Purpose To evaluate the classification criteria of triple negative breast cancer(TNBC)based on histomorphol-ogy and immunohistochemistry(IHC),and to provide theoreti-cal basis for the classification and treatment of TNBCs.Methods TNBC subtyping was performed according to the histomorphologi-cal characteristics and the expression of immune markers AR,CD8 and FOXC1,and the clinicopathological features and prog-nostic differences were compared.Results Among 93 cases of TNBC,there were 23 cases(24.7％)of luminal androgen re-ceptor subtypes,24 cases(25.8％)of immunomodulatory type,39 cases(42.0％)of basal immunosuppressive type,and 7 ca-ses(7.5％)of mesenchymal type.There were significant differ-ences in the clinicopathological features of subtypes,including pT stage(P=0.030),histological grade(P＜0.001),intersti-tial lymphocyte infiltration pattern(P＜0.001),expression of PD-L1(P＜0.001),and HER2-low(P=0.024).There was no significant difference in disease-free survival among the sub-types(P＞0.05).Univariate survival analysis showed there was significant difference in disease-free survival among the subtypes at pT1 stage(P=0.011),and other clinicopathological features were not independent prognostic factors.Conclusion The clini-copathological characteristics of TNBC subtypes are different,which are expected to be an alternative choice for complex gene expression profile analysis and to provide theoretical basis for subtypic therapy and targeted therapy.

Objective:To investigate the morphological and biological characteristics of polyploid tumor giant cells (PGCC) produced by ovarian cancer cell line SKOV3 induced by CoCl 2. Methods:Human ovarian cancer cell line SKOV3 was induced-cultured with 300 μmol/L CoCl 2 in the simulated hypoxic environment for 36 h, the live cells continued to be conventionally cultured and passaged, and the cells collected 20 days later were PGCC group; SKOV3 cell line cultured conventionally was the control group. The formation process and morphological characteristics of PGCC were observed by inverted microscope. The expression of tumor stem cell markers OCT4 and CD117 were detected by immunocytochemistry. The adipogenic differentiation and osteogenic differentiation potential of PGCC were detected by using human bone marrow mesenchymal stem cell adipogenic differentiation assay kit and human bone marrow mesenchymal stem cell osteogenic differentiation assay kit.The cell migration ability of PGCC was detected by scratch assay. PGCC group and control group SKOV3 cells were treated with 1 μmol/L paclitaxel, and the cell morphology of the two groups was observed by microscope at 0, 24 and 48 h to detect the resistance of PGCC to chemotherapy drugs. Results:A small amount of PGCC was observed in SKOV3 cell line cultured in conventional medium under the microscope. CoCl 2 can induce SKOV3 cells to form PGCC, which was nearly round in shape and lacked branching. Its volume was 3 times or more than that of SKOV3 cells, and the nuclei were usually megakaryons or multinucleates, PGCC can produce daughter cells by budding. Immunocytochemical staining showed that OCT4 was positive in some PGCC, but no CD117 was positive. Neither OCT4 nor CD117 was expressed in SKOV3 cells. When cultured with lipid-induced differentiation medium of human bone marrow mesenchymal stem cells, the formation of large vacuoles in the cytoplasm of PGCC was observed at the 3rd cycle, and orange-red, round-like lipid droplets were shown by oil red O staining. Human bone marrow mesenchymal stem cells were cultured in osteogenic induction culture medium for 20 days, and alizarin red staining showed that calcium nodules formed significantly in cells of PGCC group compared with the control group. The cell scratch assay results showed that the migration rates of PGCC cultured in serum-free medium [(59±1)%, (66±3)%] were higher than those of the control group [(11±3)%, (14±5)%] at 24 and 48 h after scratch ( t values were 32.20 and 19.55, both P < 0.001). The migration rates of PGCC cultured in 10% serum medium [(92±3)%, (100±0)%] were higher than those of the control group [(20±6)%, (59±9)%] ( t values were 16.19 and 8.00, both P < 0.001). After 1 μmol/L paclitaxel treatment for 48 h, most of the cells in the PGCC group still survived, while most of the SKOV3 cells in the control group died. Conclusions:PGCC produces daughter cells by budding. PGCC has the characteristics of tumor stem cells: it expresses tumor stem cell markers and has the potential for multidirectional differentiation and strong resistance to chemotherapy drugs.

