A randomized phase II trial of MR-guided prostate stereotactic body radiotherapy administered in 5 or 2 fractions for localized prostate cancer (FORT).

BACKGROUND: Ultra-hypofractionated regimens for definitive prostate cancer (PCa) radiotherapy are increasingly utilized due in part to promising safety and efficacy data complemented by greater patient convenience from a treatment course requiring fewer sessions. As such, stereotactic body radiation therapy (SBRT) is rapidly emerging as a standard definitive treatment option for patients with localized PCa. The commercially available magnetic resonance linear accelerator (MR-LINAC) integrates MR imaging with radiation delivery, providing several theoretical advantages compared to computed tomography (CT)-guided radiotherapy. MR-LINAC technology facilitates improved visualization of the prostate, real-time intrafraction tracking of prostate and organs-at-risk (OAR), and online adaptive planning to account for target movement and anatomical changes. These features enable reduced treatment volume margins and improved sparing of surrounding OAR. The theoretical advantages of MR-guided radiotherapy (MRgRT) have recently been shown to significantly reduce rates of acute grade ≥ 2 GU toxicities as reported in the prospective randomized phase III MIRAGE trial, which compared MR-LINAC vs CT-based 5 fraction SBRT in patients with localized PCa (Kishan et al. JAMA Oncol 9:365-373, 2023). Thus, MR-LINAC SBRT-utilizing potentially fewer treatments-is warranted and clinically relevant for men with low or intermediate risk PCa electing for radiotherapy as definitive treatment. METHODS/DESIGN: A total of 136 men with treatment naïve low or intermediate risk PCa will be randomized in a 1:1 ratio to 5 or 2 fractions of MR-guided SBRT using permuted block randomization. Randomization is stratified by baseline Expanded PCa Index Composite (EPIC) bowel and urinary domain scores. Patients undergoing 5 fractions will receive 37.5 Gy to the prostate over 10-14 days and patients undergoing 2 fractions will receive 25 Gy to the prostate over 7-10 days. The co-primary endpoints are GI and GU toxicities as measured by change scores in the bowel and urinary EPIC domains, respectively. The change scores will be calculated as pre-treatment (baseline) score subtracted from the 2-year score. DISCUSSION: FORT is an international, multi-institutional prospective randomized phase II trial evaluating whether MR-guided SBRT delivered in 2 fractions versus 5 fractions is non-inferior from a gastrointestinal (GI) and genitourinary (GU) toxicity standpoint at 2 years post-treatment in men with low or intermediate risk PCa. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04984343 . Date of registration: July 30, 2021. PROTOCOL VERSION: 4.0, Nov 8, 2022.

Absolute Percentage of Pattern 4 Disease as a Prognostic Measure for Intermediate-risk Prostate Cancer Treated with Stereotactic Body Radiotherapy.

AIMS: Intermediate-risk prostate cancer is heterogenous. The absolute percentage of biopsied tissue positive for Gleason pattern 4 disease (APP4) is a possible prognostic measure. Here we sought to determine the impact of APP4 in a prospective multi-institutional pooled analysis of men with intermediate-risk prostate cancer treated with stereotactic body radiotherapy (SBRT). MATERIALS AND METHODS: Patients with intermediate-risk prostate cancer treated with SBRT (40 Gy in five fractions or 26 Gy in two fractions) with or without androgen deprivation therapy treated on prospective clinical trials were included. Pathology reports were queried to obtain APP4, calculated as the percentage of Gleason pattern 4 disease within the tumour(s) multiplied by the percentage of total biopsied tissue positive for disease divided by 100. The optimal APP4 cut-off points for biochemical failure and distant metastasis were calculated and used as a stratification in the cumulative incidence of biochemical failure and distant metastasis. Multivariable competing risk models were developed. RESULTS: In tota, 227 patients were included. The median follow-up was 56.5 months. The optimal APP4 cut-off points were 5% for biochemical failure and 20% for distant metastasis. At 4 years, the cumulative incidence of biochemical failure was 23.6% and 2.3% for APP4 >5% and ≤ 5%, respectively (P < 0.0001). The cumulative incidence of distant metastasis was 12.5% for APP4 >20% and 1% for APP4 ≤ 20% (P = 0.02). APP4 sub-stratified favourable intermediate-risk prostate cancer and unfavourable intermediate-risk prostate cancer into groups at similarly low and similarly high risk of biochemical failure and distant metastasis. On multivariable competing risk analysis, APP4 >5% (P = 0.0004) was significantly associated with biochemical failure, but APP4 (log) was not for distant metastasis (P = 0.08). CONCLUSION: APP4 may be an easily accessible promising prognostic measure for patients with intermediate-risk prostate cancer treated with SBRT. Incorporation of APP4 into prospective trials will help to determine its value.

