly higher viability and proliferation rate compared to those sorted with FACSAria at day 7. Moreover, cells sorted by DA-technology showed less variation between experiments, highlighting a more robust performance when sorting a small number of cells. Overall, the results confirm the unique capability of the DA system to sort a small number of cells, which maintain a higher cell viability and proliferation capacity compared to those sorted with FACS. This opens up the possibility to isolate rare tumor cell clones for functional studies that can help to better understand tumor progression and response to therapy. Citation Format: Maximilian Sergio, Chiara Rossi, Valeria Stivani, Ilaria Maffei, Marco Rossi, Gianpiero Parisi, Gianni Medoro. Isolation of small number of tumor cells for proliferation studies using DEPArray technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5324.

OLIG2 Is a Determinant for the Relapse of MYC-Amplified Medulloblastoma.

Patients with MYC-amplified medulloblastoma (MB) have poor prognosis and frequently develop recurrence, thus new therapeutic approaches to prevent recurrence are needed. We evaluated OLIG2 expression in a panel of mouse Myc-driven MB tumors, patient MB samples, and patient-derived xenograft (PDX) tumors and analyzed radiation sensitivity in OLIG2-high and OLIG2-low tumors in PDX lines. We assessed the effect of inhibition of OLIG2 by OLIG2-CRISPR or the small molecule inhibitor CT-179 combined with radiotherapy on tumor progression in PDX models. We found that MYC-associated MB can be stratified into OLIG2-high and OLIG2-low tumors based on OLIG2 protein expression. In MYC-amplified MB PDX models, OLIG2-low tumors were sensitive to radiation and rarely relapsed, whereas OLIG2-high tumors were resistant to radiation and consistently developed recurrence. In OLIG2-high tumors, irradiation eliminated the bulk of tumor cells; however, a small number of tumor cells comprising OLIG2- tumor cells and rare OLIG2+ tumor cells remained in the cerebellar tumor bed when examined immediately post-irradiation. All animals harboring residual-resistant tumor cells developed relapse. The relapsed tumors mirrored the cellular composition of the primary tumors with enriched OLIG2 expression. Further studies demonstrated that OLIG2 was essential for recurrence, as OLIG2 disruption with CRISPR-mediated deletion or with the small molecule inhibitor CT-179 prevented recurrence from the residual radioresistant tumor cells. Our studies reveal that OLIG2 is a biomarker and an effective therapeutic target in a high-risk subset of MYC-amplified MB, and OLIG2 inhibitor combined with radiotherapy represents a novel effective approach for treating this devastating disease.

Key markers for diagnosis of minimal residual disease in multiple myeloma

Introduction. Therapeutic advances in recent years, the appearance on the market each time of new drugs that allow patients to achieve complete remission, a long period without progression dictate the need to control treatment by monitoring residual disease in multiple myeloma. Monitoring of multiple myeloma is the detection of a small number of tumor cells after therapy in a patient, which may be the cause of recurrence of the disease – control of minimal residual disease (MRD). This article discusses the key diagnostic markers (CD45, CD56 and CD19) of minimal residual multiple myeloma disease at initial diagnosis and after induction therapy. There are various diagnostic methods of research that can reliably assess the response to therapy and predict the occurrence of relapse. The main methods for detecting MRD are allele-specific polymerase chain reaction, next-generation sequencing and multicolor flow cytometry. The diagnosis of MRD by flow cytometry is the most widely used quite fast, quantitative, sensitive and affordable method, it is used for the primary diagnosis of multiple myeloma, as well as for monitoring MRD. It is important to identify the aberrant immunophenotype during the initial diagnosis for the correct subsequent assessment of MRD. MRD of multiple myeloma is considered an important component in the course of patient therapy before hematopoietic stem cell autotransplantation to assess the effectiveness of therapy, control and prognosis of the disease.The aim of the work MRD-study with the key diagnostic markers in multiple myeloma (CD45, CD56 and CD19).Materials and methods. The study was carried out in 59 patients with multiple myeloma. For all patients, in addition to standard methods of diagnosis and staging of the disease, bone marrow morphology (myelogram) and immunophenotype were examined. Eight-color flow cytometry with a panel of monoclonal antibodies for the diagnosis of plasma cell tumors Euro-Flow 2012 was used to diagnose MRD.Results. MRD was assessed by markers CD45, CD56 and CD19 in patients with multiple myeloma after induction therapy. The incidence of MRD-positivity for CD45 was 87.5 %, for CD56 – 97.5 %, for CD19 – 95.5 %. The incidence of MRD-negative status was for CD45 was 12.5 %, for CD56 – 2.5 % and for CD19 – 4,5 %.Conclusion. The use of a complex of these markers allows the most accurate determination of the MRD-negative status, taking into account the primary immunophenotype of malignant plasma cells.