Objective:To investigate the biological characteristics of triple negative breast cancer (TNBC) with low expression of HER2 (HER2-low).Methods:A total of 93 TNBC cases in Shanxi Cancer Hospital from 2017 to 2019 were collected and divided into HER2-negative and HER2-low groups according to HER2 expression status. The clinicopathological features and prognostic differences between the two groups were retrospectively analyzed and compared, and genetic detection of tumor tissues was performed to clarify somatic mutation status and differences between the two groups.Results:Ninety-three patients aged 26 to 86 years were enrolled, including 60 patients in the HER2-negative group and 33 patients in the HER2-low group. The distribution of HER2-low in luminal androgen receptor (LAR) subtype (14/23, 60.87%) and non-LAR subtype (19/70, 27.14%) was significantly different ( P=0.005). There were no significant differences in age, pT stage, histological grade, infiltration mode, lymph node metastasis and survival analysis. The expression of HER2-low in the tumor was heterogeneous, including different proportions of weak, weak to moderate intensity, and incomplete to intact membrane staining. With the change of the proportion of HER2-positive cells, the different distribution of those cells in the total tumor cells was noted, including cluster, mosaic and scattered patterns. The concentration and quality of DNA extracted from 71 of the 93 samples met the requirements for making libraries, including 43 in the HER2-negative group and 28 in the HER2-low group. Genetic mutations were mainly missense mutations, single nucleotide mutations, and point mutations in which base C was replaced by base T. There was no significant difference in genes with mutation frequency>3 times between the two groups. CTNNB1 and FGFR3 genes were only mutated in HER2-low group; while ALK, CYP2D6 and FAT1 genes were only mutated in HER2-negative group. HER2-low group included 18 HER2 1+ cases and 10 HER2 2+ cases. Genes with mutation frequency>3 times between the two groups included PIK3CA, TP53, SLX4, ATM and BRCA1. The mutation frequency of PIK3CA in HER2 2+ was significantly higher than that in HER2 1+ group ( P<0.05), and SLX4 gene was only mutated in HER2 1+ group. Conclusions:There are some differences of histological morphology and genetic variation between HER2-negative group and HER2-low group, and also differences in genetic variation between HER2 1+ and HER2 2+ in HER2-low group, which are helpful for more accurate stratification of TNBC and useful for finding the therapeutic target and precise treatment of HER2-low TNBC.

Objective:To explore the relationship between integrin ɑ3(ITGA3)expression and immune cell infiltration in colorectal cancer(CRC).Methods:Bioinformatic methods were used to analyze ITGA3 mRNA expression in pan-cancer and CRC tissues,as well as its associ-ation with CRC prognosis.The correlation between ITGA3 and tumor-infiltrating immune cells was also investigated.In total,233 cases of CRC diagnosed at Shanxi Provincial Cancer Hospital between January and December 2021 were included,and ITGA3,CD8,CD163,FOXP3,PD-L1,CTLA-4,and PD-1 expression in CRC tissues were determined by immunohistochemistry(IHC)to analyze the relationship between ITGA3 and infiltrating immune cells and immune checkpoints.Results:Bioinformatics analysis showed elevated ITGA3 mRNA levels in CRC.High ITGA3 expression was associated with PFS(P＜0.05).Univariate and multifactorial analyses showed that age and stage were significantly cor-related with prognosis(P＜0.05).In addition,ITGA3 upregulation was closely correlated with multiple immune cell infiltration levels in CRC.Furthermore,IHC results showed that ITGA3 expression in CRC tissues was significantly higher than that in adjacent normal tissues(P＜0.05).ITGA3 expression was associated with lymph node metastasis(P＜0.05)and correlated with the expression of immune markers,such as CD8+T-cells,PD-L1,and CTLA-4(P＜0.05).Conclusions:ITGA3 is highly expressed in CRC,which is closely related to immune cell infiltration and may regulate the tumor immune microenvironment,which provides a new idea for clinical treatment and a potential new independent predictive marker.