Four-year Prostate-specific Antigen Response Rate as a Predictive Measure in Intermediate-risk Prostate Cancer Treated With Ablative Therapies: The SPRAT Analysis.

AIMS: There is a lack of early predictive measures of outcome for patients with intermediate-risk prostate cancer (PCa) treated with stereotactic body radiotherapy (SBRT). The aim of the present study was to explore 4-year prostate-specific antigen response rate (4yPSARR) as an early predictive measure. MATERIALS AND METHODS: Individual patient data from six institutions for patients with intermediate-risk PCa treated with SBRT between 2006 and 2016 with a 4-year (42-54 months) PSA available were analysed. Cumulative incidences of biochemical failure and metastasis were calculated using Nelson-Aalen estimates and overall survival was calculated using the Kaplan-Meier method. Biochemical failure-free survival was analysed according to 4yPSARR, with groups dichotomised based on PSA <0.4 ng/ml or ≥0.4 ng/ml and compared using the Log-rank test. A multivariable competing risk analysis was carried out to predict for biochemical failure and the development of metastases. RESULTS: Six hundred and thirty-seven patients were included, including 424 (67%) with favourable and 213 (33%) with unfavourable intermediate-risk disease. The median follow-up was 6.2 years (interquartile range 4.9-7.9). The cumulative incidence of biochemical failure and metastasis was 7 and 0.6%, respectively; overall survival at 6 years was 97%. The cumulative incidence of biochemical failure at 6 years if 4yPSARR <0.4 ng/ml was 1.7% compared with 27% if 4yPSARR ≥0.4 ng/ml (P < 0.0001). On multivariable competing risk analysis, 4yPSARR was a statistically significant predictor of biochemical failure-free survival (subdistribution hazard ratio 15.3, 95% confidence interval 7.5-31.3, P < 0.001) and metastasis-free survival (subdistribution hazard ratio 31.2, 95% confidence interval 3.1-311.6, P = 0.003). CONCLUSION: 4yPSARR is an encouraging early predictor of outcome in patients with intermediate-risk PCa treated with SBRT. Validation in prospective trials is warranted.

MRI-guided radiotherapy for head and neck cancer: initial clinical experience.

PURPOSE: To report a single-institutional experience with the use of magnetic resonance imaging (MRI)-guided radiotherapy for cancers of the head and neck. MATERIALS AND METHODS: Between October 2014 and October 2016, 18 patients with newly diagnosed cancers of the head and neck were prospectively enrolled on an institutional registry trial investigating the feasibility and efficacy of external-beam radiotherapy delivered using on-board MRI. All patients had biopsy-proven evidence of malignancy, measurable disease, and the ability to provide consent. None had previously received any treatment. Median dose was 70 Gy (range 54-70 Gy). MRI scans were obtained as part of an image-guided registration protocol for alignment prior to and during each treatment. Concurrent chemotherapy was administered to 14 patients (78%). Patient-reported outcomes were assessed using the University of Washington quality of life instrument. RESULTS: Seventeen of 18 patients completed the planned intensity-modulated radiotherapy (IMRT) treatment of which 15 (83%) had a complete response and 2 (11%) had a partial response based on initial post-therapy positron emission tomography (PET) at 3 months. The 1-year estimates of progression-free survival, overall survival, and local-regional control were 95, 96, and 95%, respectively. There were no treatment-related fatalities. The incidence of grade 3+ acute toxicity was 44%. The proportion of patients rating their health-related quality of life as "very good" or "outstanding" at 6 months and 1 year after completion of radiation therapy was 60 and 70%, respectively. CONCLUSIONS: MRI-guided radiotherapy achieves clinical outcomes comparable to contemporary series reporting on IMRT for head and neck cancer.

Establishing the distribution of satellite lesions in intermediate- and high-risk prostate cancer: implications for focused radiotherapy.

