Magnus Thörn

FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans

Background Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs. Methodology/Principal Findings Human CD4+CD25hi Tregs displayed a demethylated FOXP3 promoter (1.4%±0.95% SEM methylated) in contrast to CD4+CD25lo T cells which were partially methylated (27.9%±7.1%). Furthermore, stimulated CD4+CD25lo T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-β and/or IL-10 does not induce any additional change in methylation level. Conclusions/Significance The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3+ activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

Lymphatic mapping and detection of sentinel nodes in patients with bladder cancer.

PURPOSE We examined the possibility for detecting sentinel nodes in patients with bladder cancer and whether the histopathological status of identified sentinel nodes reflected that of the lymphatic field. MATERIALS AND METHODS A total of 13 patients with bladder cancer who met the criteria qualifying them for radical cystectomy had intravesical injections of radioactive tracer and blue dye marker around the tumor followed by lymphoscintigraphy to visualize lymphatic drainage and detect sentinel nodes. Sentinel nodes were identified preoperatively by the blue color and increased radioactivity and were compared histopathologically with other routinely excised lymph nodes. RESULTS Sentinel nodes were detected in 85% (11 of 13) of patients. There were 4 patients who had sentinel nodes containing tumor cells, and each metastasis was only seen in the detected sentinel node. There were no false-negative sentinel nodes. Of the metastatic sentinel nodes 3 were located outside the normally excised lymph nodes of the obturator fossa. CONCLUSIONS Sentinel nodes can be detected in patients with bladder cancer. The histopathological status of the identified sentinel nodes was diagnostic for all other excised lymph nodes. Sentinel nodes often seem to be located outside the obturator lymphatic field, which is normally examined during preoperative staging of bladder cancer.

Increased expression of the transcription factors CCAAT-enhancer binding protein-β (C/EBβ) and C/EBPζ (CHOP) correlate with invasiveness of human colorectal cancer

Regulation of cell differentiation is most often impaired in malignant tumors and may represent a key mechanism for the progression of the disease. CCAAT‐enhancer binding protein (C/EBP) is a family of transcription factors involved in the regulation of embryonic gut development in rodents, which has also been detected in various malignancies, e.g., liposarcomas and breast and ovarian epithelial tumors. We studied the relationship between C/EBP and tumor histology (Dukes invasive stage and pathological grade) in colorectal cancer. Immunoblotting techniques were used on microdissected fresh frozen tumor specimens, and expression of C/EBPα, C/EBPβ and C/EBPζ (CHOP) was analyzed in addition to that of the cell‐cycle regulator p53 and the proliferation marker PCNA. Expression of C/EBPβ (LAP isoforms) was markedly increased in all tumors compared with normal colon mucosa. Although the inter‐patient variability was large, we found that LIP, the isoform of C/EBPβ known to inhibit transcription, was expressed at higher levels in Dukes stage B tumors compared with Dukes stage A, whereas Dukes C tumors had the lowest LIP expression. A similar relationship was seen for CHOP. The cell‐cycle regulator gene p53 was the only factor that clearly correlated with pathological grade: a decrease in p53 expression was demonstrated. Our data suggest that genetic and cellular events involving C/EBPβ and CHOP are important for tumor invasion and that these events do not appear to be related to the pathological grade of the tumor. Int. J. Cancer 86:337–343, 2000.

Second primary cancers in patients with squamous cell carcinoma of the skin : A population-based study in Sweden

We studied second primary cancer among 25,947 patients diagnosed with squamous cell carcinoma of the skin (SCC) in Sweden between 1958 and 1992. In total, 5,706 patients developed a second primary cancer at any site, compared with an expected number of 2,651 [standardized incidence ratio (SIR) = 2.15; 95% confidence interval (CI) = 2.10–2.21]. Men below 60 years of age at diagnosis of SCC had higher SIR (2.5; CI = 2.2–2.8) with the highest risk during the first year of follow‐up (SIR = 9.2; CI = 6.9–12.2). If second primary SCC was excluded, the SIR was reduced to 1.30 (CI = 1.25–1.34); the relationships by sex, age and time since diagnosis remained similar. For skin cancer, the SIR for second SCC was markedly elevated (SIR = 15.6) and the risk of malignant melanoma was elevated 3‐fold. Significantly increased risks were found for most second cancers in squamous cell epithelium: lip (SIR = 5.2), respiratory organs (SIR = 1.7), esophagus (SIR = 1.5), cervix uteri (SIR = 2.2), and vulva including vagina (SIR = 2.3). There was a generally increased risk of almost 2‐fold for second cancer in hematopoietic or lymphoproliferative tissues. Slightly increased rates (SIR = 1.0–1.5) were seen for second tumors in digestive tissues. Finally, a high SIR (SIR = 5.5) was observed for second primary cancer in salivary glands. In conclusion, patients with SCC are at increased risk to develop new primary cancer, especially in skin, squamous cell epithelial and tobacco‐related tissues. Common risk factors among the tumor types might explain our findings, however, an intrinsic susceptibility among SCC patients to develop cancer is also possible. Int. J. Cancer 80:511–515, 1999.