Pathogenesis of cutaneous T cell lymphoma: Involvement of Staphylococcus aureus.

Mycosis fungoides (MF) and Sézary syndrome (SS) are representative cutaneous lymphomas. In their early stage, a small number of tumor cells and a large number of non-malignant cells form a Th1-dominant tumor microenvironment. Increase in malignant T cells is accompanied by a decrease in CD8-positive T cells, with a shift toward a Th2-dominant milieu in advanced-stage lesions. The etiologies of MF/SS are diverse, and the underlying pathogenetic mechanisms are yet to be elucidated. Advanced MF/SS is known to be highly sensitive to Staphylococcus aureus, and the majority of deaths are caused by severe infections. The susceptibility to infection is associated with barrier dysfunction and immunosuppression, which are the main symptoms of MF. In recent years, skin-colonizing S.aureus has been identified to not only cause severe infections but also play an important role in the pathogenesis of MF/SS. Staphylococcal superantigens activate the proliferation of tumor cells and induce CD25 upregulation, FOXP3 expression, IL-17 expression, and miR-155 expression. Alpha-toxin eliminates non-neoplastic CD4-positive cells and CD8-positive cells and plays a major role in tumor cell selection. Lipoprotein may also be associated with the induction of Th2-dominant milieu. Antibiotic therapy for S.aureus eradication has been reported to cause considerable clinical improvement in the majority of individuals with advanced cutaneous T-cell lymphoma. Therefore, S.aureus may be a novel target for the treatment of advanced-stage MF/SS in the future.

Profiling of conditionally reprogrammed cell lines for in vitro chemotherapy response prediction of pancreatic cancer.

BackgroundThe establishment of patient-derived models for pancreatic ductal adenocarcinoma (PDAC) using conventional methods has been fraught with low success rate, mainly because of the small number of tumour cells and dense fibrotic stroma. Here, we sought to establish patient-derived model of PDAC and perform genetic analysis with responses to anticancer drug by using the conditionally reprogrammed cell (CRC) methodology.MethodsWe performed in vitro and in vivo tumourigenicity assays and analysed histological characteristics by immunostaining. We investigated genetic profiles including mutation patterns and copy number variations using targeted deep sequencing and copy-number analyses. We assessed the responses of cultured CRCs to the available clinical anticancer drugs based on patient responsiveness.FindingsWe established a total of 28 CRCs from patients. Of the 28 samples, 27 showed KRAS mutations in codon 12/13 or codon 61. We found that somatic mutations were shared in the primary-CRC pairs and shared mutations included key oncogenic mutations such as KRAS (9 pairs), TP53 (8 pairs), and SMAD4 (3 pairs). Overall, CRCs preserved the genetic characteristics of primary tumours with high concordance, with additional confirmation of low-AF NPM1 mutation in CRC (35 shared mutations out of 36 total, concordance rate=97.2%). CRCs of the responder group were more sensitive to anticancer agents than those of the non-responder group (P < 0.001).InterpretationThese results show that a pancreatic cancer cell line model can be efficiently established using the CRC methodology, to better support a personalized therapeutic approach for pancreatic cancer patients.Funding2014R1A1A1006272, HI19C0642-060019, 2019R1A2C2008050, 2020R1A2C209958611, and 2020M3E5E204028211

