Automatic detection method of bladder tumor cells based on color and shape features

Bladder urothelial carcinoma is the most common malignant tumor disease in urinary system,and its incidence rate ranks ninth in the world.In recent years,the continuous development of hyperspectral imaging technology has provided a new tool for the auxiliary diagnosis of bladder cancer.In this study,based on microscopic hyperspectral data,an automatic detection algorithm of bladder tumor cells combining color features and shape features is proposed.Support vector machine(SVM)is used to build classification models and compare the classification performance of spectral feature,spectral and shape fusion feature,and the fusion feature proposed in this paper on the same classifier.The results show that the sensitivity,specificity,and accuracy of our classification algorithm based on shape and color fusion features are 0.952,0.897,and 0.920,respectively,which are better than the classification algorithm only using spectral features.Therefore,this study can effectively extract the cell features of bladder urothelial carcinoma smear,thus achieving automatic,real-time,and noninvasive detection of bladder tumor cells,and then helping doctors improve the efficiency of pathological diagnosis of bladder urothelial cancer,and providing a reliable basis for doctors to choose treatment plans and judge the prognosis of the disease.

In vivo fluorescence flow cytometry reveals that the nanoparticle tumor vaccine OVA@HA-PEI effectively clears circulating tumor cells

Tumor vaccine therapy offers significant advantages over conventional treatments,including reduced toxic side effects.However,it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods.Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors,thereby limiting their clinical efficacy.In this study,we developed a nanoparticle-based tumor vaccine,OVA@HA-PEI,utilizing ovalbumin(OVA)as the presenting antigen and hyaluronic acid(HA)and polyethyleneimine(PEI)as adjuvants and carriers.This formulation significantly enhanced the proliferation of immune cells and cytokines,such as CD3,CD8,interferon-,and tumor necrosis factor-,in vivo,effectively activating an immune response against B16–F10 tumors.In vivofluorescenceflow cytometry(IVFC)has already become an effective method for monitoring circulating tumor cells(CTCs)due to its direct,noninvasive,and long-term detection capabilities.Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1(PD-1)antibody.The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone.Additionally,this combination therapy substantially reduced the number of CTCs in vivo,increased the clearance rate of CTCs by the immune system,and slowed tumor progression.Thesefindings greatly enhance the clinical application prospects of IVFC and tumor vaccines.

Prognostic value of circulating tumor cells combined with neutrophil-lymphocyte ratio in patients with hepatocellular carcinoma

BACKGROUND Circulating tumor cell(CTC)count and neutrophil-to-lymphocyte ratio(NLR)are both closely associated with the prognosis of hepatocellular carcinoma(HCC).AIM To investigate the prognostic value of combining these two indicators in HCC.METHODS Clinical data were collected from patients with advanced HCC who received im-mune therapy combined with targeted therapy at the Department of Oncology,the Affiliated Hospital of Southwest Medical University,Sichuan,China,from 2021 to 2023.The optimal cutoff values for CTC programmed death-ligand 1(PD-L1)(+)>1 or CTC PD-L1(+)≤1 and NLR>3.89 or NLR≤3.89 were evaluated using X-Tile software.Patients were categorized into three groups based on CTC PD-L1(+)counts and NLR:CTC-NLR(0),CTC-NLR(1),and CTC-NLR(2).The relationship between CTC-NLR and clinical variables as well as survival rates was assessed.RESULTS Patients with high CTC PD-L1(+)expression or NLR at baseline had shorter median progression-free survival(m-PFS)and median overall survival(mOS)than those with low levels of CTC PD-L1(+)or NLR(P<0.001).Mean-while,patients in the CTC-NLR(2)group showed a significant decrease in mPFS and mOS.Cox regression analysis revealed that alpha-fetoprotein(AFP),CTC PD-L1(+),and CTC-NLR were independent predictors of OS.The time-dependent receiver operating characteristic curve showed that the area under the curve of CTC-NLR at 12 months(0.821)and 18 months(0.821)was superior to that of AFP and CTC PD-L1(+).CONCLUSION HCC patients with high CTC PD-L1(+)or NLR expression tend to exhibit poor prognosis,and a high baseline CTC-NLR score may indicate low survival.CTC-NLR may serve as an effective prognostic indicator for patients with advanced HCC receiving immunotherapy combined with targeted therapy.