Second primary cancers in patients with cutaneous malignant melanoma: a population-based study in Sweden.

To quantify the risk of second primary cancers among patients with cutaneous malignant melanoma, we studied 20,354 patients in the Swedish Cancer Register during 1958-88. A second primary cancer was reported in 1605 patients, compared with an expected number of 1109.5 [standardised incidence ratio (SIR) = 1.45, 95% confidence interval (CI) = 1.38-1.52]. The highest risk was found among patients younger than 60 years. The greatest risk was seen during the first year after diagnosis (SIR = 1.91, CI = 1.69-2.14), but even after long-term follow-up--15 years or more--the risk was still significantly elevated (SIR = 1.56, CI = 1.35-1.79). The strongest association was found for a second primary malignant melanoma (men, SIR = 10.0, CI = 8.26-12.00; women, SIR = 8.66, CI = 7.22-10.30) and non-melanoma skin cancer (men, SIR = 3.58, CI = 2.85-4.44; women, SIR = 2.41, CI = 1.71-3.29). The risk of second cancers associated with tissues of neuroectodermal origin was increased, especially tumours of the nervous system (men, SIR = 1.73, CI = 1.10-2.60; women, SIR = 2.03, CI = 1.45-2.78). The SIR of second cancers involving the immune system was also increased. An excess risk of endometrial cancer was seen (SIR = 1.41, CI = 1.03-1.88), but no significant associations existed for cancers of the breast, ovary, testis or other endocrine glands. Among tumours of the digestive tract, only colon cancer in men had a significantly increased SIR (1.33, CI = 1.00-1.74).

Trends in tumour characteristics and survival of malignant melanoma 1960-84: a population-based study in Sweden.

In Sweden, improvement in survival rates of patients with cutaneous malignant melanoma has counteracted the increase in incidence to produce a moderate rise in mortality. Our aim was to determine the possible impact of drift in diagnostic criteria, earlier diagnosis and changing biological features of the tumours upon trends in survival. We studied a stratified sample of 528 patients diagnosed between 1960 and 1984 in a strictly defined geographical region. No evidence of drift in diagnostic criteria was found. The proportion of patients with invasion level Clark II increased from 3.2% in 1960-64 to 22.5% in 1980-84, the proportion of thin melanomas (< or = 0.75 mm) increased from 9.4% to 31.5% and the tumour thickness decreased significantly between each 5 year period of diagnosis. These changes are most likely the results of earlier diagnosis. However, changes in tumour characteristics have occurred, since the proportion of superficially spreading malignant melanoma increased from 35% in 1960-64 to 51% in 1980-84 and the proportion of acral lentiginous melanoma decreased from 11% to 2%. The proportion of nodular melanomas remained fairly constant. The proportion of tumours with lymphocytic reaction did not change, whereas those with histological regression increased slightly. Proportional hazards analyses showed a significantly lower survival in patients diagnosed in 1960-64 but no apparent trend after 1965. This finding remained after adjustment for all studied clinical and histopathological factors which point towards changes in unmeasured biological features of the disease.

Increasing Incidence Rates of Squamous Cell Carcinoma of the Skin in Sweden

The incidence of squamous cell carcinoma of the skin is increasing world-wide, and in Sweden this tumour is one of the most rapidly increasing malignancies. The aim of this study was to investigate incidence trends of squamous cell carcinoma in Sweden. For the 39,805 tumours registered in the Swedish Cancer Registry 1961-1995, incidence rates were calculated according to gender, age, anatomical site and unit surface area. Multivariate analysis was performed with the age-period-cohort model. Age-standardized incidence rates increased substantially in both men (+425%) and women (+146%) during this period. The highest rates per unit surface area were seen for chronically sun-exposed head-neck sites. Age-specific incidence rates increased in ages > or =60 years during the study period. Multivariate analyses showed that age, period and cohort effects in men could best explain the incidence rates, while in women the age-period effects model was adequate. In conclusion, a rapidly increasing incidence trend for squamous cell carcinoma was found, probably explained by increased accumulated sun exposure and increasing incidence among the elderly.