Histological and Immunohistochemical Features of Normal Histiocytes and Langerhans Cells, and Histiocytic Sarcomas in Four-Toed Hedgehogs (Atelerix albiventris)

Histiocytic sarcoma (HS) is a haematopoietic tumour of histiocyte origin that has been sporadically reported in four-toed hedgehogs (Atelerix albiventris). The present study aimed to investigate clinical, gross, histopathological and immunohistochemical features of HS in eight hedgehogs. Histological and immunohistochemical features of normal histiocytes and Langerhans cells (LCs) of hedgehogs were also investigated. HLA-DR-, Iba-1- and E-cadherin-positive LCs were observed in the epidermis, while Iba-1- and CD204-positive histiocytes were detected in the lymph nodes and spleen of normal hedgehogs. Localized HS (six cases) developed in the skin and spleen, while disseminated HS (two cases) occurred in the intestine. Tumour cells of disseminated HS were also distributed within the mesenteric lymph nodes, liver, kidney, spleen, lung and adrenal glands. Tumour cells of both localized and disseminated HS were composed of histiocytic cells, spindle to pleomorphic cells, multinucleated giant cells and erythrophagocytic cells. Most tumour cells were immunopositive for Iba-1, CD204 and lysozyme. A small number of tumour cells were positive for E-cadherin and CD208, and the tumour cells in one case were positive for HLA-DR. These results suggest that the tumour cells have variable features of histiocyte origin, including dendritic cells, LCs and macrophages. The behaviour of HS in the hedgehog was very aggressive, and 50% of cases died within 90 days of resection. The present study also highlighted the tendency for local tumour recurrence in localized cutaneous HS cases, suggesting a requirement for a long-term follow-up after excision.

Signal to Noise Ratio as a Cross-Platform Metric for Intraoperative Fluorescence Imaging.

Real-time molecular imaging to guide curative cancer surgeries is critical to ensure removal of all tumor cells; however, visualization of microscopic tumor foci remains challenging. Wide variation in both imager instrumentation and molecular labeling agents demands a common metric conveying the ability of a system to identify tumor cells. Microscopic disease, comprised of a small number of tumor cells, has a signal on par with the background, making the use of signal (or tumor) to background ratio inapplicable in this critical regime. Therefore, a metric that incorporates the ability to subtract out background, evaluating the signal itself relative to the sources of uncertainty, or noise is required. Here we introduce the signal to noise ratio (SNR) to characterize the ultimate sensitivity of an imaging system and optimize factors such as pixel size. Variation in the background (noise) is due to electronic sources, optical sources, and spatial sources (heterogeneity in tumor marker expression, fluorophore binding, and diffusion). Here, we investigate the impact of these noise sources and ways to limit its effect on SNR. We use empirical tumor and noise measurements to procedurally generate tumor images and run a Monte Carlo simulation of microscopic disease imaging to optimize parameters such as pixel size.

Intratumoral generation of 2‐fluoroadenine to treat solid malignancies of the head and neck

This report describes treatment of locoregional head and neck squamous cell carcinoma (HNSCC) by an innovative, experimental strategy involving generation of a robust anti-cancer agent (2-fluoroadenine [F-Ade]) following transduction by Escherichia coli purine nucleoside phosphorylase (PNP) in a small number of tumor cells. F-Ade works by a unique mechanism of action (ablation of RNA and protein synthesis) and confers tumor regressions of otherwise refractory HNSCC in human subjects. Clinical studies have now advanced to a pivotal (registration-directed) trial involving locoregional HNSCC, with plans to begin subject enrollment late in 2018. The present review is the first to summarize use of PNP in the context of HNSCC, and provides background regarding this emerging anti-cancer approach.

