Immune reconstitution after autologous hematopoietic stem cell transplantation in relation to underlying disease, type of high-dose therapy and infectious complications.

BACKGROUND AND OBJECTIVES: Autologous peripheral stem cell transplantation (APSCT) is increasingly used for various hematologic malignancies and solid tumors. The objective of this study was to analyze the immune reconstitution after APSCT and see if there was any correlation with diagnosis, age, type of high-dose therapy, CD34(+) selection of the autograft and double vs single APSCT. DESIGN AND METHODS: Lymphocyte subset recovery was studied in 46 consecutive patients with hematologic malignancies and breast cancer, who underwent APSCT. Eleven patients with multiple myeloma received tandem autografts. Thirty-one patients were given total body irradiation (TBI) as part of the high-dose treatment. Eighteen patients received a CD34(+) selected graft. The percentage and absolute numbers of lymphocyte populations, T-cells (CD2(+), CD3(+)), B-cells (CD19(+)), NK cells (CD56(+ )CD3(-) and CD16(+)CD3(-)) and T-cell subpopulations (CD4(+), CD8(+), CD4(+)CD45RA(+), CD4(+ )CD45RO(+), CD4(+)DR(+), CD8(+ )CD45RO(+), CD8(+)DR(+)), were monitored with flow cytometry during the first year after APSCT. RESULTS: The total B-cell (CD19(+)) and T-cell (CD3(+)) counts were reconstituted to normal levels 2-4 months after APSCT. All patients had a low CD4/CD8 ratio during the observation period, related to both a low number of CD4(+) cells and elevated numbers of CD8(+) cells. The low number of CD4(+) cells was due to a persistently low level of naive CD4(+)CD45RA(+) cells. A high proportion of the CD8+ cells displayed a phenotype compatible with activated T-cells (CD8(+)DR(+)) up to 10 months after autografting. The number of NK cells (CD56(+)3(-) or CD16(+)3(-)) reached normal values within one month post-transplant. No single variable, such as diagnoses, age, TBI as part of the high-dose treatment, tandem autografting or CD34(+) selection of the graft, influenced the immune or hematopoietic reconstitution and no correlation with documented infectious complications was found. INTERPRETATION AND CONCLUSIONS: Despite heterogeneity of diseases, age, initial treatment and high-dose regimens, lymphocyte subset analysis did not reveal any differences in hematopoietic or immune reconstitution. All patients had a low CD4(+)/CD8(+) ratio during at least the first year post-transplant, caused by a persistent increase of CD8(+) lymphocytes and a constant reduction of CD4(+) lymphocytes, making the patients susceptible to infections for a prolonged period of time post-transplant.

Bioavailability of aciclovir after oral administration of aciclovir and its prodrug valaciclovir to patients with leukopenia after chemotherapy.

The median bioavailabilities of aciclovir after administration of aciclovir and its prodrug valaciclovir were 21.5 and 70.1%, respectively, in 12 patients with malignant hematological diseases with leukopenia after chemotherapy. The interindividual variations of the bioavailability were 48.5 and 21.0% after administration of aciclovir and valaciclovir, respectively. Neither the bioavailability nor the interindividual variation of area under the concentration-time curve of oral aciclovir or valaciclovir differed from that reported in healthy volunteers.

Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines.

