Humoral stimulating activities in post-cyclophosphamide rat sera and their purified fractions.

Enhanced humoral, stimulating activities (HSAs) of post-cyclophosphamide (CY) sera and their purified fractions, acting on mitogen activated T-lymphocytes, were detected in Wistar rats after treatment by high single doses of the aplasia producing alkylating cytostatic drug CY. These activities, monitored in vitro, were partially purified from post-CY sera, collected 2, 4, and 7 days after treatment, yielding fractions with higher humoral stimulating activity. The preliminary purification included molecular cutting by Amicon-Diaflo filters (1-30 kDa pore size range) and gel-filtration on Sephadex G-50 and G-75. Results show that post-CY sera partially purified fraction (10-20 kDa), enhances the proliferation of hydrocortisone resistant (HCR) T-lymphocytes up to threefold under microculture conditions (P << 0.001); partially purified post-CY sera fraction (10-20 kDa) increases the proliferative response of T-cells in microcultures to the mitogenic 145-2C11 monoclonal antibody (mAb) up to tenfold (P << 0.001). These results show that the activity of stimulating factors is localized in the molecular weight range of 10-20 kDa.

DNA-ploidy analysis within selected regions of colorectal adenomas containing carcinoma.

In order to better understand the relationship of DNA ploidy, dysplasia, early cancer, and colorectal tumor progression, 11 colorectal adenomas containing carcinoma invading the submucosa were investigated using DNA flow cytometry. Multiple frozen samples were taken from the selected sectors corresponding to adenoma tissue with low-grade dysplasia, high grade dysplasia and early cancer. Sampling accuracy was performed under histologic examination by multiple cryostatic sections. Data were compared with previously reported results in non-cancerous adenomas and advanced carcinomas. Incidence of DNA aneuploidy among the dysplastic regions of the adenomas containing carcinomas resulted higher than that observed in non-cancerous adenomas (p=0.02). Furthermore, among the DNA aneuploid populations, the frequency of clones with high DNA Index (DI>1.3) was slightly higher in adenomas with cancer than in adenomas without cancer (p=0.07). We suggest that differences may exist in DNA aneuploidy evolution between these two types of lesions. In early cancer, the near-diploid clones were 57% with respect to 18% (p=0.01) in advanced cancer since in this latter case the majority of the DNA abnormal clones were in the near-hypertriploid region (82%). Thus, the acquisition of the invasive phenotype appears to be linked with the expansion and stabilization of high DNA aneuploid clones. Further analysis on a larger number of cases of adenomas containing carcinoma are necessary to validate these interpretations.

H-ras gene mutations in salivary gland pleomorphic adenomas.

The DNA from 17 specimens of pleomorphic adenoma of the salivary glands was screened for the presence of ras gene mutations, which are known to be involved in the pathogenesis of various human neoplasias. By a sensitive method of hybridization with synthetic oligonucleotide probes on in vitro amplified tumor DNA, point mutations, mainly in codon 12 of the H-ras gene, were detected in six tumor specimens (35%). This high incidence of mutated ras genes suggests that their alteration may play a role in the pathogenesis of pleomorphic adenomas.

Effects of a modified CMF treatment (cyclophosphamide, methotrexate and 5-fluorouracil) on hematopoietic tissues and Yoshida sarcoma in rats.

Effects of a modified CMF treatment on hematopoietic tissue and an implanted tumor were studied in rats. The modification of the treatment refers to the application of cyclophosphamide 24 h after methotrexate and 5-fluorouracil. The study was done on Wistar rats bearing Yoshida sarcoma in the ascites form. The controls were a) untreated animals bearing the tumor or b) treated conventionally with the 3 cytostatics and c) tumor-free animals under either conventional treatment or d) modified treatment. We examined survival, the appearance of metastases, and the regeneration of hematopoietic tissues. Improved survival, the absence of metastases, and improved regeneration of hematopoietic tissues was observed when modified CMF treatment was applied. These results support the importance of sequencing cytostatic protocols for basic hematological determinants and anti-tumor activity.

High incidence of H-ras oncogene mutations in squamous cell carcinoma of lip vermilion.

Nine specimens of well-differentiated squamous cell carcinoma of the lip vermilion have been analyzed for the presence of H-ras oncogene mutations, using the technique of hybridization with synthetic oligonucleotide probes on in vitro amplified tumour DNA. Five specimens harbored mutations: four in codon 12 and one in codon 13. This high incidence (55%) of mutated H-ras genes suggests that their activation may play an important role in lip tumour development and may be connected to the exposure to chemical and/or physical carcinogens.

The effect of the rat-cyclophosphamide serum on the rat hematopoietic tissue recovery, previously damaged by cyclophosphamide or by gamma-irradiation.

The effect of post-cyclophosphamide serum (post-CY serum) humoral activity in vivo, on the rat hematopoietic regeneration after damage either by cyclophosphamide (CY) or by gamma-irradiation was investigated. Three experimental groups of rats were studied. The first group was treated by a single high dose of CY (200 mg/kg), the second group was whole body irradiated by 6.5 Gy, and the third group was whole body irradiated by 3.5 Gy. Each group was subdivided into three subgroups. The first among subgroups received immediately after damage 0.5 cc post-CY serum, the second received 0.5 normal rat sera per animal. The third subgroup of rats was used as control. Post-CY sera were collected from the Wistar rats, 2.5 months old 48 hours after the treatment by high single dose by CY (300 mg/kg). Post-CY serum activity in comparison to normal serum activity was evaluated in vivo conditions. In order to take samples of peripheral blood, spleen and bone marrow, the animals were sacrificed 24 hours, 48 hours, 72 hours and seven days after treatment. The assessment of post-CY sera effect is accomplished by measuring of the proliferative capacity of splenic, as well as bone marrow cells, and by means of the RBC and WBC counts. The results obtained indicate substantial stimulative and proliferative activity of the post-CY sera, when compared to normal sera, particularly in the animals previously damaged with CY.

Labelling of leukocytes with 99mTc-HMPAO for scintigraphy of inflammatory lesions and abscesses.

A simplified and efficient procedure for 99mTc-HMPAO-labelling of leukocytes is described. For this purpose, the pH and concentration of the 99mTc-HMPAO preparation was modified. Leukocytes were isolated from a 20 mL mixture of patient blood, 5 mL ACD and 0.8 mL methylcellulose after 1 h sedimentation of erythrocytes and centrifugation (at 400 g) of the obtained plasma layer. Simultaneously, 99mTc-HMPAO was prepared (one single-dose kit for two patients) by adding 2.2 mL 99mTc-generator eluate and, after 10 min, 0.3 mL of phosphate buffer to lower the pH to 7. The isolated WBCs were then labelled by the addition of 1-1.2 mL of 99mTc-HMPAO solution and incubated for 20 min. The unbound tracer was then discarded, the labelled WBC washed and finally resuspended in autologous cell-free plasma. Leukocytes labelled by this procedure were used for scintigraphic localization of inflammatory lesions and abscesses in the gastro-intestinal tract. The labelling efficiency was 60 +/- 9%, with a separation yield of 55 +/- 11%.

Intratumor heterogeneity of K-ras2 mutations in colorectal adenocarcinomas: association with degree of DNA aneuploidy.

Detailed information about intratumor K-ras2 mutations in colorectal adenocarcinomas and a possible association with DNA content heterogeneity is still lacking. DNA diploid and aneuploid subclones, detected among multiple histologically selected primary sectors (57 superficial and 40 deep) and 9 lymph node metastases, were flow cytometrically sorted and separately submitted to codons 12-13 K-ras2 mutation spectrum analysis. DNA aneuploidy was absent among 20 near and 20 distant mucosa sites and present in 7/9 lymph node metastases and in 17/19 primary tumors (90%). Primary intratumor DNA multiclonality was approximately 50%. Degree of DNA aneuploidy (DNA Index) distribution was nonrandom and showed peaks at approximate mean DNA Index values 1.2, 1.5, and 1.8. K-ras2 mutations were detected in 0/20 mucosa cases, in 2/9 lymph node metastases, and in 9/19 adenocarcinomas (47%). No more than one mutation type per tumor was detected. Intratumor distribution of K-ras2 mutations was homogeneous in 6 and heterogeneous in 3 cases. Homogeneous distribution was associated with DNA near-diploid aneuploidy. K-ras2 mutations were strongly associated with DNA Index in the near-diploid region (83%) and almost absent (5%) among DNA near-triploid subclones (P = 0.0001). K-ras2 mutation intratumor heterogeneity indicates that sampling of the tumor may be a critical step and suggests that K-ras2 activation may be a late event in a subgroup of tumors. Our data also suggest the existence of an early process of the colorectal carcinogenesis that favors both K-ras2 mutations and DNA near-diploid aneuploidy. Onset of DNA near-triploid subclones appears, instead, to be independent from K-ras2 activation.