Celastrol activates caspase-3/GSDME-dependent pyroptosis in tumor cells by inducing endoplasmic reticulum stress Author links open overlay panel

Objective:To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved,specifically focusing on the role of the caspase-3/gasdermin E(GSDME)signaling pathway and the impact of endoplasmic reticulum(ER)stress and autophagy.Methods: Necrostatin-1(Nec-1),lactate dehydrogenase release(LDH)assay,and Hoechst/propidium iodide(PI)double staining were employed to validate the mode of cell death.Western blot was used to detect the cleavage of GSDME and the expression of light chain 3(LC3)and BIP.Results: Celastrol induced cell swelling with large bubbles,which is consistent with the pyroptotic phenotype.Moreover,treatment with celastrol induced GSDME cleavage,indicating the activation of GSDME-mediated pyroptosis.GSDME knockout via CRISPR/Cas9 blocked the pyroptotic morphology of celastrol in HeLa cells.In addition,cleavage of GSDME was attenuated by a specific caspase-3 inhibitor in celastrol-treated cells,suggesting that GSDME activation was induced by caspase-3.Mechanistically,celastrol induced endoplasmic reticulum(ER)stress and autophagy in HeLa cells,and other ER stress inducers produced effects consistent with those of celastrol.Conclusion: These findings suggest that celastrol triggers caspase-3/GSDME-dependent pyroptosis via activation of ER stress,which may shed light on the potential antitumor clinical applications of celastrol.

Circulating tumor cells in pancreatic cancer:The prognostic impact in surgical patients

Pancreatic cancer is associated with a poor prognosis,even in the early stages,mainly due to metastatic progression.New diagnostic techniques that predict unfavorable outcomes are needed in order to improve treatment strategies.Circulating tumor cells(CTCs)are showing promising results as a predictive biomarker for various tumors.In this editorial we comment on the article by Zhang et al,who published the first systematic review and meta-analysis evaluating the prognostic value of CTCs as biomarkers in early-stage pancreatic cancer patients undergoing surgery.CTCs were detected in peripheral or central venous system blood,before or during surgery.Positive CTCs showed a correlation with decreased overall survival and decreased relapse-free,disease-free and progression-free survival in this meta-analysis.However,the heterogeneity was significant.The authors suggest that this result was related to the separation methods used between studies,but other differences such as the margin status or the neoadjuvant and adjuvant treatments used are also important to consider.CTCs may be a potential prognostic biomarker in pancreatic cancer patients,but it is necessary to compare and standardize the platforms used to isolate CTCs,to compare different biomarkers from liquid biopsy and to determine the impact on prognosis when therapeutic changes are made based on CTCs levels.

Circulating tumor cells as prognostic marker in pancreatic cancer

In this editorial we comment on the article by Zhang et al published in the recent issue of the World Journal of Clinical Oncology.Pancreatic cancer is the fourth most common cause of cancer-related mortality and has the lowest survival rate among all solid cancers.It causes 227000 deaths annually worldwide,and the 5-year survival rate is very low due to early metastasis,which is 4.6%.Cancer survival increases with better knowledge of risk factors and early and accurate diagnosis.Circulating tumor cells(CTCs)are tumor cells that intravasate from the primary tumor or metastasis foci into the peripheral blood circulation system spontan-eously or during surgical operations.Detection of CTC in blood is promising for early diagnosis.In addition,studies have associated high CTC levels with a more advanced stage,and more intensive treatments should be considered in cases with high CTC.In tumors that are considered radiologically resectable,it may be of critical importance in detecting occult metastases and preventing unnecessary surgeries.

Different gap junction-propagated effects on cisplatin transfer result in opposite responses to cisplatinin normal cells versus tumor cells