BACKGROUND: In focused radiotherapy for prostate cancer (PC), a full dose of radiation is delivered to the index lesion while reduced dose is delivered to the remaining prostate to reduce morbidity. As PC is commonly multifocal, we investigated whether baseline clinical characteristics or multiparametric magnetic resonance imaging (mpMRI) may be useful to predict the actual pathologic distribution of PC in men with intermediate- or high-risk PC, which may better inform how to deliver focused radiotherapy. METHODS: A retrospective single-institutional study was performed on 71 consecutive men with clinically localized, intermediate- or high-risk PC who underwent mpMRI followed by radical prostatectomy (RP) from January 2012 to December 2012. Logistic regression analysis was performed to evaluate preoperative predictors for satellite lesions. Performance characteristics of mpMRI to detect satellite lesions and the extent of prostate disease (one hemi-gland vs both) were also evaluated. RESULTS: In all, 50.7% had satellite lesions on mpMRI. On RP specimen analysis, 66.2% had satellite lesions and 55.3% of these satellite lesions had pathologic Gleason score (pGS)⩾3+4. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for mpMRI detecting a satellite lesion being present in the RP specimen were 59.6%, 66.7%, 77.8%, 45.7% and 62.0%, respectively. The presence of MRI satellite lesions was the only preoperative predictor significantly associated with finding satellite lesions on final pathology (hazard ratio (HR), 2.95, P=0.040). There was agreement in 76.1% of the entire cohort for unilateral vs bilateral disease when incorporating both biopsy and mpMRI information and comparing with the RP specimen. CONCLUSIONS: In intermediate risk or greater PC, only the presence of mpMRI satellite lesions could predict for pathologic satellite lesions. While combining biopsy and mpMRI information may improve preoperative disease localization, the relatively high incidence of bilateral hemi-gland involvement with pGS ⩾7 satellite lesions makes it challenging to appropriately select men eligible for hemi-gland therapy.

Effects of 12-O-tetradecanoylphorbol-13-acetate on the differentiation of simian virus 40-infected human keratinocytes.

We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on the differentiation of human epidermal keratinocytes infected by the oncogenic virus Simian Virus 40. The cells in monolayers exposed to a concentration of 12-O-tetradecanoylphorbol-13-acetate as low as 5 X 10(-10) M redistributed into dense patches within 48 hr. This morphological change was accompanied by a 10- to 30-fold increase in the production of cells cytochemically stained by orange G:acid fuchsin indicating keratinization and a 2- to 3-fold increase in the exfoliation of cornified envelope-bearing cells. The induced cellular differentiation occurred in parallel with an inhibition of cell growth. The biologically inactive congener 4 alpha-phorbol-12,13-didecanoate at equimolar concentrations did not affect the growth or differentiation of these cells. Binding studies using [3H]12-O-tetradecanoylphorbol-13-acetate revealed a binding site with characteristics similar to those found for other cell types (Kd = 17 nM; 1.25 X 10(5) available sites/cell).

Simple epithelial nature of some simian virus-40-transformed human epidermal keratinocytes.

Previous studies have indicated that some Simian-virus-40-transformed human epidermal keratinocytes (SV40-HE) undergo significant changes in their growth and differentiated properties. To better understand the significance of these changes, we have characterized the keratins of SV40-HE cells by one- and two-dimensional immunoblot analysis using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that our SV40-HE cells have lost the Mr 58,000 (No. 5), Mr 56,000 (No. 6), Mr 50,000 (No. 14/15), Mr 48,000 (No. 16), and Mr 46,000 (No. 17) keratins that are expressed by cultured normal human keratinocytes. Instead, these cells express mainly Mr 52,000 (No. 8), Mr 45,000 (No. 18), and Mr 40,000 (No. 19) keratins, a set highly characteristic of simple epithelial cells. Furthermore, our SV40-HE cells have ceased to express involucrin, another marker for keratinocytes, and have a greatly diminished ability to undergo in vitro stratification. These results suggest that epidermal cells can sometimes lose their keratinocyte features as a consequence of viral transformation. This finding may have important implications regarding the mechanisms of epithelial differentiation and tumorigenesis and in the use of keratinocyte markers for tumor diagnosis.

Theophylline incorporation into the nucleic acids of theophylline-stimulated melanoma cells.