One of the most important components of G1 checkpoint is the retinoblastoma protein (pRB110). The activity of pRB is regulated by its phosphorylation, which is mediated by genes such as cyclin D1 and p16/MTS1. All three genes have been shown to be commonly altered in human malignancies. We have screened a panel of 26 oral squamous cell carcinomas (OSCC), nine premalignant and three normal oral tissue samples as well as eight established OSCC cell lines for mutations in the p16/MTS1 gene. The expression of p16/MTS1, cyclin D1 and pRB110 was also studied in the same panel. We have found p16/MTS1 gene alterations in 5/26 (19%) primary tumours and 6/8 (75%) cell lines. Two primary tumours and five OSCC cell lines had p16/MTS1 point mutations and another three primary and one OSCC cell line contained partial gene deletions. Six of seven p16/MTS1 point mutations resulted in termination codons and the remaining mutation caused a frameshift. Western blot analysis showed absence of p16/MTS1 expression in 18/26 (69%) OSCC, 7/9 (78%) premalignant lesions and 8/8 cell lines. One cell line, H314, contained a frameshift mutation possibly resulting in a truncated p16/MTS1 protein. pRB was detected in 14/25 (56%) of OSCC but only 11/14 (78%) of these contained all or some hypophosphorylated (active) pRB. In premalignant samples, 6/8 (75%) displayed pRB, and all three normal samples and eight cell lines analysed contained RB protein. p16/MTS1 protein was undetectable in 10/11 (91%) OSCCs with positive pRB. Overexpression of cyclin D1 was observed in 9/22 (41%) OSCC, 3/9 (33%) premalignant and 8/8 (100%) of OSCC cell lines. Our data suggest p16/MTS1 mutations and loss of expression to be very common in oral cancer cell lines and less frequent in primary OSCC tumours. A different pattern of p16/MTS1 mutations was observed in OSCC compared to other cancers with all the detected p16/MTS1 mutations resulting in premature termination codons or a frameshift. The RB protein was expressed in about half (44%) of OSCCs and its expression inversely correlated with p16/MTS1 expression. In conclusion, we show that abnormalities of the RB pathway are a common mechanism of oral carcinogenesis.

Prevalence of human papillomavirus infection in premalignant and malignant lesions of the oral cavity in U.K. subjects: a novel method of detection.

To evaluate the possible role of human papillomavirus (HPV) in oral neoplasms, 28 oral squamous cell carcinomas (SCC) and 12 potentially malignant lesions were analysed for the presence of HPV DNA. A nested polymerase chain reaction (PCR) approach, using two sets of HPV consensus primers to the L1 region, was used, which was able to detect a broad spectrum of HPV types. HPV DNA was detected in 14/28 (50%) carcinomas and 4/12 (33%) precancerous lesions. A novel approach based on labelling the PCR products with 32P and the separation of radioactively labelled products on an 8% polyacrylamide gel increased the sensitivity of the detection and enabled the identification of the HPV types. The typing of HPV was subsequently confirmed by direct DNA sequencing. HPV 6 and HPV 16 were the only HPV types detected and seven tumours harboured both types. Our results suggest that HPVs may be an important aetiological factor in the development of oral cancer. The detection procedure ensured sensitivity and consistency of the detection of low copy numbers of the virus DNA. The presence of HPV in 33% of premalignant tissues suggests that HPV infection may be an early event in the malignant transformation of oral SCC. There was no statistically significant association between viral infection and tumour grade or stage.

p53 aberrations in oral submucous fibrosis and oral squamous cell carcinoma detected by immunocytochemistry and PCR-SSCP.

An archival series of oral biopsies from Karachi, Pakistan, consisting of 21 cases of oral submucous fibrosis (OSF) and 27 cases of squamous cell carcinoma (SCC), of which 6 had arisen from OSF, were used to examine the aberrations in the structure and expression of the p53 tumour suppressor gene. The PCR-SSCP method was used for mutation analysis of exons 2-9, and (over)expression of p53 protein was detected by immunocytochemistry using monoclonal antibody DO 7. Positive immunostaining was observed in 15/20 (75%) of OSF specimens, 3/6 (50%) of SCC arising from OSF and 14/21 (67%) of SCC not arising from OSF. Mobility shifts in SSCP indicative of a mutation in p53 or loss of heterozygosity (deletion of a band) were seen in 13/21 cases of OSF and 15/27 cases of SCC. There was concordance between immunocytochemistry and SSCP results in a majority (33/48) of samples. Though the number of analysed SCC cases arising from OSF was limited, the results suggest that p53 mutation/protein stabilisation may play a part in the pathogenesis of OSF and its progression to SCC.