OBJECTIVE Previous work has shown that gap junction intercel ular communication(GJIC)enhances cisplatin(Pt)toxicity in testicular tumor cells but decreases it in non-tumor testicular cells.In this study,these different GJIC-propagated effects were demonstrated in tumor versus non-tumor cells from other organ tissues(liver and lung).METHODS We use several different mani pulations(no cell contact,pharmacological inhibition,and si RNA suppression)to down-regulate GJIC function.The in vivo results using xenograft tumor models were consistent with those from the above-mentioned cells.To better understand the mechanism(s)involved,we studied the effects of GJIC on Pt accumulation in tumor and non-tumor cells from the liver and lung.RESULTS The intracel ular Pt and DNA-Pt adduct contents clearly increased in non-tumor cells but decreasedin tumor cells when GJIC was downregulated.Further analysis indicated that the opposite effectsof GJIC on Pt accumulation in normal versus tumor cells from the liver were due to its different effects on copper transporter1 and multidrug resistance-associated protein2,membrane transporters attributed to intracellular Pt transfer.CONCLUSION GJIC protects normal organs from cisplatin toxicity while enhancing it in tumor cells via its different effects on intracellular Pt transfer.

Case Report: Pazopanib Treatment Response in a Patient with Metastatic Pleomorphic Dermal Sarcoma (Atypical Fibroxanthoma) with Circulating Tumor Cell-Derived Colonies as a Predictive Marker

Atypical fibroxanthomas (AFX) are rare skin tumors. These generally are superficial tumors, usually <3 cm red, fleshy, ulcerated skin lesions, that characteristically occur on sun-damaged skin, sometimes in immunocompromised or previously irradiated patients. These are part of a spectrum of more aggressive fibro-histiocytic neoplasms. In the older literature, these have been termed aggressive or metastatic AFX, but currently these have been reclassified as pleomorphic dermal sarcomas (PDS) and systemic undifferentiated pleomorphic sarcoma (UPS, formerly malignant fibrohistiocytic sarcoma, MFH). We present the case of a 64-year old woman who developed a deeply invasive PDS on the vertex of her scalp invading to the galea, with in-transit scalp metastases. Very little information is available about optimal treatment of metastatic PDS lesions. The patient was initially treated with 2 cycles of epirubicin/ifosfamide chemotherapy, resulting in life-threatening complications. A pretreatment peripheral blood sample was sent for CTC-derived colony assay. This sample grew 8 colonies from 10 ml blood. The tumor failed to respond to epirubicin and ifosfamide, and after several months of hospitalization, a second peripheral blood CTC-derived colony assay grew >376 colonies. The patient could not tolerate additional chemotherapy. She was therefore treated with the oral targeted agent pazopanib. The patient developed a dramatic biopsy-confirmed complete response. After 11 months of pazopanib treatment, a repeat CTC-derived culture sample grew only 8 colonies/10 ml blood. The complete response to pazopanib is still ongoing at over 41 months. To our knowledge, this is the first demonstration of clinical complete response of a PDS tumor following targeted therapy. An additional novel feature was the demonstration that CTC-derived colonies could be grown from the blood of a PDS patient. The number of colonies appeared to correlate with the clinical treatment response and seemed to function as a potential prognostic marker.

Circulating tumor cells in pancreatic cancer patients:Enrichment and cultivation

AIM: To investigate the feasibility of separation and cultivation of circulating tumor cells (CTCs) in pancreatic cancer (PaC) using a filtration device.

Liquid biopsy in patients with hepatocellular carcinoma: Circulating tumor cells and cell-free nucleic acids

Hepatocellular carcinoma(HCC), with its high incidence and mortality rate, is one of the most common malignant tumors. Despite recent development of a diagnostic and treatment method, the prognosis of HCC remains poor. Therefore, to provide optimal treatment for each patient with HCC, more precise and effective biomarkers are urgently needed which could facilitate a more detailed individualized decision-making during HCC treatment, including the following; risk assessment, early cancer detection, prediction of treatment or prognostic outcome. In the blood of cancer patients, accumulating evidence about circulating tumor cells and cell-free nucleic acids has suggested their potent clinical utilities as novel biomarker. This concept, so-called "liquid biopsy" is widely known as an alternative approach to cancer tissue biopsy. This method might facilitate a more sensitive diagnosis and better decision-making by obtaining genetic and epigenetic aberrations that are closely associated with cancer initiation and progression. In this article, we review recent developments based on the available literature on both circulating tumor cells and cell-free nucleic acids in cancer patients, especially focusing on Hepatocellular carcinoma.