A comprehensive review on Phyllanthus derived natural products as potential chemotherapeutic and immunomodulators for a wide range of human diseases

Abstract Treatment options for most cancers are still insufficient, despite developments and technology advancements. It has been postulated that the immune response to progressive tumors is insufficient due to a deficiency in afferent mechanisms responsible for the development of tumor-reactive T cells. Many patients treated for cancer will have their cancer recurrence, often after a long remission period. This suggests that there are a small number of tumor cells that remain alive after standard treatment(s) – alone or in combination and have been less effective in combating metastasis that represents the most elaborate hurdle to overcome in the cure of the disease. Therefore, any new effective and safe therapeutic agents will be highly demanded. To circumvent many plant extracts have attributed for their chemoprotective potentials and their influence on the human immune system. It is now well recognized that immunomodulation of immune response could provide an alternative or addition to conventional chemotherapy for a variety of disease conditions. However, many hurdles still exist. In recent years, there has been a tremendous interest either in harnessing the immune system or towards plant-derived immunomodulators as anticancer agents for their efficacy, safety and their targeted drug action and drug delivery mechanisms. This review discusses Phyllanthus Schum. & Thonn. their chemopreventive and immunomodulatory properties over the past few years. Although, as many as 500 important bioactive phytochemical compounds have been isolated from Phyllanthus their chemotherapeutic, immunomodulatory properties, molecular targets and modes of action are yet to be enlightened in detail. Hence, the theme of this review is very useful for further research on Phyllanthus species. because many phytochemicals from these plants have demonstrated preclinical therapeutic efficacy for a wide range of human diseases.

P2.09-32 Detached Epithelial Cell Cluster Size in Lung Adenocarcinoma is a Marker of Poor Prognosis

Various types of detached epithelial cell clusters (DECs) are occasionally seen within lung adenocarcinoma (L-ADC): small clusters containing a small number of tumor cells (type 1); medium-sized clusters comprising 5-20 tumor cells (type 2); and large clusters comprising more than 20 tumor cells (type 3). In general, type 2 DEC is considered a micropapillary pattern (MP-p). Although MP-p is known to be a sign of worse prognosis than other growth patterns, the prognostic significance of type 1 and 3 DEC structures are not well known. This study aimed to analyze the prognostic significance of DEC by histologically reviewing 921 resected L-ADCs. We investigated the presence of DEC, the DEC pattern (type 1, 2, or 3), and their associations with clinicopathological parameters. Furthermore, we studied the prognostic significance of DEC in resected L-ADCs. Two hundred sixty-four tumors (28.6%) were DEC-positive. These DEC-positive tumors were significantly associated with higher levels of T factor (P < 0.001), node positivity (P < 0.001), higher tumor stage (P < 0.001), lymphovascular invasion (P < 0.001), and the presence of spread through air spaces (STAS) (P < 0.001). On the other hand, no association was observed between DEC positivity and EGFR, KRAS, or ALK gene alterations. Patients with DEC-positive tumors had significantly worse prognoses than those with DEC-negative tumors, as assessed by both overall survival and disease-free survival (P < 0.001 for both). Furthermore, patients with tumors showing type 3 DEC had the worst prognoses, followed by patients type 2 DEC. Patients with type 1 or no DEC had significantly better prognoses (P < 0.001). The present study indicated that DECs are significant prognostic markers of L-ADC outcomes. These results suggest that patients with DEC in resected L-ADCs may require additional treatment after resection.

Histopathologic and Immunohistochemistry Findings in Feline Renal Cell Carcinoma.