Objective:To investigate the correlation between KRAS, NRAS and BRAF V600E gene mutations and the clinicopathological characteristics of patients with colorectal cancer.Methods:Specimens from 217 patients with colorectal cancer who underwent surgical resection and were pathologically confirmed in Shanxi Province Cancer Hospital from January 2020 to December 2021 were selected, and the clinical data of the patients were retrospectively analyzed. The mutation status of KRAS, NRAS and BRAF V600E genes were detected in the paraffin specimens of surgically-resected tissues by direct sequencing. The mutation rates of KRAS, NRAS and BRAF V600E were compared among patients with different clinicopathological characteristics.Results:The mutation rates of KRAS, NRAS and BRAF V600E in 217 patients with colorectal cancer were 48.4% (105/217), 4.1% (9/217) and 3.7% (8/217), of which 1 patient (0.5%) had both KRAS and NRAS mutations. NRAS gene mutation was not correlated with gender, age, tumor size, tumor location, pathological type, degree of differentiation, depth of invasion, lymph node metastasis, distant metastasis, TNM stage, hemangioma thrombus/nerve invasion (all P>0.05); KRAS mutation rate in patients ≥ 60 year old was higher than that in patients < 60 year old [55.3% (63/114) vs. 40.8% (42/103), χ2 = 4.55, P = 0.033),and there was no correlation between KRAS gene mutation and other clinicopathological features (all P > 0.05); the mutation rate of BRAF V600E gene in colorectal cancerpatients with distant metastasis was higher than that in patients without distant metastasis [16.7% (4/24) vs. 2.1% (4/193), P = 0.006], and there was no correlation between BRAF V600E gene mutation and other clinicopathological features (all P > 0.05). Conclusions:Older colorectal cancer patients may be prone to KRAS gene mutation, and the BRAF V600E gene mutation rate is higher in patients with distant metastasis, and there is no correlation between NRAS gene mutation and clinicopathological characteristics.

Objective:To analyze the correlation between 21-gene recurrence score (RS) and clinicopathological characteristics of patients with Lumina type breast cancer, and to explore its significance in individualized treatment.Methods:The clinicopathological data of 59 patients with surgical resection and pathological diagnosis of Lumina type breast cancer in Shanxi Provincial Cancer Hospital from May 2018 to May 2019 were retrospectively analyzed. Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of 21 gene and RS was calculated. According to the 21-gene RS, the patients were divided into low recurrence risk group (RS < 18 points), intermediate recurrence risk group (RS 18-31 points) and high recurrence risk group (RS > 31 points). Univariate and multivariate logistic regression analyses were made to evaluate the correlations between different recurrence risk and clinicopathological characteristics of patients and their influence on the choice of adjuvant chemotherapy.Results:Based on the 21-gene RS, 29 patients were in low recurrence risk group, 22 cases were in intermediate recurrence risk group, and 8 cases were in high recurrence risk group. Single-factor analysis showed that age ( P = 0.012), maximum mass diameter ( P = 0.031), histological grade ( P = 0.036), progesterone receptor (PR) level ( P = 0.015), Ki-67 positive index ( P = 0.049) and molecular typing ( P = 0.010) were influencing factors of 21-gene RS recurrence risk. Multivariate logistic regression analysis showed that the age and Ki-67 positive index were negatively correlated with 21-gene RS recurrence risk (both P < 0.05). After grouping according to the 21-gene RS, 17 patients in the intermediate recurrence risk group (according to the traditional postoperative recurrence risk grouping method for breast cancer) were classified as low recurrence risk group, and 4 patients in the low recurrence risk group were classified as intermediate recurrence risk group ( χ2 = 4.535, P = 0.033). After grouping based on 21-gene RS, the number of patients who needed chemotherapy in individualized treatment decreased. Of the 17 cases, 11 cases did not undergo postoperative chemotherapy, and the remaining patients received chemotherapy. The postoperative follow-up period was 11-22 months. As of March 2020, there was no recurrence or disease progress. Conclusion:The 21-gene RS can provide objective basis for the individualized precise treatment and prognosis prediction for patients with early-stage Lumina type breast cancer.

