Changes of histological and biological features by serial passages in a human adenoid cystic carcinoma line transplantable in nude mice

The basic histologic patterns of adenoid cystic carcinoma (ACC) are classified into three types (tubular, cribriform and solid), but clinical significance of the histological type is unclear. We have successfully established a human tumor line derived from ACC that is serially transplantable in nude mice. This tumor showed an increased growth rate as the passage levels proceeded, and the histological type was changed from a cribriform pattern in the initial stage to a solid one. In this study, we investigated the relationship between histological type and biological characteristics by analyzing the serially transplantable ACC tumor model. As a result, the tumor growth rate at the 15th passage level was increased approximately 5-fold compared with that at the initial passage level. In the histological type, approximately 30% of the cribriform pattern in the initial level was changed to a solid one at the 15th passage level, and the PCNA labeling index was elevated 4-fold. Concomitant with this, expression of Ki-67, p53 and bcl-2 proteins was increased, and apoptotic cells were decreased as demonstrated by the TUNEL method. From these findings, it was suggested that cell proliferation and histological change of this ACC tumor are related to the inhibition of apoptosis. This tumor line would provide a useful model for investigating the biological behavior of ACC.     

The soft agar clonogenicity and characterization of cells obtained from human solid tumors by mechanical and enzymatic means

Summary A two-step procedure for releasing cells from solid tumors has been applied to specimens of human melanoma, sarcoma, lung, colon, and breast carcinoma. The first population released mechanically has been compared with the population subsequently released enzymatically in tests of dye exclusion, ribonucleoside triposphate pool sizes, intactness of DNA, and clonogenicity in soft agar. While greater numbers of dye-excluding cells are released in the enzymatic step, and these cells have higher ribonucleoside triphosphate pools and more intact DNA, both populations contain clonogenic cells in approximately equal numbers.Several semisolid media were employed in tests of clonogenicity, and all methods employing an agar underlayer appeared satisfactory and approximately equivalent in cloning efficiency. The methyl cellulose upper layer system facilitated implanting of pooled colonies into nude mice, which resulted in growth in the nude host and marked increase in cloning efficiency when the cells were replanted into soft agar-methyl cellulose plates.A comparison of four different areas of individual tumor specimens was made with cells released enzymatically and measuring cell yield, dye exclusion, ATP pool size, and uptake and metabolism of 5-fluoropyrimidines. Only relatively small variations were seen from one area to the next, with trypan blue exclusion exhibiting the least variation, and metabolism of fluorinated pyrimidines showing the most.     

Ultrasound findings and histological features of ductal carcinoma in situ detected by ultrasound examination alone

Background

With the increasing use of high-resolution ultrasound (US) examination, many breast carcinomas that cannot be identified by mammography (MMG) alone have been detected. Many of these carcinomas are ductal carcinoma in situ (DCIS) and small-sized invasive carcinomas. Until date, DCISs have often been described as palpable masses with calcifications on MMG, but what are the characteristics of DCISs that are detectable by US alone?

Methods

One hundred fifty cases with DCIS that we experienced at our clinic from 2003 to 2007 were classified into 47 cases (echo group) diagnosed by US alone and 103 cases (MMG/PE group) diagnosed by MMG or clinically.

Results

US findings of the echo group showed cystic or solid lesions in 37 cases (79%). The mean age of the echo group was significantly higher than that of the MMG/PE group (59.6 vs. 51.2 years; P < 0.01). Tumor sizes detected by US were 5.7 + 2.8 and 11.5 + 10.8 mm (P < 0.001), respectively. The tumor sizes of the echo group were, therefore, approximately half that of the MMG/PE group. Extensive intraductal components were significantly fewer in the echo group, and tumor grades of the echo group were significantly low (Van Nuys classification). In the echo group, all cases with a tumor size <5 mm were grade 1 by Van Nuys classification. In addition, cases with ≥5 mm tumor size had a significantly lower tumor grade in the echo group than in the MMG/PE group.

Conclusions

Cystic or solid lesions accounted for approximately 80% of US findings of DCISs detected by US alone, and most were similar to benign forms. Moreover, most DCISs detected by US alone were localized and of low grade (Van Nuys classification).