Molecular analysis of mutations in the CSB (ERCC6) gene in patients with Cockayne syndrome.

Cockayne syndrome is a multisystem sun-sensitive genetic disorder associated with a specific defect in the ability to perform transcription-coupled repair of active genes after UV irradiation. Two complementation groups (CS-A and CS-B) have been identified, and 80% of patients have been assigned to the CS-B complementation group. We have analyzed the sites of the mutations in the CSB gene in 16 patients, to determine the spectrum of mutations in this gene and to see whether the nature of the mutation correlates with the type and severity of the clinical symptoms. In nine of the patients, the mutations resulted in truncated products in both alleles, whereas, in the other seven, at least one allele contained a single amino acid change. The latter mutations were confined to the C-terminal two-thirds of the protein and were shown to be inactivating by their failure to restore UV-irradiation resistance to hamster UV61 cells, which are known to be defective in the CSB gene. Neither the site nor the nature of the mutation correlated with the severity of the clinical features. Severe truncations were found in different patients with either classical or early-onset forms of the disease.

p53 alterations and HPV infections are common in oral SCC: p53 gene mutations correlate with the absence of HPV 16-E6 DNA.

To examine the association between HPV infections and p53 gene aberrations, a panel of 28 oral squamous cell carcinomas (SCC) and 12 potentially malignant oral mucosal lesions were analysed for p53 mutations in exons 2-9. p53 protein was analysed by immunocytochemistry using DO7 antibody. The same panel was also examined for the possible presence of HPV infection. p53 overexpression was detected in 13/26 (50%) malignant and 2/9 (22%) premalignant lesions. Mutations in the coding region of the p53 gene were found in 10 malignant samples. None of the premalignant lesions were shown to have p53 mutations. The total number of p53 mutations in 10 samples were 14 of which 12 (85%) were in exon 5 suggesting the presence of hot spots in exon 5 for carcinogens involved in the transformation of oral epithelial cells. The presence of HPV DNA was first screened with consensus primers to the L1 region and nested PCR approach. HPV 6 and HPV 16 were detected in 14/28 (50%) oral SCC and 4 of 12 (33%) precancerous lesions, 7 tumours harboured both types. The samples were then examined for the presence of E6 oncogenic sequence of HPV16 using E6 specific primers. 7/27 (26%) SCC and 5/9 (55%) premalignant lesions harboured E6 DNA of which 6 (3 SCC and 3 premalignant) were negative with L1 primers suggesting possible integration of the specific viral genes or loss of other viral DNA sequences after integration of larger viral fragments. 9/10 (90%) SCC with p53 mutations were negative for E6 DNA. Our results show that both p53 alterations and HPV infection may be important etiological factors in the development of oral cancer. However, there is: i) No concordance between p53 mutations and its overexpression. ii) the presence of HPV capsid DNA (L1) does not necessarily indicate the presence of HPV oncogenic genes. iii) p53 gene mutations, but not overexpression, correlate with the absence of HPV 16-E6 and not L1 gene.

Molecular and biochemical characterization of xrs mutants defective in Ku80.

The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.

Correlated mutagenesis of bcl2 and hprt loci in blood lymphocytes.