Objective:To study the expression of GATA3 and bcl-11b in peripheral T-cell lymphoma (PTCL) and their correlation with clinicopathological features.Methods:The Oncomine and GEO databases were used for analyzing the expression levels of GATA3 and bcl-11b mRNA in PTCL. Immunohistochemistry was used to detect the expression of GATA3 and bcl-11b proteins in 127 cases of PTCL diagnosed at Shanxi Provincial Cancer Hospital from January 2010 to June 2020, as well as 40 cases of lymph node with reactive hyperplasia.Results:The data in Oncomine and GEO databases showed that the expression of GATA3 and bcl-11b mRNA in PTCL was lower than that in normal tissues ( P<0.05). Immunohistochemistry showed that the positive rates of GATA3 in PTCL and lymph nodes with reactive hyperplasia were 60.6% (77/127) and 85.0% (34/40, P<0.05), respectively. The expression rates of bcl-11b in PTCL and lymph nodes with reactive hyperplasia were 55.1% (70/127) and 75.0% (30/40, P<0.05), respectively. The expression of GATA3 was related to the pathological classification of the patients with PTCL, and was inversely related to the Ann Arbor stage of the patient, while the expression of bcl-11b was inversely correlated with the IPI score of the patient ( P<0.05). The expression of GATA3 and bcl-11b was related to the patients′ age, gender, LDH level, and B symptoms. Other clinicopathological characteristics were irrelevant. Spearman correlation analysis shows that the expression of GATA3 protein was associated with that of bcl-11b protein in PTCL. Conclusion:GATA3 and bcl-11b are closely related to the prognosis of PTCL, and may be important factors involved in the occurrence and development of PTCL.

Objective:To analyze the value of droplet digital polymerase chain reaction (ddPCR) in detecting epidermal growth factor receptor (EGFR) mutations in peripheral blood circulating tumor DNA (ctDNA) of patients with non-small cell lung cancer (NSCLC).Methods:Peripheral blood samples of 63 patients with NSCLC who were treated in Shanxi Provincial Cancer Hospital from August 2018 to March 2019 were collected, and EGFR sensitive mutations in peripheral blood of patients were detected by ddPCR, and the results were compared with the results of amplification refractory mutation system polymerase chain reaction (ARMS-PCR). Kappa test was used to analyze the consistency of the two methods.Results:The EGFR sensitive mutations were found in 31 cases (49.2%) by ddPCR in peripheral blood of 63 patients with NSCLC. Among them, 1 case (1.6%) had G719X, 12 cases (19.0%) had E19-Del, 11 cases had T790M (17.5%), 7 cases (11.1%) had L858R. Seven cases (22.6%) of the patients had double mutations. In comparison, the above 4 mutations were found in 26 cases (41.3%) by the ARMS-PCR method, with 0 case, 12 cases (19.0%), 6 cases (9.5%), and 8 cases (12.7%), respectively, including 5 cases (19.2%) with double mutations. L858R in one case was positive when detected by ARMS-PCR, while it was negative when detected by ddPCR. The consistency rate of the two methods was 90.3% (κ = 0.8, P < 0.05). The median abundance of EGFR mutations in peripheral blood ctDNA of 31 cases was 1.7% (range 0.04%-23.60%). The median abundance of E19-Del was 2.50% (0.35%-22.70%), that of T790M was 0.6% (0.04%-14.00%), and that of L858R was 2.3% (0.20%-23.60%). Ten cases with the abundance of EGFR mutations < 1% when detected by ddPCR, accounting for 32.6% (10/31) of total patients with mutations, but only 5 cases of them were detected by ARMS-PCR. The detection rate of T790M by ddPCR in patients who had received tyrosine kinase inhibitor (TKI) and had acquired drug resistance was 57.9% (11/19), while it was 0 in patients without TKI treatment. Among patients with T790M mutation, 1 case had a mutation abundance < 0.1%, 7 cases had a mutation abundance of 0.1%-2.0%, 3 cases had a mutation abundance > 2.0%.Conclusions:The ddPCR provides a non-invasive, highly sensitive and absolutely quantitative method for detecting EGFR mutations in peripheral blood ctDNA of NSCLC patients. It provides a new detection method for EGFR-TKI targeted therapy in NSCLC patients with difficult sampling or with acquired drug resistance who need to repeatedly sample. The approach provides an important basis for the individualized targeted therapy.

Objective:To investigate the expression of CDK6 and FOXM1 in peripheral T-cell lymphoma (PTCL), and its correlation with clinicopathologic features and patient prognosis.Methods:The Oncomine was used for data mining and analyzing the expression levels of CDK6 and FOXM1 in PTCL. Immunohistochemistry (IHC) of EnVision method was used to detect the expression of CDK6 and FOXM1 proteins in 166 cases of PTCL diagnosed at Shanxi Provincial Cancer Hospital from January 2016 to December 2018, and 30 cases of lymph node with reactive hyperplasia as control.Results:Among the PTCL patients, 104 were male and 62 were female, aged from 3 to 85 years, with an average age of 53 years. Analyses of the Oncomine 4.5 database showed that mRNA expression of CDK6 and FOXM1 in PTCL tissues was significantly higher than that in normal tissue ( P<0.05). IHC staining showed the positive rates of CDK6 and FOXM1 proteins in PTCL tissues were 27.7% (46/166) and 80.7% (134/166), respectively. The expression was mainly present in the nuclei of tumor cells, showing a diffuse, strongly positive pattern. The positive rates of CDK6 and FOXM1 proteins among the 30 cases of lymph-node reactive hyperplasia were 0 (0/30) and 30% (9/30), respectively. The expression of FOXM1 was mainly found in the lymphoid follicle germinal center, and not present in the T-zone cells. CDK6 protein, FOXM1 protein and the co-expression of CDK6 and FOXM1 proteins in PTCL were associated with higher Ann Arbor staging and international prognostication index score ( P<0.05), and inversely associated with overall survival ( P<0.05). Meanwhile, CDK6 protein expression was positively correlated with FOXM1 protein expression ( P<0.05). Conclusions:CDK6 and FOXM1 may be new targets for the diagnosis and treatment of PTCL. The overexpression of CDK6 may lead to enhanced function of the transcription factor FOXM1, while the overexpression of CDK6 and FOXM1 also promotes the development and progression of PTCL.

Objective: To understand the consistency of ALK Ventana-D5F3 immunohistochemistry (IHC) interpretation in Chinese lung adenocarcinoma among histopathologists from different hospitals, and to recommend solution for the problems found during the interpretation of ALK IHC in real world, with the aim of the precise selection of patients who can benefit from ALK targeted therapy. Methods: This was a multicenter and retrospective study. A total of 109 lung adenocarcinoma cases with ALK Ventana-D5F3 IHC staining were collected from 31 lung cancer centers in RATICAL research group from January to June in 2018. All cases were scanned into digital imaging with Ventana iSCANcoreo Digital Slide Scanning System and scored by 31 histopathologists from different centers according to ALK binary (positive or negative) interpretation based on its manufacturer′s protocol. The cases with high inconsistency rate were further analyzed using FISH/RT-PCR/NGS. Results: There were 49 ALK positive cases and 60 ALK negative cases, confirmed by re-evaluation by the specialist panel. Two cases (No. 2302 and No.2701) scored as positive by local hospitals were rescored as negative, and were confirmed to be negative by RT-PCR/FISH/NGS. The false interpretation rate of these two cases was 58.1% (18/31) and 48.4% (15/31), respectively. Six out of 31 (19.4%) pathologists got 100% accuracy. The minimum consistency between every two pathologists was 75.8%.At least one pathologist gave negative judgement (false negative) or positive judgement (false positive) in the 49 positive or 60 negative cases, accounted for 26.5% (13/49), 41.7% (25/60), respectively, with at least one uncertainty interpretation accounted for 31.2% (34/109). Conclusion There are certain heterogeneities and misclassifications in the real world interpretation of ALK-D5F3 IHC test, which need to be guided by the oncoming expert consensus based on the real world data.