     

The biological features of PanIN initiated from oncogenic Kras mutation in genetically engineered mouse models

Pancreatic intraepithelial neoplasia (PanIN) is the most common premalignant lesion of the pancreas. Further understanding of the biological behavior and molecular genetic alterations in the stepwise progression of PanINs is necessary toward the development of pancreatic ductal adenocarcinoma (PDAC) interventions. In this study, we analyzed the morphological characteristics, molecular alterations, and biological behavior of pancreatic wild-type and neoplasia tissues, including analysis of PanIN cell line SH-PAN (isolated from Pdx-1-Cre; LSL-Kras^G12D/+ mouse) and PDAC cell line DT-PCa (isolated from Pdx1-Cre; LSL-Kras^G12D/+; LSL-Tp53^R172H/+ mouse). Results show that Kras^G12D induces ductal lesion PanINs. Increased expression of EGFR, Her-2/Neu, p-MAPK and β-Catenin was observed in low-grade PanINs. Tp53 was not expressed in wild-type and low-grade PanINs, however, increased expression was observed in high-grade PanINs. Furthermore, SH-PAN cells did not exhibit any colony formation and showed significantly lower migration and invasion ability compared with DT-PCa cells. Notably, we first found PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) expression was significantly higher in SH-PAN cells than DT-PCa cells, and was high in 96 of 172 peritumoral normal human pancreatic tissues and 20 of 36 human low- or middle-grade PanIN tissues, whereas, was weak or negligible in 12 of 20 human high-grade PanIN tissues and 124 of 172 human PDAC tissues post-operation. The expression of PPP2R2A appears to be correlated with clinical survival. Taken together, Kras^G12D – driven PanIN showed the tumorigenic ability, however, did not undergo a malignant transformation, and decreased expression of PPP2R2A in PDACs may provided a new target for pancreatic carcinoma intervention.     

Discrimination of metabolic profiles of pancreatic cancer from chronic pancreatitis by high-resolution magic angle spinning 1H nuclear magnetic resonance and principal components analysis

The metabolic profiles of Sprague-Dawley rat pancreases were investigated by high-resolution magic angle spinning proton magnetic resonance spectroscopy ((1)H NMR) combined with principal components analysis (PCA) to discriminate pancreatic cancer from chronic pancreatitis. Intact pancreatic tissue samples were obtained from Sprague-Dawley rats with histologically proven pancreatic cancer (n = 5), chronic pancreatitis (n = 5), and two matched controls (n = 5 per group). Two (1)H NMR experiments, single-pulse and Carr-Purcell-Meiboom-Gill, were carried out separately. Increases in phosphocholine and glycerophosphocholine levels and decreases in leucine, isoleucine, valine, lactate and alanine levels were observed in chronic pancreatitis, whereas the opposite trends were observed in pancreatic cancer. Increasing taurine and decreasing betaine were found both in chronic pancreatitis and in pancreatic cancer. Additionally, the lipid content in pancreatic cancer was higher than that in chronic pancreatitis. PCA was carried out for the single-pulse and Carr-Purcell-Meiboom-Gill (1)H NMR spectra, respectively, to visualize separation among the samples and to extract characteristic metabolites of pancreatic cancer and chronic pancreatitis. Decreased phosphocholine and glycerophosphocholine were suggested as unique metabolite indicators of pancreatic cancer. Furthermore, even with the disturbance of various quantities of lipid contents pancreatic cancer and chronic pancreatitis could be differentiated well by the combination of high-resolution magic angle spinning (1)H NMR and PCA. Thus this combination was demonstrated to have the potential to improve magnetic resonance spectroscopy for positive early diagnosis of pancreatic cancer in clinical settings.     