The biological behavior and immunohistochemical features of feline renal cell carcinoma (RCC) have not been well characterized. In the present study, immunohistochemical examinations were performed in 12 feline cases of RCC. The RCC consisted of solid ( n = 2), solid-tubular ( n = 2), tubular ( n = 3), papillary ( n = 2), tubulopapillary ( n = 2), and sarcomatoid ( n = 1) type lesions. Of the cases with RCC, 1 developed metastatic disease and 6 cases had no evidence of recurrence at 80 to 2292 days after surgery. One papillary-type tumor had cuboidal cells with scant cytoplasm and monomorphic nuclei, and the other had pseudostratified columnar cells with abundant cytoplasm. Immunohistochemistry revealed that the tumor cells in most cases were positive for cytokeratin (CK)7, CK20, KIT, and CD10, with the exception of cases of the solid type with clear cytoplasm (solid anaplastic), papillary type with columnar cells, and sarcomatoid types. A small number of tumor cells in the solid anaplastic and in the sarcomatoid types were positive for aquaporin-1. Increased expression of N-cadherin and Twist along with nuclear accumulation of β-catenin were observed in the sarcomatoid type. These results indicated that CK, KIT, and CD10 are relatively strongly expressed in most feline RCC. The solid anaplastic RCC exhibited CD10 expression with the absence of distal tubule marker expression. Although immunohistochemistry profiles were relatively consistent with those described in human RCC, the histopathologic features were different from those seen in humans. Epithelial-mesenchymal transition (EMT) marker expression in the current cases may suggest the involvement of an EMT-like mechanism in the development of sarcomatoid RCC in cats.

Sulfated fucans and a sulfated galactan from sea urchins as potent inhibitors of selectin-dependent hematogenous metastasis.

Metastasis is responsible for the majority of cancer-associated deaths, though only a very small number of tumor cells are able to efficiently complete all the steps of that process. Tumor cell survival in the bloodstream is one of the limiting aspects of the metastatic cascade. The formation of tumor cell-platelet complexes that promote tumor cell survival is facilitated by the binding of P-selectin on activated platelets to sialyl Lewis-containing oligosaccharides on the surface of tumor cells. Inhibition of this interaction has been shown to attenuate metastasis. Heparin is a potent selectin inhibitor and is capable to block platelet-tumor cell complex formation, thereby attenuating metastasis. Similarly, other sulfated polysaccharides isolated from marine invertebrates attenuate metastasis by a P-selectin-mediated mechanism. In this work, we investigated the selectin-dependent antimetastatic activity of sea urchin sulfated polysaccharides with slight structural differences: a sulfated fucan from Strongylocentrotus franciscanus; a sulfated fucan from Strongylocentrotus droebachiensis; and a sulfated galactan from Echinometra lucunter. The results demonstrate that these fucans and the galactan have different antiselectin activities despite being very similar molecules. Therefore, they may be interesting tools for studies on the structure-function relationship or even for future treatments.

Pembrolizumab and its role in relapsed/refractory classical Hodgkin's lymphoma: evidence to date and clinical utility.

Immune evasion is a critical mechanism of malignant cell survival, and relies in part on molecular signaling through the programmed cell death 1 (PD-1)/PD-1 ligand (PD-L1) axis that contributes to T cell exhaustion. Immune modulatory therapy with monoclonal antibodies against PD-1 designed to enhance antitumor immune response have shown promise in the treatment of advanced solid tumors and hematologic malignancies. Classical Hodgkin's lymphoma (cHL), a unique B cell malignancy characterized by an extensive but ineffective immune cell infiltrate surrounding a small number of tumor cells, has shown significant response to anti-PD-1 directed therapy. The anti-PD-1 monoclonal antibodies nivolumab and pembrolizumab have shown similarly remarkable activity in relapsed/refractory cHL and have been approved by the Food and Drug Administration for treatment of this disease. In this article we review the rationale of targeting the PD-1/PD-L1 axis in cHL and the pharmacology of pembrolizumab, and summarize the data on activity and safety profile of this agent in the treatment of relapsed/refractory cHL. We also discuss the potential benefits and pitfalls of using PD-1 blockade in the setting of allogeneic stem-cell transplantation, and summarize ongoing prospective trials of single-agent pembrolizumab and combination strategies as well as future directions.

Low frequency of p53 and k-ras codon 12 mutations in non-small cell lung carcinoma (NSCLC) tumors and surgical margins.