Comparative study on radiosensitivity of various tumor cells and human normal liver cells

AIM:To investigate the radiation response of various human tumor cells and normal liver cells. METHODS: Cell lines of human hepatoma cells (SMMC-7721), liver cells (L02), melanoma cells (A375) and cervical tumor (HeLa) were irradiated with 60Co γ-rays. Cell survive was documented by a colony assay. Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A. RESULTS: Linear quadratic survival curve was observed in all of four cell lines, and dose-dependent increase in radiation-induced chromatid and isochromatid breaks were observed in GB2B phase. Among these four cell lines, A375 was most sensitive to radiation, while, L02 had the lowest radiosensitivity. For normal liver cells, chromatid breaks were easy to be repaired, isochromatid breaks were difficult to be repaired. CONCLUSION: The results suggest that the y-rays induced chromatid breaks can be possibly used as a good predictor of radiosensitivity, also, unrejoined isochromatid breaks probably tightly related with cell cancerization.

Down-regulation of Hsp90 could change cell cycle distribution and increase drug sensitivity of tumor cells

AIM:To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs.METHODS:A 1.03kb cDNA sequence of Hsp90beta was obtained from the primary plasmid phHSP90 by EcoR I and BamH I nuclease digestion and was cloned to the EcoR I and BamH I site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90beta expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protection assay, and the expression of Hsp90 protein determined with western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to Adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry.RESULTS:In EcoR I and BamH I restriction analysis, the size and the direction of the cloned sequence of Hsp90beta remained what had been designed and the gene constructs were named pcDNA-Hsp90.AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/VCR and AH-Ec109 cells, G(1) phase cells were increased; S phase and G(2) phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G(1)phase cells were decreased, G(2) phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G(1), S and G(2) phase cells remained unchanged as compared with their parental cell lines. The sensitivity of AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P 【 0.05).CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells.

;~lasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth

Persistent activation of Survivin and its overexpression contribute to the formation, progression and metastasis of several different tumor types. Therefore, Survivin is an ideal target for RNA interference mediated-growth inhibition. Blockade of Survivin using specific short hairpin RNAs （shRNA） can significantly reduce prostate tumor growth. RNA interference does not fully ablate target gene expression, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Survivin-specific shRNA, we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 （GRIM-19）. Then, the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium （S. typhimurium） to treat prostate cancer in vitro and in vivo. We found that the co-expressed Survivin-specific shRNA and GRIM-19 synergistically and more effectively inhibited prostate tumor proliferation and survival, when compared with treatment with either single agent alone in vitroand in vivo. This study has provided a novel cancer gene therapeutic approach for prostate cancer.

Internalization of NK cells into tumor cells requires ezrin and leads to programmed cell-in-cell death

Cytotoxic lymphocytes are key players in the orchestration of immune response and elimination of defective cells. We have previously reported that natural killer （NK） cells enter target tumor ceils, leading to either target cell death or self-destruction within tumor cells. However, it has remained elusive as to the fate of NK cells after internalization and whether the heterotypic cell-in-cell process is different from that of the homotypic cell-in-cell event recently named entosis. Here, we show that NK cells undergo a cell-in-cell process with the ultimate fate of apoptosis within tumor cells and reveal that the internalization process requires the actin cytoskeletal regulator, ezrin. To visualize how NK cells enter into tumor cells, we carried out real-time dual color imaging analyses of NK cell internalization into tumor cells. Surprisingly, most NK cells commit to programmed cell death after their entry into tumor cells, which is distinctively different from entosis observed in the homotypic cell-in-cell process. The apoptotic cell death of the internalized NK cells was evident by activation of caspase 3 and DNA fragmentation. Furthermore, NK cell death after internalization is attenuated by the caspase inhibitor, Z-VAD-FMK, confirming apoptosis as the mode of NK cell death within tumor cells. To determine protein factors essential for the entry of NK cells into tumor cells, we car- ried out siRNA-based knockdown analysis and discovered a critical role of ezrin in NK cell internalization. Impor- tantly, PKA-mediated phosphorylation of ezrin promotes the NK cell internalization process. Our findings suggest a novel regulatory mechanism by which ezrin governs NK cell internalization into tumor cells.