The prognostic implication of clinical and histological features in Ph1+ chronic myelocytic leukaemia (CML)

A study on clinical and histological features of prognostic significance was performed in 45 patients with Ph1+ -CML who showed median survival of 36 months. The histological features were evaluated by morphometry of iliac crest biopsies. Among the variables correlated with prognosis, we eliminated those without primary importance by mutual univariate retrospective stratification. Thus a clinical and a histological set of important prognostic criteria was established. Their influence on prognosis proved to be independent of each other and therefore could be used for separate classifications. Clinical classification yielded two groups with different prognosis: compared to the rest of the patients, the prognosis was much worse for those with a spleen size greater than 10 cm, a liver size greater than 2 cm (below costal margin) and greater than 5% circulating blasts plus promyelocytes. The histomorphological classification consisted of three subgroups: a better than average prognosis was found for patients with pseudo-Gaucher cells in the bone marrow, while in the remaining cases the prognosis was worse in patients with a high number of megakaryocytes (greater than 70/mm2) and a low volume ratio of granulopoiesis: megakaryocytes (less than 15). Since liver size was correlated with the duration of prediagnostic symptoms, the clinical classification probably reflects different disease stages, i.e. a later CML diagnosis. However, the histological set of prognostic factors is independent of the length of the prediagnostic period. Consequently, this morphological classification seems to discriminate different subgroups. Another important prognostic factor, marrow fibrosis, was independent of other histomorphological features, and correlated with duration of symptoms. It obviously also indicates more advanced disease.     

AFP基因转染的树突状细胞的生物学特性及靶向性CTL细胞毒作用

目的:观察基因转染的树突状细胞生物学特性及靶向性CTL对肿瘤细胞的杀伤作用。方法:用重组IL-4和GM-CSF诱导扩增小鼠骨髓来源的单个核细胞,将表达AFP基因的重组腺病毒转染DC(dendriticcells,DC),构建基因转染的DC瘤苗,并免疫C57BL/6小鼠。用流式细胞术(FCS)检测转染前后DC细胞表面分子MHCI、MHCII、LFA、ICAM、B7.1、和B7.2等的变化。取免疫小鼠脾细胞诱导细胞毒性CTL,LDH非放射性细胞毒性试验检测其对不同肿瘤细胞的杀伤作用。用ELISA法检测CTL诱导过程中TNF-α和IFN-γ分泌变化规律。结果:AFP基因转染后的DC分子表面高表达MHCI、MHCII、LFA、ICAM、B7.1、和B7.2等分子,尤其是MHCII、ICAM和LFA分子,与转染前比较具有显著性差异(P<0.05)。与对照组比较,AFP抗原诱导的靶向性CTL对AFP分泌性肿瘤细胞具有更强的杀瘤活性,且在CTL诱导过程中TNF-α和IFN-γ分泌水平较对照组也有显著性差异。结论:AFP基因重组腺病毒修饰的DC能表达高水平的MHCI、MHCII、LFA、ICAM、B7.1、和B7.2等分子,为CTL进一步活化提供了共刺激信号;用该瘤苗免疫C57BL/6J小鼠,能诱导出针对AFP抗原的靶向性CTL,从而实现对AFP分泌性肿瘤细胞的特异性杀伤作用。     

Prediction of lymph node metastases in breast cancer by clinicopathological and biological features of the primary tumor

Background  Although histologically confirmed nodal status continues to be the most important and reliable prognostic factor, preoperative assessment of nodal status has been required in order to avoid unnecessary surgery. Methods  The present review was carried out to elucidate the relationship between nodal status and various histologic/biologic factors of the primary tumor, and to evaluate their nodal predictabilities. Results  The incidence of nodal metastases increases with increasing tumor size. Nodal metastases, however, are quite uncommon in Tla (tumor sized 5 mm or less) cancers and in ductal carcimona in situ (DCIS). The use of various promising biological markers for predicting nodal status, however, seems to be still far from clinical use. Conclusion  The probability of nodal metastases is quite low in patients with Tl a cancer or DCIS. Therefore, routine axillary dissection for these patients has no diagnostic or therapeutic value.     

Histopathologic features of retinoblastoma and its relation with in vitro drug resistance measured by means of the MTT assay.

