Jan Pontén

A High Frequency of Sequence Alterations Is Due to Formalin Fixation of Archival Specimens

Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.

Consistent high viral load of human papillomavirus 16 and risk of cervical carcinoma in situ: a nested case-control study

BACKGROUND Persistent infection with certain types of human papillomavirus (HPV) is believed to be a prerequisite for the development of cervical neoplasia. Persistence may depend on certain characteristics, such as viral load, which has so far been given little attention. We investigated the association between HPV 16 viral load and cervical carcinoma in situ. METHODS We did a nested case-control study of women participating in cytological screening in Sweden. We used a sensitive quantitative PCR assay to estimate HPV 16 load in multiple smears for each woman, taken during a period of up to 26 years before diagnosis. We calculated C, values, which decrease as the number of viral DNA copies increases. FINDINGS 2081 smears from 478 cases and 1754 smears from 608 controls were tested. Among cases, we found a consistently increased load of HPV 16 already 13 years or more before diagnosis, and when many smears were still cytologically normal. Women with high HPV 16 viral loads were at least 30 times the relative risk of HPV-16-negative women more than a decade before diagnosis. The increase in relative risk was constant over time. About 25% of women (95% CI 0.12-0.32) infected with a high viral load before age 25 years developed cervical carcinoma in situ within 15 years. INTERPRETATION Cervical carcinoma in situ associated with HPV 16 occurs mainly in HPV-16-positive women who have consistently high viral loads long term. Women at high risk could be identified by use of a quantitative HPV test in addition to cytological screening.

International incidence rates of invasive cervical cancer after introduction of cytological screening

Because Pap-smear screening can detect pre-invasive cervical cancer,such screening can markedly reduce the occurrence of invasive cancer.However, its impact in different populations is uncertain. This studycompares the changes in cervical cancer incidence at different ages after theintroduction of screening in different populations, and addresses the impactof organized and opportunistic smear taking. We identified 17 cancerregistries large enough and existing long enough to analyze screeningeffects. For each registry, we calculated the relative reduction inage-specific incidence rates and in incidence rates age-standardized to theworld population after the introduction of cytologic screening. In 11 of the17 populations, age-standardized incidence rates declined markedly from 27percent in Norway and to 77 percent in Finland. Age-specific declines wereconfined to women aged 30 to 70 years old with a nadir around ages 40 to 55.In six other populations, age-standardize d incidence rates declined lessthan 25 percent, an amount too small to provide unambiguous evidence of ascreening effect. In several populations, cytologic screening had a morepronounced effect than is generally recognized. Because age-specific declinesin cervical cancer incidence rates were strikingly similar in populationswith widely different screening practices, organized screening may not bemarkedly superior to opportunistic screening. The reduction in reportedcancer incidence because of screening is smaller in younger and older women.

The division cycle and RNA-synthesis in diploid human cells at different passage levels in vitro

Abstract The division cycle and RNA synthesis were analysed in early and late passage cells of a human fibroblastic strain (WI-38). Cells were exposed to tritium labeled thymidine or uridine. Cultures after the 40th passage were found to be very heterogeneous regarding the length of the division cycle. The fraction of cells involved in DNA synthesis after a short pulse was smaller and the generation time was prolonged in late passage cells. Evidence is presented that the cause for a prolonged generation time was mainy due to retardation in the G1 and G2 periods. The rate of synthesis of RNA was found to be decreased in late passage cells. However, the pattern of incorporation of labeled precursor was the same in both types of cells and active RNA synthesis occurred in 100 per cent of the phase III cells.

Inhibition of the division cycle in confluent cultures of human fibroblasts in vitro

Abstract DNA synthesis was studied in a strain of human embryonic fibroblasts at different passage levels in vitro, between the subcultivation and the post-logarithmic resting stage. The cultures were labeled with 3H-TdR and DNA-synthesis was measured with autoradiographic techniques. The percentage of labeled interphases was found to decrease when the cell counts increased. This event was simultaneous with the appearance of an increasing number of cells blocked in the G2 period. This block was reversible by seeding the cultures at a lower cell density. In 48th passage cultures most cells that synthesized DNA after subcultivation did not divide during that passage.

Smoking and oral contraceptives as risk factors for cervical carcinoma in situ.

Human papillomavirus (HPV) is probably a necessary but definitely not a sufficient cause of cervical carcinoma. However, it remains unclear which factors, in addition to HPV, are important for the development of cervical carcinoma and its precursor lesions. To address this issue, we conducted a case‐control study nested in a population‐based cohort consisting of women participating in cytological screening in one Swedish county, any time during 1969 through 1995. Detailed information on sexual practice, smoking habits and oral contraceptive (OC) use were collected through telephone interviews with 422 case patients diagnosed with cervical carcinoma in situ and 422 control subjects. All cytological smears were analyzed for presence of HPV16/18 by a polymerase chain reaction (PCR)‐based method. Odds ratios (OR) were used as measures of relative risk. After multivariate adjustment, a 2‐fold higher risk was observed among current smokers compared with never smokers [OR 1.94; 95% confidence interval (CI 1.32–2.85)], an association apparently confined to women younger than 45 years. Current use of OCs was associated with a 4‐fold increased risk overall (OR 3.64; 95% CI 1.91–6.93) with a monotonic increase with increasing duration of use (p for trend < 0.001). The number of sexual partners was significantly, positively associated with risk among HPV 16/18‐negative (p for trend < 0.005) but not among HPV 16/18‐positive women. Our data confirm the association between smoking and cervical carcinoma in situ, which might be age‐dependent. Our results further indicate a relation with OC use and the risk for cervical carcinoma in situ. Int. J. Cancer 81:357–365, 1999.