In vivo measurement of human somatic mutations may be a valuable biodosimeter of exposure to carcinogens and of cancer risk. We have surveyed translocations at the bcl2 locus in B lymphocytes, and mutations at hprt in T lymphocytes, in 120 individuals with varying exposure to radon and cigarette smoke. bcl2 t(14:18) translocation is the commonest chromosomal alteration observed in non-Hodgkins lymphoma (NHL). We observed a significantly larger range of bcl2 translocation frequency (range: 0-372 x 10(-6), median: 1.9 x 10(-6)) than of hprt mutation frequency (range: 0-76.4 x 10(-6), median: 11.1 x 10(-6)), which is likely the result of clonal proliferation of deathless B cell mutants. We observed that the frequencies of these two distinct lymphocytic mutations are significantly correlated. Although some of the correlated variation is explained by age, a significant correlation of bcl2 mutagenesis persists after age adjustment. Correlated mutagenesis at distinct loci in distinct cell types could be explained by the existence of a mutator phenotype or by variation in exposure to environmental mutagens. NHL is commoner in men than in women, and our data indicate a trend toward higher bcl2 mutagenesis in males than females. There is mounting epidemiological evidence for a worldwide increase in NHL, which may have an environmental basis; molecular epidemiological analysis of bcl2 mutagenesis in exposed populations might be especially relevant to the identification of putative environmental causes. Given the relative ease of the bcl2 assay versus the hprt assay, and the consistency with which data are reproduced from laboratory to laboratory, it is likely that the bcl2 assay will be soon added to the array of assays used in human mutational surveillance.

Loss of heterozygosity on chromosome 5q in ovarian cancer is frequently accompanied by TP53 mutation and identifies a tumour suppressor gene locus at 5q13.1-21.

Forty-nine ovarian tumours were examined for loss of heterozygosity (LOH) on chromosome 5 using eight microsatellite markers spanning both arms, including one at the APC locus. LOH on 5q was a frequent event, detectable in 23 of 49 (47%) tumours, whereas 5p LOH was detected in only 1 of 22 tumours (5%). Six tumours showed partial LOH on 5q, enabling the candidate region to be localised to a 22 cM region proximal to APC, flanked by D5S424 and D5S644. An association was found between 5q LOH and TP53 mutation, with 18 of 23 (78%) tumours with LOH on 5q also harbouring a TP53 mutation. LOH on 5q was observed in 6 of 18 (33%) stage I tumours, suggesting that it may be an early event in the molecular pathogenesis of certain ovarian carcinomas.

Development of new molecular procedures for the detection of genetic alterations in man.

The Restriction Site Mutation (RSM) procedure is a DNA-based method for detecting mutations at any unselected locus. Mutations are identified as alterations of the DNA sequence at a chosen restriction site. DNA from cells exposed to mutagenic treatment is exhaustively digested with the restriction enzyme (RE). Sequences containing the mutated target site are specifically amplified using the polymerase chain reaction (PCR), whereas DNA without mutations at this site will have been cleaved and can not therefore provide a substrate for PCR. We have developed this procedure using both bacterial and mammalian cells. With bacteria, in plasmid reconstruction experiments we were able to detect mutations at a frequency of 10(-6) at an EcoRI site in the AraA locus of Salmonella typhimurium. The detection limit with an RsaI site in the lacI gene of Escherichia coli was 10(-5), and we were able to detect DNA damage and repair after treatment with N-methyl-N-nitrosourea (MNU). With mammalian cells, we have detected mutations induced by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells. In extensive studies with normal and repair-deficient human cells, we have detected and sequenced mutations induced by UV-C or UV-B in fibroblasts and lymphoblastoid cells from repair-deficient xeroderma pigmentosum (XP) donors. Similar results were obtained at TaqI sites in three genes, hprt, c-Ha-rasI and p53. These results demonstrate that the system is able to detect and analyse mutations induced at high frequencies. In our extensive attempts to extend the work to conditions of lower mutation frequencies, we have encountered several obstacles, the most serious being false-positive mutant DNA in totally untreated cells. This appeared to be a cell-line specific phenomenon, which we have not been able to eliminate by altering conditions. We propose therefore that, at present, RSM is a suitable method for studying high mutation frequencies at different loci and could be used for mutagen testing with repair-deficient cells. As yet, however, its sensitivity and specificity is not sufficient for population monitoring.

U.v.-hypermutability of xeroderma pigmentosum cells demonstrated with a DNA-based mutation system.