Theophyllin, an inhibitor of cAMP-degrading phosphodiesterase, stimulates melanin biosynthesis in cultures of RPMI 3460 hamster melanoma cells. Although theophylline does produce an initial transient elevation of intracellular cAMP levels, long-term treatment with theophylline produces a significant decrease in cAMP content. There is an inhibition of the theophylline stimulation by dibutyryl-cAMP; this is apparently caused by interference of dibutyryl-cAMP with the uptake and incorporation of theophylline, as shown by experiments with 3H-theophylline. An alternative theory is that theophylline, being a methylxanthine compound, is metabolized by the cell and possibly causes melanotic stimulation by becoming incorporated into cellular nucleic acids or by altering the normal nucleic acid metabolism. The following observations are consistent with this theory: (u) 3H-theophylline was incorporated into both trichloroacetic acid (TCA)-soluble and TCA-insoluble cell fractions; most of the insoluble label became soluble after digestion with ribonuclease and deoxyribonuclease. (2) These nuclease digests of the 3H-theophylline-labeled TCA-insoluble cell fractions contained 3H-labeled material that chromatographed differently from normal nucleotides on ion exchange thin layer sheets. (3) The acid-soluble pool of 3H label disappeared rapidly while both the insoluble label and the induction of melanogenesis remained stable for 50 hr after the removal of exogenous 3H-theophylline.

Transformation and immortalization of human keratinocytes by SV40.

We have studied the appearance of transformed properties following infection of human epidermal keratinocytes by the oncogenic virus SV40. Shortly after infection, only a small fraction of the cells are positive for SV40 T antigen by immunofluorescence; this fraction progressively increases upon serial subcultivation concomitant with an increase in plating/colony-forming efficiency and growth rate. The capacity of the cells to differentiate progressively decreases, as indicated by cytochemical staining and cornified cell-envelope formation induced by suspension in methyl cellulose. The infected cells enter a period of growth crisis characterized by cytopathology and cell death as the level of T antigen synthesis reaches about 90 percent positive cells at about the tenth serial passage. Viable cells emerging from the crisis period are found to exhibit anchorage-independent growth, as indicated by the formation of viable colonies in semisolid media, but there is considerable variability in colony formation among clones isolated from anchorage-independent populations. The emergent population also manifests phenotypic instability in terms of the appearance of variants, which, in contrast to uninfected cells, expresses a well-defined actin cytoskeleton. The infected cells eventually become "immortalized," as evidenced by an indefinite lifespan, i.e., replication capability maintained well beyond the ordinary time of senescence for uninfected cells. We present these findings in the context of a stage-specific model of epithelial transformation in vitro.

Expression of pemphigoid antigen by SV40-transformed human keratinocytes.

The expression and properties of pemphigoid antigen of SV40-transformed human keratinocytes were studied. By indirect immunofluorescence, SV40-transformed keratinocytes in passage 80-85 expressed the pemphigoid antigen as coarsely granular perinuclear fluorescence. To characterize this antigen, NP40 extracts of cells labeled with [14C]amino acids were immunoprecipitated using sera of 8 patients: bullous pemphigoid (6 patients), chronic localized pemphigoid (1 patient), and drug-induced lichen planus pemphigoides (1 patient). These immunoprecipitates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then fluorographed. All 8 sera precipitated a protein of Mr 240K, while normal human sera did not precipitate this protein. These results indicate that SV40-transformed human keratinocytes synthesize pemphigoid antigen, and that autoantibodies in the sera of pemphigoid patients with different clinical features identify the same antigen of Mr 240K in these cells.

SSA/Ro antigen expression in simian virus 40-transformed human keratinocytes.

SSA/Ro antigen is a soluble cellular component to which antibodies are frequently produced in patients with Sjögren's syndrome and systemic lupus erythematosus. Its exact location within the cell has yet to be determined. In this study we report the expression of SSA/Ro antigen in simian virus 40 (SV40)-transformed keratinocytes. The locations of SSA/Ro, U1RNP, and DNA antigens were studied by indirect immunofluorescence using monospecific antibodies. SSA/Ro antigen was detected in both the nucleus and cytoplasm of SV40-transformed keratinocytes tested with three monospecific sera. Primary cultured keratinocytes derived from adult human skin showed localized immunofluorescent staining within the nucleus. When Ca++ concentration of the medium was switched to 0.05 mM, these cells expressed cytoplasmic SSA/Ro antigens within 48 h. Depletion of the antibody activity with insolubilized human spleen extract abolished the staining. Surface expression of this antigen could not be detected in either primary or transformed cells. Localization of U1RNP and DNA was not altered. These results indicate that expression of SSA/Ro antigen in human keratinocytes is modulated by SV40 infection and that this antigen is expressed to a greater degree in cells that are less differentiated, transformed, or proliferating.