BACKGROUND: Retinoblastoma is frequently treated with chemotherapy to facilitate intraocular therapy, as well as to diminish or delay radiotherapy in invasive disease. It is also used more extensively in patients with dissemination to the central nervous system and/or the bone marrow. Once the disease has spread, the prognosis is poor. Radiotherapy is effective in ocular retinoblastoma, but is associated with facial deformation and a higher chance for second primary tumors in the irradiation field. These sequelae emphasize the need to determine more effective chemotherapy schedules and local treatment. The aim of this study is to investigate the relation between in vitro drug resistance for ten cytostatic drugs and histopathologic features in primary retinoblastoma. MATERIALS AND METHODS. Forty-four fresh samples of primary retinoblastoma were tested for in vitro drug resistance using the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The histopathologic features for differentiation, invasion and intra-ocular extension, necrosis, mitosis, and apoptosis were scored. RESULTS: The differentiation of the tumors revealed 24 poorly differentiated, 14 intermediately differentiated, and 6 well differentiated tumors. Tumor infiltration showed 3 minimal and 3 massive choroideal invasions, as well as 21 prelaminary and 2 postlaminary optic nerve invasions. The tumor was unifocal in 16 eyes and multifocal in 28 eyes, with extensive retinal involvement in 10 eyes and tumor seeding in 21 eyes. The MTT assay was successful in 82% of the samples after enzymatic handling of the tumor cells was omitted. Undifferentiated tumors were more sensitive to carboplatin (p = 0.034) and doxorubicin (p = 0.025), thiotepa (p = 0.051) and ifosfamide (p = 0.075) in comparison to differentiated tumors. Type of retinal involvement, invasion, focality, and seeding did not show a relationship with drug resistance. Calcified tumors were more resistant to actinomycin D and ifosfamide and more sensitive to vincristine; conversely, apoptotic tumors were more sensitive to ifosfamide and more resistant to vincristine (p = 0.027). Necrotic tumors were more sensitive to actinomycin D (p = 0.004), and mitotic tumors were more sensitive to idarubicin (p = 0.026). In 90% of the tumors extreme drug resistance to cytarabin was present. CONCLUSIONS: In retinoblastoma many histopathologic features are related to in vitro drug resistance. Undifferentiated tumors are more sensitive to several cytostatic drugs. Calcification and apoptosis show an inverse relation with in vitro drug resistance to ifosfamide and vincristine. Extreme drug resistance to cytarabin is observed; this drug should not be used in retinoblastoma treatment.     

CC chemokine ligand 25 enhances resistance to apoptosis in CD4+ T cells from patients with T-cell lineage acute and chronic lymphocytic leukemia by means of livin activation

We investigated CD4 and CD8 double-positive thymocytes, CD4(+) T cells from typical patients with T-cell lineage acute lymphocytic leukemia (T-ALL) and T cell lineage chronic lymphocytic leukemia (T-CLL), and MOLT4 T cells in terms of CC chemokine ligand 25 (CCL25) functions of induction of resistance to tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. We found that CCL25 selectively enhanced resistance to TNF-alpha-mediated apoptosis in T-ALL and T-CLL CD4(+) T cells as well as in MOLT4 T cells, but CD4 and CD8 double-positive thymocytes did not. One member protein of the inhibitor of apoptosis protein (IAP) family, Livin, was selectively expressed in the malignant cells at higher levels, particularly in T-ALL CD4(+) T cells, in comparison with the expression in CD4 and CD8 double-positive thymocytes. After stimulation with CCL25 and apoptotic induction with TNF-alpha, the expression levels of Livin in these malignant cells were significantly increased. CCL25/thymus-expressed chemokine (TECK), by means of CC chemokine receptor 9 (CCR9) ligation, selectively activated Livin to enhance resistance to TNF-alpha-mediated apoptosis in c-jun-NH(2)-kinase 1 (JNK1) kinase-dependent manner. These findings suggested differential functions of CCR9/CCL25 in distinct types of cells. CD4 and CD8 double-positive thymocytes used CCR9/CCL25 for migration, homing, development, maturation, selection, cell homeostasis, whereas malignant cells, particularly T-ALL CD4(+) T cells, used CCR9/CCL25 for infiltration, resistance to apoptosis, and inappropriate proliferation.     

