B cells and tertiary lymphoid structures are associated with survival in papillary thyroid cancer.

PURPOSE: The function of B cells in papillary thyroid cancer (PTC) is controversial. The role of B-cell-related tertiary lymphoid structures (TLSs) is still unclear. Whether B cells exert their anti-tumor effect through forming TLS in PTC needs further investigation. METHODS: We detected the percentage of B cells in PTC tissues by multi-parameter flow cytometry. Paraffin-embedded tumor tissues of 125 PTC patients were collected and stained with Haematoxylin-Eosin (H&E) for inflammatory infiltration analysis in combination with clinical features. Multiplexed immunohistochemistry (mIHC) was performed to verify the TLSs in above inflammatory infiltration. Correlation of B cells and TLSs with prognosis was analyzed using the TCGA database. RESULTS: We observed that PTC patients with higher expression of B lineage cell genes had improved survival and the percentage of B cells in the PTC tumor tissues was variable. Moreover, PTC tumor tissues with more B cells were surrounded by immune cell aggregates of varying sizes. We furtherly confirmed the immune cell aggregates as TLSs with different maturation stages. By analyzing PTC data from TCGA database, we found the maturation stages of TLSs were associated with genders and clinical stages among PTC patients. Moreover, patients with high TLSs survived longer and had a better prognosis. CONCLUSION: B cells are associated with the existence of TLSs which have different maturation stages in PTC. Both B cells and TLSs are associated with the survival rate of PTC. These observations indicate that the anti-tumor effects of B cells in PTC are associated with TLSs formation.

[The screening of potential biological markers of seizure onset zone in focal cortical dysplasia based on bioinformatics analysis].

Three patients diagnosed with focal cortical dysplasia (FCD) in the First Hospital of Peking University from September to November 2020 were recruited in the study. Based on stereotactic electroencephalogram (SEEG) or electrocorticogram (ECoG) analysis to localize the seizure onset zone (SOZ), RNA sequencing (RNA-seq) analysis was performed for the SOZ and para-SOZ tissue obtained from surgery. The differentially expressed genes between SOZ and para-SOZ samples were analyzed by performing Go (Gene ontology) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis."Extracellular matrix"was significantly enriched, which included collagen synthesis genes (e.g., COL1A1)."Ether lipid metabolism"was enriched in the KEGG pathway enrichment analysis. These differences could be the potential biological markers for SOZ localization.

[mTOR signaling pathway-mediated autophagy involved in inhibition of osteoblast differentiation induced by cadmium in human bone marrow mesenchymal stem cells].

Objective: To investigate the role of autophagy mediated by mTOR signaling pathway in the inhibition of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) induced by cadmium. Methods: HBMSCs were divided into 0, 2.5 or 5.0 µmol/L groups according to the exposure dose of cadmium chloride (CdCl2), and each group was treated for 1 day, 4 days and (or) 7 days. The ALP activity and mRNA and protein expression levels of osteogenesis markers (ALP, RUNX2 and OSTERIX), autophagy-related proteins (LC3 and Beclin-1) and mTOR signaling pathway related proteins (mTOR, p-mTOR and p-p70S6K) expression, alkaline phosphatase staining and alizarin red staining were detected. MHY 1485 was selected as the signaling pathway activator. The control group, CdCl2 group (5.0 µmol/L), MHY 1485 group and CdCl2+MHY 1485 combined treatment group were set. After 7 days of treatment, the expression levels of autophagy related proteins and mTOR signaling pathway related proteins of hBMSCs in each group were detected. Results: There was no significant difference in ALP activity between 0, 2.5 and 5.0 µmol/L groups on day 1 and 4 (P>0.05); On day 7, compared with the 0 µmol/L group, the ALP activity, expression of osteogenic markers (ALP, RUNX2, OSTERIX) and mTOR signaling pathway related proteins (mTOR, p-mTOR, p-p70S6K) expression decreased in the 2.5 and 5.0 µmol/L group (P<0.05). Compared with the 0 µmol/L group, the staining of the 2.5 and 5.0 µmol/L groups became lighter, and the formation of ALP and mineralized nodules was reduced. Compared with the CdCl2 group, the autophagy related protein expression in the CdCl2+MHY 1485 combined treatment group decreased, and the mTOR signaling pathway related protein expression increased. The difference was statistically significant (P<0.05). Conclusion: The inhibition of osteogenic differentiation of hBMSCs by cadmium may be related to autophagy mediated by mTOR signaling pathway.