NM1 keratinocyte line is cytogenetically and biologically stable and exhibits a unique structural protein.

We have previously described a human keratinocyte line, NM1, which had been carried for more than 400 doublings, was trisomic for chromosome 8, and appeared to make a number of structural proteins characteristic of keratinocytes. This line has now been carried for more than 800 doublings and grows with the same vigor. It reaches confluence in 7 to 10 days and can be grown without a feeder layer for more than 15 passages. Its karyotype has remained 47,XY, +8. The current NM1 cells make readily detectable amounts of 67 kd and 48 kd keratins, and it has been established that the previously poorly resolved 58 kd band actually consists of 58 kd and 59 kd bands. We have also found that the apparent 56 kd band consists of the 56 kd and 56.5 kd bands. A unique basic polypeptide precursor of the cornified envelope has been discovered in the NM1 line. Although similar in charge to one in normal cells it is lower in molecular weight.

Expression of proliferating cell nuclear antigen/cyclin in human keratinocytes.

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, in human keratinocytes was examined by using the serum obtained from a SLE patient and a murine monoclonal antibody against PCNA/cyclin. In the normal epidermis, few of the nuclei were labeled with anti-PCNA/cyclin. This was in contrast to the positive nuclear staining seen in active lesions of psoriasis. In a primary culture of human keratinocytes growing as a monolayer, 20%-30% of cells expressed PCNA/cyclin. SV40-transformed human keratinocytes showed positive nuclear staining in about 40% of the cell population. In stratified keratinocytes cultured in a high Ca++ medium, PCNA/cyclin expression was decreased and only the cells in the basal and suprabasal layers showed positive staining. These results indicate that the expression of PCNA/cyclin correlates with the proliferating state in human keratinocytes and may not be associated with the mechanism of differentiation in keratinocytes.

Integration of the SV40 promoter/enhancer sequences in an anchorage-independent clonal subline of SV40-infected human keratinocytes.

Long-term cultures of SV40-infected human keratinocytes contain integrated, but structurally altered copies of the viral sequences. The presence of these sequences is required for expression of properties associated with the transformed phenotype including immortalization. The integrated viral sequences in an anchorage-independent line of viral-transformed human keratinocytes have been found to be contained on two BamHI fragments of about 6.9 and 5.2 kb. In the larger fragment the viral sequences were present as two tandemly repeated subgenomic fragments containing the viral origin/promoter region with nucleotide alterations that affect enhancer function, the origin of replication and T antigen binding site 1. The viral early gene promoter in one integrant gave rise to an unspliced fusion transcript comprised of a short portion of the viral early gene leader sequence and the flanking human sequences. The smaller fragment consisted of full-length SV40 containing a nine-nucleotide insertion in the C-terminal portion of the SV40 T antigen, a region involved in the regulation of viral host range.

Characterization of a 1.8-kb DNA segment located upstream from the human Ha-ras protooncogene and possibly regulating its function.

We have cloned a 1.8-kb segment of DNA just adjacent to the 5' end of the 6.4-kb BamHI fragment which contains the Ha-ras oncogene (ras1; GenBank notation HUMRASH). Electrophoretic mobility shift assays (EMSA) indicate the presence of multiple nuclear protein-binding sites within the 1.8-kb segment. At least three putative regulatory protein-binding sites have been identified in a 206-bp subfragment by DNase I footprint analysis. Within the subfragment containing the DNase-protected regions, there is also a segment containing a number of consensus sequences for DNA-binding proteins and two sub-sequences that exhibit strong sequence homology to at least two previously characterized enhancers. These data suggest that a novel set of regulatory elements may lie as far as 2 kb upstream from the normal Ha-ras transcription start points.

Posttreatment follow-up of radiation oncology patients in a managed care environment.