组蛋白去乙酰化酶在食管鳞癌中的表达及其表达下调对EC9706细胞生物特性的影响

目的:探讨组蛋白去乙酰化酶(histone deacetylase2,HDAC2)在食管鳞癌组织中的表达,并研究其表达下调对食管鳞癌EC9706细胞增殖、细胞周期和细胞凋亡的影响,以及分析其相关的分子机制。方法:采用免疫组织化学法检测食管鳞癌组织中HDAC2蛋白的表达。将特异性针对HDAC2基因的小分子干扰RNA(small interfering RNA,siRNA)和对照siRNA分别转染食管鳞癌EC9706细胞,实验分3组:未处理组、对照siRNA组和HDAC2siRNA组。蛋白质印迹法检测各组EC9706细胞中HDAC2蛋白的表达。CCK-8计数法检测转染前后细胞的增殖情况。FCM法检测细胞周期和细胞凋亡的变化。蛋白质印迹法检测与细胞增殖、细胞周期和细胞凋亡相关蛋白的表达变化。结果:HDAC2蛋白在食管鳞癌组织中表达的阳性率为79.71%,显著高于癌旁不典型增生组织的51.11%和正常食管黏膜组织的23.19%,3者之间差异具有统计学意义(χ2=44.121,P=0.000);此外,HDAC2蛋白表达与患者的年龄和性别无关(P均>0.05),但是与组织学分级、浸润深度、TNM分期和淋巴结转移均显著相关(P均<0.05)。HDAC2siRNA能有效下调食管鳞癌EC9706细胞中HDAC2蛋白的表达,明显抑制食管鳞癌EC9706细胞的增殖,促使细胞周期静止在G0/G1期,并诱导细胞凋亡。蛋白质印迹法检测结果显示,HDAC2表达下调能明显提高p21和凋亡相关蛋白bax的表达量,同时降低细胞周期蛋白cyclin D1和bcl-2的表达量。结论:HDAC2可能在食管鳞癌的发生、发展中具有重要作用,其表达下调介导的食管鳞癌细胞增殖抑制、细胞周期静止以及细胞凋亡可能与p21、bax的表达升高和cyclin D1、bcl-2表达降低密切相关。     

The importance of orthotopic implantation to the isolation and biological characterization of a metastatic human clear cell renal-carcinoma in nude-mice

We established a new human renal cell carcinoma system to study some properties of metastatic renal cancer cells and the influence of the organ environment on their metastatic potential. Renal cell carcinoma obtained from a surgical specimen was dissociated enzymatically. Cells were injected into the subcutis, kidney, cecal wall, and spleen of nude mice. Tumors grew in the subcutis and kidney. Only kidney tumors produced distant metastasis. Subcutaneous tumors were avascular and encapsulated, whereas kidney tumors were highly vascularized and invaded the kidney parenchyma. Cell lines were also established from several spontaneous lung metastases. The most metastatic cell line (LM-6) expressed higher levels of basic fibroblast growth factor, gelatinase, and urokinase activity. These results show that human neoplasms are heterogeneous for biologic properties, that orthotopic implantation is essential for the selection, growth, and metastasis of human renal cell carcinoma cells, and that metastatic cells must possess multiple properties to enable them to complete the process.     

The histological appearance of tumours derived from rat embryo cells transformed in vitro spontaneously and after treatment with nitrosomethylurea

Wistar rat embryo cells were treated in vitro with either 25 μg/ml of nitrosomethylurea (NMU) or phosphate buffered saline. Both groups showed morphological transformation by the 13th passage but their ability to grow in soft agar did not occur until at least passage 23; plating efficiencies indicated that NMU had reduced transformation. However, both control and treated cells gave rise to fibrosarcomata after similar latent periods following inoculation into syngeneic recipients. The fibrosarcomata had “myxoid” and “leiomyomatous” areas, and two resembled haemangiopericytomata; for the most part the tumours were transplantable. Inoculation of cloned NMU-treated cells produced fibrosarcomata with a high proportion of giant cells but only after a very long latent period. No virus particles were detected in tumour samples by electron microscopy.     