Trends in colorectal cancer incidence in Sweden 1959-93 by gender, localization, time period, and birth cohort

Objectives: This study examined invasive colorectal cancer incidence-rates in Sweden from 1959 through 1993 (n = 134,643 cases). Methods: Age-standardized rates were calculated using the Swedish population in 1970 as a reference. Results: In right-sided colon cancer (ascending and transverse colon including right and left flexures), male age-standardized rates rose from 8.0 to 15.0 (1.8 percent annually, 95 percent confidence interval [CI] = 1.3-2.4) and female rates increased from 9.1 to 14.4 (1.5 percent annually, CI = 1.0-2.0). For left-sided colon cancer (descending and sigmoid colon), the rates have been stable recently. For rectal cancer, the rates among men rose from 18.8 to 23.0 and among women from 10.7 to 14.7. For both men and women, the relative risk (RR) of right-sided colon cancer had been increasing in successive generations, until leveling-off in those born after 1930. The RR of left-sided colon cancer had been almost constant for cohorts born before 1930 but steadily decreasing in later-born cohorts. The RR of rectal cancer was slightly increasing in successive cohorts. Conclusions: Changes in lifestyle or carcinogenic exposures during early life probably explain Swedish colorectal cancer incidence-trends better than improved diagnostic activities.

The association between anatomic site and survival in malignant melanoma. An analysis of 12,353 cases from the Swedish cancer registry

The relationship between site and survival in cutaneous malignant melanoma was investigated by a follow-up of 12,353 Swedish patients diagnosed in 1960-1982. In males, the poorest prognosis was found for tumors located on the scalp-neck region (5-year relative survival rate, RS51%), followed by the lower extremity (RS66%) and trunk (RS68%). Among females, the poorest prognosis was noted for tumors located on the external ear (5-year RS 71%), trunk (RS 78%) and scalp-neck (RS 78%). The prognosis varied considerably between the sites of the head-neck region--for eyelid and facial lesions the prognosis was good, but for external ear and scalp-neck tumors it was poor. Multivariate analysis taking into account age and year of diagnosis showed the highest relative hazards (RH) for female lesions of the trunk (1.40) and male scalp-neck tumors (1.65), with the upper extremity used as reference (RH = 1.00). Except for lesions of the trunk, no significant differences in RH were found between the various sites after 4 years of observation.

CpG Methylation of the IFNG Gene as a Mechanism to Induce Immunosupression in Tumor-Infiltrating Lymphocytes

The execution of appropriate gene expression patterns during immune responses is of eminent importance where CpG methylation has emerged as an essential mechanism for gene silencing. We have charted the methylation status of regulatory elements in the human IFNG gene encoding the signature cytokine of the Th1 response. Surprisingly, human naive CD4+ T lymphocytes displayed hypermethylation at the IFNG promoter region, which is in sharp contrast to the completely demethylated status of this region in mice. Th1 differentiation induced demethylation of the IFNG promoter and the upstream conserved nucleotide sequence 1 enhancer region, whereas Th2-differentiated lymphocytes remained hypermethylated. Furthermore, CD19+ B lymphocytes displayed hypomethylation at the IFNG promoter region with a similar pattern to Th1 effector cells. When investigating the methylation status among tumor-infiltrating CD4+ T lymphocytes from patients with colon cancer, we found that tumor-infiltrating lymphocytes cells are inappropriately hypermethylated, and thus not confined to the Th1 lineage. In contrast, CD4+ T cells from the tumor draining lymph node were significantly more demethylated than tumor-infiltrating lymphocytes. We conclude that there are obvious interspecies differences in the methylation status of the IFNG gene in naive CD4+ T lymphocytes, where Th1 commitment in human lymphocytes involves demethylation before IFNG expression. Finally, investigations of tumor-infiltrating lymphocytes and CD4+ cells from tumor draining lymph node demonstrate methylation of regulatory regions within key effector genes as an epigenetic mechanism of tumor-induced immunosupression.