PURPOSE: Health care delivery in the United States is in the midst of a structural revolution called managed care. Demands for cost control within the managed care environment force radiation oncologists to defend the need and obligation to follow their patients. METHODS AND MATERIALS: We have analyzed this follow-up requirement from six potential justifications: patient care, medical-legal, quality assurance, outcome measurement, cost, and improvement of care. RESULTS: Practical recommendations for discussing the need for follow-up with the medical directors and primary care physicians of managed care entities are given. Follow-up without valid documentation of benefit is hard to justify in this era of managed care. CONCLUSIONS: Collaborative follow-up between the referring physician, the treating radiation oncologist, and the other oncologic specialists will allow for outcome measurement and improvement in practice without driving up cost or exposing the patient to undue risk.

Enhancement of PCRs by partial restriction digestion of genomic templates.

Genomic DNA preparations derived from mammalian cells can often exhibit poor template activity in PCR, particularly when carried out on target sequences present at low copy number. Using genomic DNA bearing SV40 sequences integrated into host chromosomal DNA at low copy number as a target, we show that template efficiency can be dramatically enhanced after treatment of the genomic template with restriction enzymes for varying periods of time. Also, our results indicate that, while template activity was enhanced by all of the restriction enzymes tested, optimal digestion time varied for each enzyme.

Synthesis of epidermolysis bullosa acquisita antigen by simian virus 40-transformed human keratinocytes.

The synthesis of epidermolysis bullosa acquisita (EBA) antigen in simian virus 40 (SV40)-transformed human epidermal keratinocytes was studied. Indirect immunofluorescent staining of SV40-transformed keratinocytes employing a serum sample from an EBA patient as a source of antibodies decorated EBA antigen as a perinuclear granular fluorescence. This staining pattern was similar to that of nontransformed epidermal keratinocytes grown in a low Ca2+ medium. In contrast, stratified primary cultures of keratinocytes stained after growth in a high Ca2+ medium showed only small amounts of the antigen localized in the substrate-attached basal cells. To demonstrate biosynthesis of the EBA antigen by SV40-transformed keratinocytes, cells were metabolically labeled with 14C-amino acids and the cell lysates were immunoprecipitated with EBA antiserum. Analysis of immunoprecipitates by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography revealed that the EBA serum precipitated a protein with an apparent molecular weight of 290 kD from extracts of these cells. These results indicate that SV40 induces the synthesis of the 290 kD EBA antigen. Expression of this antigen may be a general feature of nonstratifying, proliferating epidermal cells.

Effectiveness of screening instruments in detecting substance use disorders among prisoners.

This study examined the effectiveness of several screening instruments in detecting substance use disorders among prison inmates. A sample of 400 male inmates were administered eight different substance abuse screening instruments and the Structured Clinical Interview for DSM-IV (SCID-IV), Version 2.0, Substance Abuse Disorders module. The latter was used as a diagnostic criterion measure to determine the presence of substance use disorders. Based on positive predictive value, sensitivity, and overall accuracy, the Texas Christian University Drug Screen, the Simple Screening Instrument, and a combined instrument-Alcohol Dependence Scale/Addiction Severity Index-Drug Use section were found to be the most effective in identifying substance abuse and dependence disorders.

Proliferating cell nuclear antigen/cyclin in cultured human keratinocytes.

Expression of proliferating cell nuclear antigen (PCNA)/cyclin in cultured human keratinocytes was studied using an antibody from an SLE patient as the reagent. By indirect immunofluorescence staining, SV40-transformed human keratinocytes expressed PCNA/cyclin in 40-45% of the cells as a nulcear granular fluorescence. After synchronization of these cells, their nuclear distribution pattern during the S phase was sequential and showed a clear correlation with DNA synthesis. Primary cultured keratinocytes grown in high Ca+ medium expressed PCNA/cyclin in 10-15% of the cells with a similar staining pattern. These positively stained cells were confined to the basal and immediate suprabasal layers of the stratified culture sheet. The keratinocytes disaggregated by trypsin were separated according to cell size through a screen of Nitex monofilament cloth. The cells smaller than 15 microns in diameter synthesized abundant PCNA/cyclin, while the larger cells expressed very low levels. These results indicate that the expression of PCNA/cyclin correlates with DNA synthesis in cultured keratinocytes, but is not associated with their differentiation process.