The importance of clear margins in myxofibrosarcoma: Improving local control by means of staged resection and reconstruction

IntroductionMyxofibrosarcomas are associated with a locally infiltrative growth pattern, making a clear-margin resection margin challenging. This leads to high local recurrence rates. While immediate wound closure and adjuvant radiotherapy has been proposed to mitigate incomplete excisions, we present our experience treating myxofibrosarcomas with staged excisions until clear margins are obtained, prior to reconstruction.MethodsAll patients with myxofibrosarcomas treated with a curative intent at our centre between 2009 and 2019 were identified. Patient demographics, tumour characteristics, number of resections, method of reconstruction, adjuvant therapy, complications, local recurrence rates, length of hospital stay and overall survival were assessed.Results97 consecutive eligible patients were identified. Forty-six (47%) had positive margins reported following a first resection. The median number of resections required to obtain clear margins was two and the median time from first excision to definitive wound closure was 15 days. Local recurrence rate for the whole cohort was 14%. Patients who had staged resection until clear margins were obtained had a significantly lower rate of local recurrence compared to those who had positive margins at time of reconstruction (p-value = 0.001). The estimated 5-year disease-specific survival for the whole cohort was 93%.DiscussionObtaining clear margins in myxofibrosarcoma via staged resections was associated with lower local recurrence rates for patients who had an initial resection with positive margins. The outcomes of performing staged resections are equivalent to patients for whom a clear margin were obtained in the first instance.     

The benefit of microsatellite instability is attenuated by chemotherapy in stage II and stage III gastric cancer: Results from a large cohort with subgroup analyses

We previously reported that the prognosis of microsatellite instability high (MSI‐H) gastric cancer is similar to that of MSI‐low/microsatellite stable (MSI‐L/MSS) gastric cancer. The reason for this seemed to be related to the effects of chemotherapy. To verify this hypothesis, we expanded the study population and reanalyzed the prognosis of MSI‐H gastric cancer. Data from 1,276 patients with Stage II and III gastric cancer who underwent gastrectomy with curative intent between January 2005 and June 2010 were reviewed. The prognosis of MSI‐H tumors in comparison with MSI‐L/MSS tumors was analyzed, according to the administration of chemotherapy and other clinicopathologic features. A total of 361 (28.3%) patients did not receive chemotherapy (MSI‐H = 47 and MSI‐L/MSS = 314), whereas 915 (71.7%) patients did receive chemotherapy (MSI‐H = 58 and MSI‐L/MSS = 857). The hazard ratio of MSI‐H versus MSI‐L/MSS was 0.49 (95% confidence interval: 0.26–0.94, p = 0.031) when chemotherapy was not received and 1.16 (95% confidence interval: 0.78–1.71, p = 0.466) when chemotherapy was received. In subgroup analyses, the prognosis of MSI‐H was better in Stage III, women, with lymph node metastasis, and undifferentiated histology subgroups when chemotherapy was not received. However, in patients treated with chemotherapy, prognosis was worse for MSI‐H tumors in Stage III, undifferentiated histology, and diffuse type subgroups of gastric cancer. In conclusion, MSI‐H tumors were associated with a good prognosis in Stage II and III gastric cancer when patients were treated by surgery alone, and the benefits of MSI‐H status were attenuated by chemotherapy.     

RT-PCR测定乳癌及腋窝淋巴结微量组织MUC1及GST-π基因的表达

目的研究MUC1及GST-π mRNA在乳癌腋窝清扫淋巴结中表达与乳癌发生、发展、预后转归及临床耐药的相关性.方法用RT-PCR技术检测20例乳腺癌病人的108个HE染色阴性淋巴结MUC1及GST-π mRNA的表达.结果 MUC1及GST-π mRNA在HE染色阴性淋巴结中阳性检出率分别为66%及39%,在乳癌组织、HE阳性淋巴结及正常乳腺组织中阳性率分别为100%/88%、100%/71%及100%/0.结论 HE阴性淋巴结中出现MUC1阳性表达表明存在肿瘤微转移灶,MUC1mRNA检测可提高淋巴结微转移诊断率;GST-π在肿瘤启动和促进阶段可表达,是一种重要的乳腺癌生物学标志物;乳癌组织中耐药性普遍存在;MUC1及GST-π mRNA在腋淋巴结的表达可能是预后不良的标志,二者联合检测对乳癌早期诊断及预后判断有重要意义.