We have developed a DNA-based system, to detect mutations at restriction sites without any selection in culture. DNA is exhaustively digested with a restriction enzyme. Primers flanking a chosen site for this enzyme are used in the polymerase chain reaction (PCR). Only DNA molecules mutated at the chosen site are resistant to digestion and can serve as templates for the PCR. We have initially used this system to demonstrate the generation of mutations by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells, and by u.v.-C irradiation at a TaqI site in the hprt gene of human fibroblasts. In repair-deficient xeroderma pigmentosum (XP) cells the u.v.-induced mutant frequency was greatly enhanced. We have been able to detect and analyse mutations in XP cells at TaqI sites in three different genes, hprt, p53 and c-Ha-ras1. Both u.v.-C and u.v.-B irradiation have been used as mutagenic agents with both lymphoblastoid and fibroblast cells from XP patients from complementation group G. The mutant DNA molecules have been sequenced. Following u.v.-C-irradiation, the majority of mutations analysed were GC-->AT transitions, but several double and tandem mutations were also found.

Mutations in the xeroderma pigmentosum group D DNA repair/transcription gene in patients with trichothiodystrophy.

DNA repair defects in the xeroderma pigmentosum (XP) group D complementation group can be associated with the clinical features of two quite different disorders; XP, a sun-sensitive and cancer-prone disorder, or trichothiodystrophy (TTD) which is characterized by sulphur-deficient brittle hair and a variety of other associated abnormalities, but no skin cancer. The XPD gene product, a DNA helicase, is required for nucleotide excision repair and recent evidence has demonstrated a role in transcription. We have now identified causative mutations in XPD in four TTD patients. The patients are all compound heterozygotes and the locations of the mutations enable us to suggest relationships between different domains in the gene and its roles in excision repair and transcription.

Molecular analysis of mutations in the hprt gene in circulating lymphocytes from normal and DNA-repair-deficient donors.

Circulating lymphocytes from patients with the DNA-repair-deficient disorders, xeroderma pigmentosum (XP) and ataxia telangiectasia (A-T) have elevated frequencies of mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus. We have analysed the DNA sequence of the hprt gene in mutants from normal donors, and compared them with mutants from XP and A-T individuals. In normal donors we found a range of mutations including principally transitions (40%), transversions (32%) and small deletions (20%). In an excision-deficient XP donor from complementation group C the mutation spectrum was similar to that from normal donors, whereas in an XP variant there was a significantly higher frequency (44%) of small deletions. In the two A-T donors, there was a high frequency of large deletions (22 and 75%) compared with only 4% in normal donors.

Mutant sequences in the rpsL gene of Escherichia coli B/r: mechanistic implications for spontaneous and ultraviolet light mutagenesis.

Mutants able to grow in the presence of 1.2 mg/ml streptomycin were isolated from Escherichia coli WP2 after exposure to ultraviolet light (UV) or in the absence of any treatment (spontaneous), and from a umuC derivative after exposure to UV and delayed photoreversal. These mutants, characterized as streptomycin resistant (Smr) or dependent (Smd), carry mutations in the rpsL gene. This gene was amplified using the polymerase chain reaction and sequenced. Mutations induced by UV were largely (76%) of the Smr phenotype, all of which were changes at an A:T base pair at codons 42 or 87. Mutations induced by UV plus delayed photoreversal in the non-UV-mutable umuC122 derivative of WP2 were exclusively of the Smd phenotype and all occurred at G:C base pairs at codons 41, 90 or 91. These results are consistent with current understanding of the mechanism of mutagenesis by UV and delayed photoreversal. A broader spectrum of mutations was seen in the spontaneous series including three-base deletions leading to amino acid loss (2 of codon 93, 1 of codon 87). Of particular note was the number of intragenic second site mutations in the spontaneous series, most if not all of which appeared to be silent with respect to streptomycin phenotype. It is necessary to postulate a high rate of formation of such mutations at some stage during the experiment. One possibility is that spontaneous mutation may often occur in bursts when an error correction mechanism (eg., proofreading, mismatch correction) is temporarily inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.

A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA.