A 64-year-old man who had taken acute myelogenous leukemia (AML-M2) in 1989 have relapsed with t(12; 17) (p13; q11.2-21) chromosomal abnormality and presenting marked infiltration to the skin in 1994. Blasts were seen on his peripheral blood smear (15%) and bone marrow examination showed increased leukemic cells (56%), with maturation. Leukemic cells expressed CD13 and CD33 antigen but not HLA-DR. Although leukemic cells had not promyelocytic feature morphologically, detection of PML/RAR alpha infusion signal of peripheral leukemic cells were positive for 8% (1% for control) by fluorescence in situ hybridization method. Because he did not response to standard combination chemotherapy and because we considered the possibility that t(12; 17) (p13; q11.2-21) observed in this case are t(15; 17) variant, we tried all trans retinoic acid (ATRA) to him. Interestingly, ATRA was very effective for skin lesion but hematologically it had no effect at all, and he died because of bacterial pneumonia.

Within the recent four years, there have been substantial advances in understanding the molecular pathogenesis of HNPCC. As one of the result of investigation, microsatellite instability has been observed in hereditary non-polyposis colorectal cancer (HNPCC) and other sporadic cancers. Moreover, there is strong supporting evidence that mismatch repair genes play a role in HNPCC. Here, we present our investigational results and discuss possible molecular mechanisms governing DNA mutation and genomic instability, leading to the development of neoplasm. We investigated replication error (RER) of 4 microsatellite markers (dinucleotide repeats) in 131 patients with colorectal cancer (10 met the criteria of HNPCC group B), using fluorescence-based DNA sequencer. We detected RER positivity (at more than two loci) in 12 of 131 patients (9.2%). PCR-SSCP and direct sequencing of the mismatch repair genes, hMSH2 and hMLH1, revealed that two patients in HNPCC group B had germline mutations of hMSH2. The fluorescence-based techniques, such as the present RER analysis do not require radioactive materials and specialized rooms, and can easily be performed in a clinical laboratory.

Although adenocarcinoma of the prostate is recently becoming one of most common malignancies in Japanese men, it still poses many questions regarding its etiology, pathology, pathogenesis and clinical management. Many reports have been made on oncogene and tumor suppressor gene, however, frequent genetic alterations have not been identified during prostate cancer development. Loss of heterozygosity (LOH) on 8p might be an important event in the early stage of prostatic carcinogenesis, whereas alteration in 17p is now considered a late event. Numerous reports about the androgen receptor (AR) gene have revealed that mutations in the coding region of AR possibly results in an acquired resistance to androgen blockade therapy and anti-androgen withdrawal syndrome. It has been also shown that shorter CAG repeats of AR gene are associated with a higher risk of prostate cancer. Regarding molecular diagnosis, prostate-specific membrane antigen (PSM) appears to be a new molecule with many potentially valuable applications. PSM-reverse transcriptase-polymerase chain reaction (RT-PCR) is probably more sensitive and specific than PSA-RT-PCR to predict micrometastatic disease. Gene therapy based on the above molecular aspect is currently under investigation but not generally used yet.

By using three hematopoietic cell lineages including myelomonocytic, erythroblastic and megakaryocytic differentiation, downregulation of telomerase activity was found to be a general response to the induction of differentiation. The decrease in telomerase activity occurred as early as 24h when HL-60 and K562 cells were cultured in the presence of VD3, ATRA and hemin and completely disappeared after 3 days. On the other hand MEG-01 cells showed dramatic inhibition of telomerase activity after 6 days of culture with TPA. Analysis of telomeric DNA in HL-60 cells and K562 cells demonstrated no remarkable loss of telomeric DNA with cellular differentiation while loosing telomerase activity. The repression of telomerase is one of many molecular events during the complex process of cellular differentiation, and testing of additional cell lines that are capable of differentiation will be helpful for understanding the mechanisms of telomerase control.