Aneuploidy of chromosome 8 in circulating tumor cells correlates with prognosis in patients with advanced gastric cancer

Objective： Previous work indicated that aneuploidy of chromosome 8 in circulating tumor cells（CTCs）correlated with therapeutic efficacy for advanced gastric cancer（AGC） patients. In this follow-up study performed on the same population of AGC patients, we investigated whether and how aneuploidy of chromosome 8 in CTCs correlates with patients＇ clinical prognosis.Methods： The prospective study was performed on 31 patients with newly diagnosed AGC. Previously established integrated subtraction enrichment（SE） and immunostaining-fluorescence in situ hybridization（i FISH）platform was applied to identify, enumerate and characterize CTCs. Quantification of CTCs and analysis of their aneuploidy of chromosome 8 were performed on patients before and after therapy.Results： CTCs were measured in 93.5% of AGC patients, and two CTC subtypes with diverse threshold values were identified, multiploid CTCs with the threshold of ≥2 per 7.5 m L and multiploid plus triploid CTCs with the threshold of ≥4, which were found to significantly correlate with poor progression-free survival（PFS） and overall survival（OS）. In particular, patients with ≥10% increased multiploid CTCs after an initial 6 weeks of therapy had poor PFS and OS, whereas improved PFS and OS were observed on those who had ≥10% decreased multiploid CTCs. After adjusting for clinically significant factors, ≥10% increased post-therapy multiploid CTCs was the only independent predictor of PFS and OS.Conclusions： Aneuploidy of CTCs correlates with prognosis of AGC patients. Quantitative comparison monitoring multiploid CTCs before and after therapy may help predict improved or inferior prognosis and chemoresistance.

Liquid biopsy of gastric cancer patients:Circulating tumor cells and cell-free nucleic acids

To improve the clinical outcomes of cancer patients, early detection and accurate monitoring of diseases are necessary. Numerous genetic and epigenetic alterations contribute to oncogenesis and cancer progression, and analyses of these changes have been increasingly utilized for diagnostic, prognostic and therapeutic purposes in malignant diseases including gastric cancer (GC). Surgical and/or biopsy specimens are generally used to understand the tumor-associated alterations; however, those approaches cannot always be performed because of their invasive characteristics and may fail to reflect current tumor dynamics and drug sensitivities, which may change during the therapeutic process. Therefore, the importance of developing a non-invasive biomarker with the ability to monitor real-time tumor dynamics should be emphasized. This concept, so called &#x0201c;liquid biopsy&#x0201d;, would provide an ideal therapeutic strategy for an individual cancer patient and would facilitate the development of &#x0201c;tailor-made&#x0201d; cancer management programs. In the blood of cancer patients, the presence and potent utilities of circulating tumor cells (CTCs) and cell-free nucleic acids (cfNAs) such as DNA, mRNA and microRNA have been recognized, and their clinical relevance is attracting considerable attention. In this review, we discuss recent developments in this research field as well as the relevance and future perspectives of CTCs and cfNAs in cancer patients, especially focusing on GC.

Prognostic value of circulating tumor cells in esophageal cancer

AIM To perform a meta-analysis of the related studies to assess whether circulating tumor cells(CTCs) can be used as a prognostic marker of esophageal cancer.METHODS Pub Med, Embase, Cochrane Library and references in relevant studies were searched to assess the prognostic relevance of CTCs in patients with esophageal cancer. The primary outcome assessed was overall survival(OS). The meta-analysis was performed using the random effects model, with hazard ratio(HR), risk ratio(RR) and 95% confidence intervals(95%CIs) as effect measures.RESULTS Nine eligible studies were included involving a total of 911 esophageal cancer patients. Overall analyses revealed that CTCs-positivity predicted disease progression(HR = 2.77, 95%CI: 1.75-4.40, P < 0.0001) and reduced OS(HR = 2.67, 95%CI: 1.99-3.58, P < 0.00001). Further subgroup analyses demonstrated that CTCs-positive patients also had poor OS in different subsets. Moreover, CTCs-positivity was also significantly associated with TNM stage(RR = 1.48, 95%CI: 1.07-2.06, P = 0.02) and T stage(RR = 1.44, 95%CI: 1.13-1.84, P = 0.003) in esophageal cancer.CONCLUSION Detection of CTCs at baseline indicates poor prognosis in patients with esophageal cancer. However, this finding relies on data from observational studies and is potentially subject to selection bias. Prospective trials are warranted.