Molecular pathology in basal cell cancer with p53 as a genetic marker.

Human basal cell cancer (BCC) has unique growth characteristics with virtual inability to metastasize. We investigated clonality and genetic progression using p53 mutations as marker. Sampling was done through microdissection of frozen immunohistochemically stained 16 μm slices of tumors. From 11 BCC tumors 78 samples were analysed. Direct DNA sequencing of exons 5 – 8 was performed, haplotypes were determined after cloning of p53 exons and loss of heterozygosity (LOH) ascertained by microsatellite analysis. All tumors had p53 mutations and in a majority both p53 alleles were affected, commonly through missense mutations. Microdissection of small parts (50 – 100 cells) of individual tumors showed BCC to be composed of a dominant cell clone and prone to genetic progression with appearance of subclones with a second and even third p53 mutation. Samples from normal immunohistochemically negative epidermis always showed wild type sequence, except for a case of previously unknown germline p53 mutation. Our analysis also included p53 immunoreactive patches i.e. morphologically normal epidermis with a compact pattern of p53 immunoreactivity. Mutations within those were never the same as in the adjacent BCC. This detailed study of only one gene thus uncovered a remarkable heterogeneity within a tumor category famous for its benign clinical behavior.

Regulation of proliferation and movement of human glialike cells in culture

Abstract The control of DNA synthesis and locomotion have been investigated in vitro in serially propagated astrocyte-like lines from normal adult human brain. DNA synthesis was measured autoradiographically as the percentage of cells showing uptake of tritiated thymidine during a 24 h period. In the period after subcultivation when the cell number increased rapidly, about 40–80% of the cells participated in DNA synthesis. After 10–15 days when the number of cells had stabilized at a level of about 60,000/cm 2 , the percentage of labeled cells dropped to 1% or less, indicating strict control of DNA synthesis in a complete monolayer. Addition of fresh medium caused a slight temporary increase of DNA synthesis in stationary layers. Analysis of the length of the cell cycle periods gave the following values: T, 22 h, G1, 8.5 h, S, 8 h, G2, 4.5 h, M, 1 h. There was no evidence for division by amitosis. Cell locomotion was analysed by time-lapse cinematography. Glia cells were mobile in a sparse culture, but had a tendency to form adhesions along areas of mutual contact. These were not always sufficiently strong to prevent cytoplasmic and even nuclear overlapping. When a complete monolayer had formed, the cells were essentially immobile and no ruffled membranes were seen. The results indicate that adult human glia cells have unusually well developed growth control mechanisms in vitro and should lend themselves well to the experimental study of such controls.

Perinatal characteristics in relation to incidence of and mortality from prostate cancer.

Abstract Objective: To test the hypothesis that factors causing morbidity and mortality from prostate cancer may operate in utero. Design: Matched case-control study of singleton men born between 1874 and 1946 at one hospital. Setting: Uppsala University Hospital. Subjects: 250 patients with prostate cancer and 691 controls, including 80 patients who died from prostate cancer and their 196 matched controls. Main outcome measures: Mothers age at menarche, parity, pre-eclampsia or eclampsia before delivery, age at delivery and socioeconomic status; case or controls birth length and weight, placental weight, prematurity derived from gestational age, and presence of jaundice. Results: Both pre-eclampsia (odds ratio 0, 95% confidence interval 0 to 0.71) and prematurity (0.31, 0.09 to 1.04) were inversely associated with incidence of prostate cancer. Among subjects born full term, placental weight, birth weight, and ponderal index (weight/height*RF 3*) showed non-significant positive associations with prostate cancer incidence, and stronger associations with mortality. Conclusion: Prenatal exposures that are likely correlates of pregnancy hormones and other growth factors are important in prostate carcinogenesis and influence the natural course as well as the occurrence of this cancer. Key messages Recent evidence suggests that events in utero may affect the risk of prostate cancer later in life In this study 250 men born since 1874 at one hospital and who developed prostate cancer were identified and compared with 691 controls Pre-eclampsia and prematurity were associated with a reduced risk of prostate cancer and ponderal index was associated with an increased risk of mortality from prostate cancer Prenatal exposures that are likely correlates of pregnancy hormones and other growth factors seem to be of importance in the prostate carcinogenesis

Residual bodies and “aging” in cultured human glia cells: Effect of entrance into phase III and prolonged periods of confluence☆

Abstract Normal cells in vitro show progressive loading with secondary lysosomes of the residual body variety as they approach the end of their life span. The present investigation was undertaken to study the mechanism(s) responsible for this accumulation. In one series of experiments the ultrastructure of (‘young’) phase II cells kept in confluence for long periods of time was comared with that of (‘old’) cells brought into phase III by repeated subcultivation. Great numbers of large, irregularly shaped residual bodies were found both in cells which did not divide because they were in phase III and in phase II cells kept in stationary phase. In the former system, a conspicuous occurrence of residual bodies was first observed when the cells approached phase III while there was a continuous accumulation of such organelles in the contact-inhibited cells. These findings suggest that the accumulation of residual bodies in phase III cells is more a function of insufficient dilution due to limited potential for division than to impaired metabolism related to intracellular damage. In a second experiment, clones of cells were allowed to grow until the peripheral cells approached phase III (about 2 months). Following release of contact inhibition, the central (contact-inhibited) cells retained their capacity for mitosis. Hence, it was concluded that the quantity of residual bodies present after this time did not abolish the ability for mitosis and, further, that ‘metabolic time’ is less important than the number of mitoses in order to reach phase III. The electronmicroscopic findings indicated that the residual bodies resulted from cellular autophagocytosis.