In order to clarify the relationship between the telomeric length of human female breast carcinoma cells and patient age, and between telomeric length and the histological type of carcinoma, we examined 64 patients (aged 20-89 years) with breast carcinoma by histological and southern blot analysis. No difference in the telomeric length was recognizable among the three major histological types: papillotubular, solid tubular and scirrhous (7.9-8.7 kilobase pairs (kbp)). Mean telomere lengths in the groups aged under 35 (8 patients), 36-50 (10 patients), 51-70 (17 patients), 71-80 (19 patients), and over 81 years (10 patients) were 11.0, 9.9, 7.0, 7.7 and 7.6 kbp, respectively. There was no significant evidence for telomeric shortening in breast carcinoma according to patient age. Two peaks of telomeric length were observed in three carcinomas comprising a medullary carcinoma and two solid tubular carcinomas showing very prominent lymphocyte infiltration histologically.

The purpose of this study was to evaluate the correlation between alteration in telomere length and prognosis in patients with pathological stage I-II non-small cell lung cancer. We measured telomeric repeat length and telomerase activity by use of southern blot analysis of terminal restriction fragments and a non-radioactive ELISA-based assay, respectively.

Of a total of 21 surgically resected lung adenocarcinomas (15 men and 6 women with an average age of 59.6 yr) well, moderately and poorly differentiated types were 6, 7 and 8 cases, respectively. Telomerase activity determined by the TRAP assay was classified into four types as follows; negative = Score 0, weakly positive = 1, moderately positive = 2, strongly positive = 3. Chromosome aberrations were also classified into four groups: Score 0, 1, 2, 3 according to the number of marker chromosomes, 0, 1-5 6-15 and over 16 respectively. CT-Indices (chromosome aberration x telomerase activity) of well, moderately, and poorly differentiated types were 1, 4.3, 5.1 respectively. Thus, CT-Indices were correlated with cell differentiation in the lung adenocarcinomas (p < 0.01).

Telomerase activity and terminal restriction fragment (TRF) length were examined in hepatocellular carcinoma (HCC). Telomerase activity was assayed by telomeric repeat amplification protocol (TRAP) connected with an internal telomerase assay standard (ITAS). The incidence of strong telomerase activity (highly variable level compared with the activity of non-cancerous liver tissue) was 79% in well, 84% in moderately, and 100% in poorly differentiated HCC, while 0% in non-cancerous liver tissues. The incidence of TRF length alteration (reduction or elongation) was 53% in HCC. The incidence of TRF alteration was significantly higher in HCC exceeding 3 cm in diameter, moderately or poorly differentiated in histology. Telomerase activity was not associated with TRF length alteration in HCC. In conclusion, strong telomerase activity and TRF length alteration increased with HCC tumor progressions.

In this study, we investigated the correlation between the telomerase activity and the microsatellite instability in colorectal cancers and adenomas. Telomerase activity was detected in 75% of cancers and in 33% of adenomas. Microsatellite instability was detected in 24% of sporadic cancers, in 54% of multiple cancers, and in 22% of adenomas. Telomerase activity and microsatellite instability were independent events in colorectal carcinogenesis. The patients with telomerase activity and microsatellite instability showed poor prognosis. These results indicate that colorectal cancers with both telomerase activity and microsatellite instability may have more malignant potential, and adenomas with both them may be more intensive precursor of colorectal cancers.

We analyzed telomere length and telomerase activity in intestinal metaplasia (IM), adenoma, and cancer of the stomach and studied the stages at which the cells acquire telomerase activity in carcinogenesis and also the correlation between telomerase activity and telomere length. Telomerase activity was detected in 15%, 45%, 89% of IM, adenomas, and cancers. Telomere lengths shortened as normal mucosa changed into IM and more into adenoma. Gastric cancers showed a broad range of telomeric length. The shortest telomere length was found among gastric adenomas. These results suggest that telomerase is expressed during early phase of gastric carcinogenesis but the activity at that stage is not strong enough to fully restore the reduced telomeric DNA.

Stable maintenance of chromosomes needs the functional telomeres at the chromosomal ends. It has been proposed that the loss of telomeric function (LTF) plays a major role in the production of abnormal chromosomes, such as the telomere association (TA) and the jumping translocation (JT). We analyzed TA and JT to evaluate the involvement of LTF in chromosomal instability. A shortened telomeres was identified at the fusion point of the JT. We also developed a method to examine TA based on PCR and found relationships between telomere shortening and products of PCR. Our findings strongly suggest that LTF may cause chromosomal instability and contribute to cancer cell evolution.