[Predictive factors of pathological complete response after neoadjuvant chemoradiotherapy for middle-low rectal cancer].

Objective: To explore the predictive factors of pathological complete response (pCR) after neoadjuvant chemoradiotherapy for middle-low rectal cancer. Methods: A case-control study was conducted. The inclusion criteria were as follows: (1) colonoscopy, digital examination or magnetic resonance imaging (MRI) showed a distance from the lower edge of the tumor to the dentate line of no more than 10 cm; (2) complete clinicopathological data were available; (3) preoperative biopsy revealed adenocarcinoma; (4) preoperative pelvic MRI or endorectal ultrasonography was performed; (5) no distant metastasis was found. Exclusion criteria: (1) preoperative radiotherapy and chemotherapy were not administrated according to the standard; (2) simultaneous multiple primary cancer and familial adenomatous polyposis were observed. According to the above criteria, clinicopathological data of 245 patients with middle-low rectal cancer undergoing preoperative neoadjuvant chemoradiotherapy in Changhai Hospital of Navy Medical University from January 2012 to December 2019 were retrospectively collected. Univariate analysis and multivariate logistic analysis were used to identify the clinical factors predicting pCR. pCR is defined as complete disappearance of cancer cells under the microscope in cancer specimens (including lymph nodes) after neoadjuvant chemoradiotherapy. Results: A total of 72 patients with pCR were enrolled in this study. Univariate analysis showed that preoperative T stage, tumor circumference, tumor morphology, carbohydrate antigen (CA) 19-9, interval between the end of neoadjuvant therapy and operation were associated with pCR (all P<0.05). The above 5 variables were included in multivariate logistic analysis and the results revealed that the T stage (OR=5.743, 95% CI: 2.416-13.648, P<0.001), tumor circumference (OR=7.754, 95% CI: 3.822-15.733, P<0.001), tumor morphology (OR=0.264, 95% CI: 0.089-0.786, P=0.017) and the interval between the end of neoadjuvant therapy and operation (OR=0.303, 95% CI: 0.147-0.625, P=0.001) were independent predictive factors of pCR, while CA 19-9 level was not an independent factor (OR=1.873, 95% CI:0.372-9.436, P=0.447). Conclusion: By knowing the clinical features of preoperative T stage, tumor circumference, tumor morphology and the interval between neoadjuvant chemoradiotherapy and operation, patients with higher likelyhood of pCR after neoadjuvant chemoradiotherapy may be identified.

[Prognostic significance of early phase donor chimerism after allogeneic peripheral blood stem cell transplantation].

Objective: To evaluate the prognostic significance of early phase full donor chimerism (FDC) after myeloablative allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Methods: The clinical data of 72 hematological patients received myeloablative allo-PBSCT from Feb. 2016 to Jul. 2017 were analyzed retrospectively. The median age was 36.5 years (range 4-59), 44 were males and 28 females. Of the donors, there were 35 HLA matched sibling donors, 27 haploidentical donors and 10 unrelated donors. Polymerase chain reaction amplification of short tandem repeat sequence (PCR-STR) was used to detect donor cell chimerism (DC) rate of recipient bone marrow at one, two and three months after transplantation. Results: The median follow-up was 462 d (range: 47-805 d), 55 cases were still alive, and 45 cases were disease-free survival (DFS) at the end of follow-up. The 2-year overall survival (OS) and DFS were (68.9±7.7)% and (59.5±6.3)%, respectively. A number of 16 cases underwent relapses, with 2-year cumulative incidence of (24.1±5.3)%. The median time of recurrence was 157(32-374) d. Forty cases (55.6%) developed acute graft-versus-host diseases (aGVHD), with median time of 35.5 (13-90) d. Chronic GVHD (cGVHD) occurred in 23 patients (31.9%), with median time of 169 (94-475) d. Univariate analysis found the following factors were not related to OS, DFS or relapse rate (RR), including age, sex, blood type and sex of donor-recipient, occurrence of aGVHD and cGVHD. The OS and DFS in cases reached FDC and no FDC at two months after transplantation were (85.2±6.9)% vs (66.1±7.7)% (P=0.051) and (76.7±7.7)% vs (48.9±8.1)% (P=0.021), respectively. The RR rate in FDC group was lower than that in no FDC group [(16.6±6.8)% vs (30.4±7.8)%, P=0.187, respectively]. Conclusion: The present study confirmed the important value for predicting the prognosis with whether or not the patients reached FDC at the early phase after allo-PBSCT. The OS and DFS in cases with FDC at two months after transplantation were significantly higher than those of no FDC patients.

[Hypoxia-inducible factor-1α is involved in arsenite-induced epithelial-mesenchymal transition and malignant transformation of human liver epithelial cells via regulating Snail].

Objective: To investigate the role of hypoxia-inducible factor-1α (HIF-1α) in arsenite-induced epithelial-mesenchymal transition (EMT) and malignant transformation of human liver epithelial cells (L-02 cells). Methods: After the L-02 cells were chronic treated with 2.0 µmol/L NaAsO(2) for 0 (reference), 10, 20, or 30 passages, con siRNA or HIF-1α siRNA was transfected into arsenite-transformed L-02 (T-L-02) cells by lipofectamine(TM)2000 and were set as T-L-02+con siRNA group and T-L-02+HIF-1α siRNA group as well as L-02 group and T-L-02 group, EMT index and levels of HIF-1α were detected by western blots. The reporter assays were performed to determine if HIF-1α directly regulate Snail transcriptional activity, and soft agar colony formation and Transwell assay were used to detect the malignancy, invasion, and migration ability of cells. Results: When L-02 cells were treated for 10 generations with 2 µmol/L NaAsO(2), relative expressions of E-cadherin were gradually increased compared to control cells, while the levels of N-cadherin, Snail, and HIF-1α were gradually increased in the L-02 cells compared to control cells, showing the longer the treatment time was, the more obvious the change was (P<0.05) . Down regulating the level of HIF-1α by siNRA caused E-cadherin levels to rise compared to T-L-02 group, while the levels of N-cadherin and Snail fall back compared to T-L-02 group (P<0.05) . Double luciferase reporter gene assays showed that HIF-1α directly targeted Snail to regulate its expression. Soft agar colony formation and Transwell assays showed that the numbers of formed colonies, invasion cells, and metastasis cells of cells in T-L-02 group were all lower than those in L-02 group (P<0.05) . Conclusion: HIF-1α is involved in arsenite-induced EMT and malignant transformation of human liver epithelial cells via regulating Snail.

Expressions and effects of G250, Bax and Bcl-2 in rats with renal clear cell carcinoma.

OBJECTIVE: To investigate the expressions and effects of G250, B-cell lymphoma-2 associated X protein (Bax) and Bcl-2 in rats with renal clear cell carcinoma (RCCC). MATERIALS AND METHODS: A total of 66 male Sprague-Dawley (SD) rats were selected, among which 56 were selected to establish RCCC rat model and the remaining 10 were selected as control group. Three weeks after modeling, 4 rats failed in the modeling. Expressions of G250 in RCCC rat model group and healthy rat model control group were detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR); expressions of Bcl-2 and Bax in each group were detected by Western blot and their effects were analyzed. RESULTS: The positive expression rates of G250 in 52 RCCC rats in model group and 10 healthy rats in control group were 83.3% and 0%, respectively. The results showed that expression of G250 had a certain correlation with the pathological changes of RCCC (p < 0.01). Expressions of Bax and Bcl-2 were up-regulated in the RCCC group, while expressions were down-regulated in the healthy control group (p < 0.05). CONCLUSIONS: G250, as a new specific marker of renal cell carcinoma, is involved in the pathological changes of renal cell carcinoma. Joint detection of Bax and Bcl-2 can be used as an important index for the diagnosis of RCCC.

Radiation pneumonitis in non-small-cell lung cancer patients treated with helical tomotherapy.

OBJECTIVE: In this study, we investigated the incidence of radiation pneumonitis (RP) in non-small-cell lung cancer (NSCLC) patients undergoing helical tomotherapy (HT) and the clinical and dosimetric factors associated with it. MATERIALS AND METHODS: We analyzed data from the treatment protocols of 62 NSCLC patients. The median total radiation dose was 64 Gy (range 57.6-66 Gy) at 1.8-2.2 Gy/fraction. Thirty-four of these patients underwent HT alone and 28 underwent HT in combination with chemotherapy. Treatment-related pneumonitis was graded according to the Common Terminology Criteria for Adverse Events, version 3.0. RESULTS: We found that RP grades 1, 2, 3 and 5 occurred in 29 (46.8%), 23 (37.1%), 8 (12.9%), and 2 (3.2%) patients, respectively. Using univariate analyses, we found that a grade ≥3 RP was associated with poor performance status (PS), age, planning target volume, mean lung dose, and relative V5through V25, in increments of 5 Gy (P < 0.005). We determined that PS and V5V15were the most significant factors associated with grade ≥3 RP using multivariate analysis. CONCLUSIONS: We found that poor PS and V5-V15 were the risk factors associated with grade ≥3 RP in NSCLC patients treated with HT. Thus, for NSCLC patients treated with HT, the volume of total lung with low-dose region (V5-V15) should be carefully regulated and the use of HT should be restricted in patients with Eastern Cooperative Oncology Group ≥2.

Secreted protein acidic and rich in cysteine expression in human colorectal cancer predicts postoperative prognosis.

OBJECTIVE: Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix glycoprotein involved in cell proliferation, migration and angiogenesis. The aim of this study was to assess its expression in colorectal cancer, see whether and how it correlates with clinicopathological features, and evaluate its potential prognostic significance. PATIENTS AND METHODS: SPARC expression was detected by microarrays containing 847 immunohistochemically stained specimens, and further correlated with the clinicopathological and prognostic data. The prognostic significance of its expression was assessed using Kaplan-Meier survival with log-rank tests. Multivariate regression utilizing Cox's proportional hazard model was used to evaluate prognostic factors. RESULTS: SPARC expression in the normal colorectal mucosa and colorectal cancer tissue was significantly different (p < 0.001). Low SPARC expression was found to be associated with poor prognosis, and it was unfavorably correlated with overall survival and disease-free survival in colorectal cancer patients. In addition, SPARC expression in surrounding mesenchymal and stromal cells, bowel wall invasion, lymph node metastasis, and distant metastasis were independent prognostic factors for overall survival and disease-free survival. CONCLUSIONS: Reduced expression of SPARC in colorectal cancer tissue is associated with poor prognosis and aggressive clinicopathological features. Therefore, SPARC expression could potentially be used as a prognostic predictor for colorectal cancer patients.

Rapid genomic DNA changes in allotetraploid fish hybrids.

Rapid genomic change has been demonstrated in several allopolyploid plant systems; however, few studies focused on animals. We addressed this issue using an allotetraploid lineage (4nAT) of freshwater fish originally derived from the interspecific hybridization of red crucian carp (Carassius auratus red var., ♀, 2n=100) × common carp (Cyprinus carpio L., ♂, 2n=100). We constructed a bacterial artificial chromosome (BAC) library from allotetraploid hybrids in the 20th generation (F20) and sequenced 14 BAC clones representing a total of 592.126 kb, identified 11 functional genes and estimated the guanine-cytosine content (37.10%) and the proportion of repetitive elements (17.46%). The analysis of intron evolution using nine orthologous genes across a number of selected fish species detected a gain of 39 introns and a loss of 30 introns in the 4nAT lineage. A comparative study based on seven functional genes among 4nAT, diploid F1 hybrids (2nF1) (first generation of hybrids) and their original parents revealed that both hybrid types (2nF1 and 4nAT) not only inherited genomic DNA from their parents, but also demonstrated rapid genomic DNA changes (homoeologous recombination, parental DNA fragments loss and formation of novel genes). However, 4nAT presented more genomic variations compared with their parents than 2nF1. Interestingly, novel gene fragments were found for the iqca1 gene in both hybrid types. This study provided a preliminary genomic characterization of allotetraploid F20 hybrids and revealed evolutionary and functional genomic significance of allopolyploid animals.

[Studies on retinoids. IV. Design, synthesis and structure-activity relationships of di-t-butylphenyl compounds].

Retinoic acid and its analogues play important roles in modulating cell growth, differentiation, immunity and apoptosis. Clinically they are used for cancer chemoprevention and chemotherapy. Based upon the moiety of 3,5-di-t-butyl-4-hydroxy phenyl ring, a series of substituted aromatic amide, ester and chalcones were designed and synthesized, which mimic the molecular shape, size, and spacial disposition of functional groups of retinoic acid. The general structure is as follows: [formula: see text] where R stands for hydrogen atom or methyl group, Y is the linkage -CONH-, -NHCO-, -COO-, -COCH = CH-, or a member of a heterocycle, X represents various substituents at different positions. The SAR indicates that the presence of hydrophobic group(s) at one end of the molecule, and a carboxyl group at the other end, and a conjugative system of molecule are necessary and full prerequisite for exhibiting activity. Loss of any one factor of them will abolish the activity. Being obligatory for anti-oxidative effect, the phenolic hydroxy group does not convey biological activity, because after methylation of the hydroxy group the compound increases the differentiation-inducing activity and loses the anti-oxidative effect, indicating that there is no correlation between the two activities. With a stable conformation of two phenyl rings with cis-conformation N-methylated acyl amide (No. 30) features in bent shape of the molecule, instead of an extended conformer, which is taken by the non-N-methylated partner and all-trans retinoic acid. A bent conformer of No. 30 accounts for the inactivity. In this paper compounds No. 4f, 4g, 5a, 7, 13, 32, 37, and 38 exhibited significant activity among them 4-[3-(3, 5-di-t-4-methoxyphenyl)-3-oxo-1-propenyl] benzoic acid (No. 38) showed high activity comparable to that of retinoic acid. The pharmacological action of No. 38 is under investigation.

The origin of isochromosomes and their significance in tumorigenesis of human lung cancer.

Isochromosomes related to chromosomes 1, 5, 6, 8, and 9 were frequently observed in 23 cases of lung cancer. Their existence in tumor cells might be nonrandom. Premature centromere separation (PCS) was found in the PGT-131 cell line of a lung cancer. It is suggested that PCS may play a role in the formation of isochromosomes in lung cancer.

Direct chromosome analysis of 50 primary breast carcinomas.

Direct chromosome analyses were performed in 50 cases of primary breast carcinoma. Thirty-six cases had modal chromosome counts in the diploid range; the other 14 cases were polyploid. Of the 22 cases with detailed G-banding analyses, the most frequent structural changes involved chromosome 1 (15 of 22), 3 (13 of 22), and 6 (13 of 22). Deletion of chromosome 1p was noted in nine cases, and both 3p- and 6q- were noted in 10 cases.

Cytogenetic abnormalities in a squamous cell carcinoma of the penis.

Cytogenetic findings in a squamous cell carcinoma (SCC) of the penis in a 60-year-old patient was observed for the first time. The stemline karyotype of the tumor was 46,XY,del(2) (q33q36),der(4)t(4;?)(p16;?),der(5;15)(q10;q10),der(8)t(8;?13)(p21 ;?),-13,- 13,-15,+3mar.

[Establishing orthotopic transplanted models of human pancreatic cancer in nude mice and study on their biological properties].

Two cell lines of human pancreatic cancer have been established, which can be successfully transplanted into pancreas of nude mice, the first of this kind of cell lines in China. Fresh specimens human of pancreatic cancer taken surgically were transplanted in the pancreas of pure line BALB/C-nu/nu nude mice. The transplanted tumours grew and reproduced successfully, and were named PINMP-1 and PINMP-2, respectively. So far, 9 generations of PINMP-1 and 6 generations of PINMP-2 were obtained. Their biological properties, ways of invasion and metastasis and morphological characteristics under light and electron microscope were studied. The results showed a 95%-100% transplanting success rate, with the success rate of transplanting from tissues revivified from the liquid nitrogen preservation being 100%. Both of the lines could produce large amount of CEA, and chromosome analysis confirmed that they had retained a karyotype of the human cancer cells. In nude mice transplanted with the tumours, metastasis could be found in the lymph nodes, lungs and livers. Metastasis via lymphatic channels and blood vessels were also demonstrated. The pathological and ultrastructural examination confirmed that the transplanted tumours had identical characteristics as their donor tumours. The transplanted cells grew independently in the pancreas of the nude mice, making a better model for study on tumour invasion and metastasis than subcutaneously transplanted tumours. This indicated that the microenvironment in the transplantation site had certain influence on the biological behavior of the transplanted tumours. The models could be used in the study on the invasion, metastasis and experimental therapy of pancreatic carcinomas.

Direct chromosome analysis of seven primary colorectal carcinomas.

Chromosome studies were performed on direct preparations of seven cases of primary colorectal carcinomas. Two cases had relatively simple chromosome changes: 48,XY,+8,+21/51, XY,+8,+9,+10,+i(17q),+21, and 47,der(X)t(X;14)(q11;q11)-Y,t(6;18)(p22;q24)+7,+8,der(19)t (19;?)(q13;?). The five others had complicated deletions and translocations; 1p- was noted in five cases, and i(17q) was noted in three cases.

Cytogenetic studies of eight primary gastric cancers.

Cytogenetic analysis was performed on eight primary gastric cancers. Three of them had simple chromosome changes: 47,XX,+X/48,XX,+X,+X; 48,XX,+8,+19,t(3;5) (q21;q31) and 47,XY,+del(7)(q22). The five others had complicated chromosome changes; both 3p- and 7q- were noted in four cases and i(5p) was noted in two cases.

The three-dimensional structure of the aspartyl protease from the HIV-1 isolate BRU.

The crystal structure of the aspartyl protease encoded by the gene pol of the human immunodeficiency virus (HIV-1, isolate BRU) has been determined to 2.7 A resolution. The enzyme, expressed as an insoluble denatured polypeptide in inclusion bodies of Escherichia coli has been renatured and crystallized. It differs by several amino acid replacements from the homologous enzymes of other HIV-1 isolates. A superposition of the C alpha-backbone of the BRU protease with that of the SF2 protease gives a roots mean square positional difference of 0.45 A. Thus, neither the denaturation/renaturation process nor the amino acid replacements have a noticeable effect on the three-dimensional structure of the BRU protease or on the detailed conformation of the catalytic site, which is very similar to that of other aspartyl proteases.

Cytogenetic studies of five primary esophageal cancers.

In direct preparation of five primary esophageal cancers, chromosomes 1, 3, and 12 were most frequently involved in structural abnormalities; 12p- was consistently seen in all five cases and, thus, could be considered as one of the characteristic chromosome changes in esophageal cancer. Moreover, 2p-, 4p-, and 7q- were seen in three cases.