Protein Expression of BLM Gene and Its Apoptosis Sensitivity in Hematopoietic Tumor Cell Strains

Patients with Bloom syndrome （BS） show an immunodeficiency, an enhanced sister chromatid exchanges （SCEs）, a strong genetic instability and an increased predisposition to all. In order to investigate the differential expression of BLM protein in hematopoietic tumor cell strains and study the effects of BLM gene on ultraviolet （UV）- or hydroxyurea （HU）-induced apoptosis, Western blot was used to detect the expression of BLM protein in normal human bone marrow mononuclear cells and 4 kinds of hematopoietic tumor cell strains. The 4 kinds of hematopoietic tumor cells were exposed to UV light with a germicidal UV lamp or treated with 2 mmol/L hydroxyurea and the apoptotic rate was detected by using AnnexinV-FITC. The results showed that these tumor cells expressed BLM protein higher than the normal human bone marrow mononuclear cells （P〈0.01）. In the 4 hematopoietic tumor cells, BLM protein was all specially cleaved in response to UV- or HU-induced apoptosis. The increase of BLM protein expression may play an important role in the development of these tumors, and BLM proteolysis is likely to be a general feature of the apoptotic response.

Combined peripheral natural killer cell and circulating tumor cell enumeration enhance prognostic efficiency in patients with metastatic triple-negative breast cancer

Objective：Triple-negative breast cancer（TNBC）is a heterogeneous disease with poor prognosis.Circulating tumor cells（CTCs）are a promising predictor for breast cancer prognoses but their reliability regarding progression-free survival（PFS）is controversial.We aim to verify their predictive value in TNBC.Methods：In present prospective cohort study,we used the Pep@MNPs method to enumerate CTCs in baseline blood samples from 75 patients with TNBC（taken at inclusion in this study）and analyzed correlations between CTC numbers and outcomes and other clinical parameters.Results：Median PFS was 6.0（range：1.0–25.0）months for the entire cohort,in whom we found no correlations between baseline CTC status and initial tumor stage（P=0.167）,tumor grade（P=0.783）or histological type（P=0.084）.However,among those getting first-line treatment,baseline CTC status was positively correlated with ratio of peripheral natural killer（NK）cells（P=0.032）,presence of lung metastasis（P=0.034）and number of visceral metastatic site（P=0.037）.Baseline CTC status was predictive for PFS in first-line TNBC（P=0.033）,but not for the cohort as a whole（P=0.118）.This prognostic limitation of CTC could be ameliorated by combining CTC and NK cell enumeration（P=0.049）.Conclusions：Baseline CTC status was predictive of lung metastasis,peripheral NK cell ratio and PFS in TNBC patients undergoing first-line treatment.We have developed a combined CTC-NK enumeration strategy that allows us to predict PFS in TNBC without any preconditions.

Circulating tumor cells(CTCs)in breast cancer:a diagnostic tool for prognosis and molecular analysis

Metastatic breast cancer （MBC） is characterized by a combination of tumor growth, proliferation and metastatic progression and is typically managed with palliative intent. The benefit of standard systemic therapies is relatively limited and the disease is considered incurable suggesting the need to investigate the biological drivers of the various phases of the metastatic process in order to improve the selection of molecularly driven therapies. The detection, enumeration and molecular analysis of circulating tumor cells （CTCs） provide an intriguing opportunity to advance this knowledge. CTCs enumerated by the Food and Drugs Administration-cleared CellSearchTM system are an independent prognostic factor of progression- free survival （PFS） and overall survival （OS） in MBC patients. Several published papers demonstrated the poor prognosis for MBC patients that presented basal CTC count _〉5 in 7.5 mL of blood. Therefore, the enumeration of CTCs during treatment for MBC provides a tool with the ability to predict progression of disease earlier than standard timing of anatomical assessment using conventional radiological tests. During the metastatic process cancer cells exhibit morphological and phenotypic plasticity undergoing epithelial- mesenchymal transition （EMT）. This important phenomenon is associated with down regulation of epithelial marker （e.g., EpCAM） with potential limitations in the applicability of current CTCs enrichment methods. Such observations translated in a number of investigations aimed at improving our capabilities to enumerate and perform molecular characterization of CTCs. Theoretically, the phenotypic analysis of CTCs can represent a ＂liquid＂ biopsy of breast tumor that is able to identify a new potential target against the metastatic disease and advance the development and monitoring of personalized therapies.