Site-specific recombination of the TAL1 gene was analyzed by Southern blotting and polymerase chain reaction (PCR) in 44 cases of childhood T-cell acute lymphoblastic leukemia (T-ALL), 20 cases of childhood T-cell non-Hodgkin's lymphoma (T-NHL) and 35 cases of adult T-cell malignancies. This recombination was found in 10 (22.7%) of 44 childhood T-ALL patients, but in none of the T-NHL or adult T-cell malignancies. Recombination of the TAL1 gene was therefore suggested to be specific for childhood T-ALL. The immunophenotypic features of the 10 T-ALL patients with this recombination were CD1-, CD2+, CD4-, CD7+, CD10-, and they had a significantly better outcome than other T-ALL cases without the recombination. The PCR technique revealed minimal residual disease (MRD) in 2 patients. One showed persistent MRD, while in the other MRD was recognized only at initial diagnosis. Further investigation is needed whether T-ALL with this recombination constitutes a distinct clinical subgroup among childhood T-ALL patients.

Cells with multidrug resistance (MDR) phenotype express P-glycoprotein (P-gp) on the cell membrane, which functions as a drug-efflux pump. To quantify the expression of the gene encoding P-gp (multidrug resistance 1; MDR1) and assess P-gp function, we developed competitive reverse transcription-polymerase chain reaction (RT-PCR) assay using heterologous competitor RNA and flow cytometric analysis using rhodamine 123 (Rh123; an artificial substrate for P-gp), respectively. First, we adjusted the assays by analyzing leukemia sublines showing various levels of MDR1 expression. The MDR1 expression in leukemia sublines quantified by competitive RT-PCR assay showed linearity over a wide range, and the results were parallel with those of MDR1 expression measured by Northern blot analysis, the P-gp antigen expression measured by flow cytometric analysis using MRK16, P-gp function analysis by Rh123 dye-efflux assay, and MDR phenotype. Then, we applied these assays to leukemic cells from patients with hematological malignancies. All 69 samples from 64 patients were successfully assayed, and the range of MDR1 expression was from 1.6 to 100 amol/microgram RNA. Since subpopulations of normal lymphocytes show a low degree of P-gp function, strict gating of leukemia cells was mandatory for dye-efflux assay of clinical samples. MDR1 expression in normal lymphocytes was below 8 amol/microgram RNA. By comparison to MDR1 expression quantified by competitive RT-PCR assay with P-gp function assessed by Rh123 dye-efflux assay in gated leukemic cells, more than 8 amol/microgram RNA was regarded as positive MDR1 expression in the leukemic cells themselves. These data suggest that these assays are suitable for evaluating P-gp expression and function in clinical samples when the proper cut-off value is used and may provide insights into the contribution of P-gp to drug resistance in hematological malignancies.

A 13-year-old girl and her older sister were referred to our hospital because her father and another older sister and been suffering from medullary thyroid carcinoma (MTC). Physical examinations revealed no thyroid mass. Ultrasonography (US) detected a small hypoechoic lesion in the right thyroid lobe, 3 mm in size. The serum calcitonin level was slightly elevated for their age. One and half year later, US finding showed a slightly larger mass, 7 mm in size, which was not palpable. Genetic testing for ret protooncogene mutation revealed a point mutation at codon 634 (Cys-->Arg) in exon 11. Surgical treatment was recommended, however, the patient wanted to receive it only after the admission to senior high school. Her sister had no mutation in ret protooncogene. The patient underwent total thyroidectomy with modified neck dissection at 16 years of age. At that time, a new mass emerged in the left thyroid lobe and the serum calcitonin level increased to 171 pg/ml. MTC was found in each lobe with positive staining of CEA and carcitonin. No lymph node metasis was found. Her postoperative course was uneventful. Genetic testing for ret mutation is effective for early diagnosis and treatment of patient with FMTC, and is recommended as standard management for hereditary MTC in affected families.