Lung cancer is one of the most common cancers and has became a predominant cause of cancer-related death throughout the world. The k-ras codon 12 mutation, which is the most common lung cancer mutation, is found in 15 to 30% of all lung cancers. Furthermore, the p53 gene has a very important role in the biological properties of tumor cells, and it is mutated in about 50% of non-small cell lung cancers. Residual tumor cells remain in surgical margins diagnosed as tumor free by histopathological techniques, and they can play a role in forming any local recurrence. Molecular methods may be exploited that are sensitive enough to detect small numbers of tumor cells. In the present study, we examined p53 gene mutations and k-ras codon 12 mutations from the tumor samples and surgical margins of 34 non-small-cell lung cancer patients. P53 gene mutations were analyzed by single strand conformational polymorphism analysis, heterodublex analysis and DNA sequencing. K-ras codon 12 mutations were analyzed by the mutagenic PCR-restricted fragment length polymorphism method. A p53 mutation was detected only in primary tumors of 3 out of 34 patients (8.82%). These mutations were clustered in exon 5. Moreover, a k-ras codon 12 mutation was detected in both the primary tumor and the surgical margin tissues of 2 out of 34 patients (5.88%). The detected mutation rate was low, in the range given in the literature. We think that different mechanisms related to other genes and individual genetic differences might play a role in cancer formation in our study group. We believe that molecular studies are necessary to identify biomarkers and to determine genetic alterations in histopathologically normal surgical margins.

Extracting microtentacle dynamics of tumor cells in a non-adherent environment.

During metastasis, tumor cells dynamically change their cytoskeleton to traverse through a variety of non-adherent microenvironments, including the vasculature or lymphatics. Due to the challenges of imaging drift in non-adhered tumor cells, the dynamic cytoskeletal phenotypes are poorly understood. We present a new approach to analyze the dynamic cytoskeletal phenotypes of non-adhered cells that support microtentacles (McTNs), which are cell surface projections implicated in metastatic reattachment. Combining a recently-developed cell tethering method with a novel image analysis framework allowed McTN attribute extraction. Full cell outlines, number of McTNs, and distance of McTN tips from the cell body boundary were calculated by integrating a rotating anisotropic filtering method for identifying thin features with retinal segmentation and active contour algorithms. Tethered cells behave like free-floating cells; however tethering reduces cell drift and improves the accuracy of McTN measurements. Tethering cells does not significantly alter McTN number, but rather allows better visualization of existing McTNs. In drug treatment experiments, stabilizing tubulin with paclitaxel significantly increases McTN length, while destabilizing tubulin with colchicine significantly decreases McTN length. Finally, we quantify McTN dynamics by computing the time delay autocorrelations of 2 composite phenotype metrics (cumulative McTN tip distance, cell perimeter:cell body ratio). Our automated analysis demonstrates that treatment with paclitaxel increases total McTN amount and colchicine reduces total McTN amount, while paclitaxel also reduces McTN dynamics. This analysis method enables rapid quantitative measurement of tumor cell drug responses within non-adherent microenvironments, using the small numbers of tumor cells that would be available from patient samples.

Rozpoznawanie i leczenie chorych na chłoniaka Hodgkina

Hodgkin lymphoma (HL) is a rare B-cell derived lymphoid malignancy. Its typical feature is the presence of small number of tumor cells surrounded by immune cell infiltrate of immunosuppressive character. Two entities of HL are distinguished: classical HL and nodular lymphocyte predominant HL. Assessment of clinical advancement stage and prognostic factors is important for tailoring appropriate therapy based on chemotherapy and radiotherapy. Prognosis in HL is good with cure rate reaching almost 80%. About 10% of patients (especially in advanced stages) does not achieve complete remission. In 20–30% patients, who initially achieved a response, relapses occur. For these patients’, besides chemotherapy, treatment with brentuximab vedotin, PD-1 inhibitors, autologous or allogeneic hematopoietic stem cell transplantation, or participation in clinical trial comprise poten¬tial therapy options. In this publication, we discuss the guidelines of diagnosis and treatment of HL.