Pancreatic intraductal sampling during ERCP in patients with chronic pancreatitis and pancreatic cancer: cytologic studies and k-ras-2 codon 12 molecular analysis in 47 cases.

BACKGROUND: A preoperative tissue diagnosis of pancreatic cancer is desirable but difficult to obtain. METHODS: Pancreatic brush cytology, salvage cytology, and collection of pancreatic juice were attempted prospectively during ERCP in 34 patients with pancreatic cancer and 11 with chronic pancreatitis. K-ras-2 codon 12 was analyzed for presence and type of point mutations. RESULTS: Brush cytology coupled with salvage cytology had a sensitivity of 74%. The addition of cytologic analysis of pancreatic juice did not substantially improve sensitivity (76%). K-ras-2 was mutated in both cancer (87%) and pancreatitis (40%). The specificity for cytology was 100% and for K-ras-2 mutations 60%. Combining cytology with mutation analysis increased sensitivity to 93% but reduced the positive predictive value. The negative predictive value never exceeded 75%. None of the patients with chronic pancreatitis had cancer develop (median follow-up 60 months). CONCLUSIONS: Pancreatic ductal brushing with salvage cytology is useful in the diagnosis of cancer, whereas cytologic analysis of pancreatic juice can be abandoned. At present, K-ras-2 mutation is not useful for differentiating pancreatic cancer from chronic pancreatitis or the identification of patients with chronic pancreatitis at risk for malignant transformation.

K-ras-2 G-C and G-T transversions correlate with DNA aneuploidy in colorectal adenomas.

BACKGROUND/AIMS: K-ras-2 mutations and DNA content heterogeneity represent early events of human colorectal tumor progression. The aim of the study was to investigate if specific K-ras-2 mutations in 58 human sporadic adenomas were correlated with DNA aneuploidization and cell proliferation. METHODS: Multiparameter flow cytometry, based on scatter parameters and DNA content, was performed using 4,6-diamidino-2-phenilindole-2-hydrochloride-stained nuclei obtained from adenoma fragments with either mild-moderate or severe dysplasia. K-ras-2 polymerase chain reaction and spectrum analysis were performed using sorted DNA specific epithelial subclones. RESULTS: We detected six G-A transitions, and four G-C and two G-T transversions. The DNA aneuploid subclones were 25 with DNA index values in the near diploid region (DNA index < 1.3) for the vast majority of cases (80%). DNA aneuploidy among the mutated adenomas with G-A transitions was 1 of 6 (17%) and 6 of 6 (100%) among G-C and G-T transversions. Although DNA aneuploidy and high S-phase values were also present among K-ras-2 wild-type adenomas, their statistical associations with K-ras-2 status were P < 0.005 and P < 0.05, respectively. CONCLUSIONS: The present series of sporadic colorectal adenomas indicates that codon 12 G-C and G-T K-ras-2 transversion mutations and DNA aneuploidy are correlated. The underlying mechanisms that explain such association remain to be investigated.

K-ras2 activation and genome instability increase proliferation and size of FAP adenomas.

The possible role of K-ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP) adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%), low grade dysplasia (63%), and were preferentially located in the right colon (60%). Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K-ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO) hybridization, while flow cytometry (FCM) was used for evaluating degree of DNA ploidy and S-phase fraction. Overall, incidences of K-ras2 mutations, DNA aneuploidy and high S-phase values (>7.2%) were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K-ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K-ras2 and size, DNA ploidy and size and K-ras2 and S-phase (p < 0.001). In particular, among the wild type K-ras2 adenomas, high S-phase values were detected in 8% of the cases versus 57% among the K-ras2 mutated adenomas (p = 0.0005). The present series of FAP adenomas indicates that K-ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.