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Background Non-seminoma testicular cancer survivors (TCS) have an increased risk of developing colorectal cancer (CRC) when they have been treated with platinum-based chemotherapy. Previously we demonstrated that among Hodgkin lymphoma survivors (HLS) there is enrichment of rare mismatch repair (MMR) deficient (MMRd) CRCs with somatic hits in MMR genes. We speculate that this phenomenon could also occur among other cancer survivors. We therefore aim to determine the MMR status and its underlying mechanism in CRC among TCS (TCS-CRC). Methods Thirty TCS-CRC, identified through the Dutch pathology registry, were analysed for MMR proteins by immunohistochemistry. Next-generation sequencing was performed in MMRd CRCs without MLH1 promoter hypermethylation (n = 4). Data were compared with a male cohort with primary CRC (P-CRC, n = 629). Results MMRd was found in 17% of TCS-CRCs vs. 9% in P-CRC (p = 0.13). MMRd was more often caused by somatic double or single hit in MMR genes by mutation or loss of heterozygosity in TCS-CRCs (3/30 (10%) vs. 11/629 (2%) in P-CRCs (p < 0.01)). Conclusions MMRd CRCs with somatic double or single hit are more frequent in this small cohort of TCS compared with P-CRC. Exposure to anticancer treatments appears to be associated with the development of these rare MMRd CRC among cancer survivors.

Background:In human papillomavirus (HPV)-based screening, a repeat HPV test is often recommended for HPV-positive women with normal cytology (HPV-pos/cyt-neg), but its absolute risk of cervical precancer (CIN3+) over two screening rounds needs to be assessed.Methods:We compared the 5-year risk of HPV infection and CIN3+ in HPV-pos/cyt-neg women with a negative repeat HPV test to the risk in HPV-negative women with normal cytology (double negatives) in the POBASCAM cohort. We obtained histology data from the Dutch pathology registry (PALGA).Results:Human papillomavirus infection risk was 20.4% (19 of 93) in HPV-pos/cyt-neg, repeat HPV-negative women and 3.2% (294 of 9186; P

Background: Patients with peritoneal metastases (PMs) originating from colorectal carcinoma (CRC) are curatively treated by cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C (MMC). We aim to improve patient selection for HIPEC by predicting MMC sensitivity. Methods: The MMC sensitivity was determined for 12 CRC cell lines and correlated to mRNA expression of 37 genes related to the Fanconi anaemia (FA)–BRCA pathway, ATM–ATR pathway and enzymatic activation of MMC. Functionality of the FA–BRCA pathway in cell lines was assessed using a chromosomal breakage assay and western blot for key protein FANCD2. Bloom syndrome protein (BLM) was further analysed by staining for the corresponding protein with immunohistochemistry (IHC) on both CRC cell lines (n=12) and patient material (n=20). Results: High sensitivity correlated with a low BLM (P=0.01) and BRCA2 (P=0.02) at mRNA expression level. However, FA–BRCA pathway functionality demonstrated no correlation to MMC sensitivity. In cell lines, weak intensity staining of BLM by IHC correlated to high sensitivity (P=0.04) to MMC. Low BLM protein expression was significantly associated with an improved survival in patients after CRS and HIPEC (P=0.04). Conclusions: Low BLM levels are associated with high MMC sensitivity and an improved survival after HIPEC.

BACKGROUND: Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the interaction between p53 and its negative feedback regulator MDM2.METHODS: We examined whether nutlin-3 enhances apoptosis induction by rhTRAIL and the DR5-selective TRAIL variant D269H/E195R in wild-type p53-expressing ovarian, colon and lung cancer cell lines and in an ex vivo model of human ovarian cancer.RESULTS: Nutlin-3 enhanced p53, p21, MDM2 and DR5 surface expression. Although nutlin-3 did not induce apoptosis, it preferentially enhanced D269H/E195R-induced apoptosis over rhTRAIL. Combination treatment potentiated the cleavage of caspases 8, 9, 3 and PARP. P53 and MDM2 siRNA experiments showed that this enhanced apoptotic effect was mediated by wild-type p53. Indeed, nutlin-3 did not enhance rhTRAIL-induced apoptosis in OVCAR-3 cells harbouring mutant p53. Addition of the chemotherapeutic drug cisplatin to the combination further increased p53 and DR5 levels and rhTRAIL- and D269H/E195R-induced apoptosis. As a proof of concept, we show that the combination of D269H/E195R, nutlin-3 and cisplatin induced massive apoptosis in ex vivo tissue slices of primary human ovarian cancers.CONCLUSION: Nutlin-3 is a potent enhancer of D269H/E195R-induced apoptosis in wild-type p53-expressing cancer cells. Addition of DNA-damaging agents such as cisplatin further enhances DR5-mediated apoptosis.

Background: Little is known about the factors that drive metastasis formation in colorectal cancer (CRC). Here, we set out to identify genes and proteins in patients with colorectal liver metastases that correlate with early disease recurrence. Such factors may predict a propensity for metastasis in earlier stages of CRC. Methods: Gene expression profiling and proteomics were used to identify differentially expressed genes/proteins in resected liver metastases that recurred within 6 months following liver surgery vs those that did not recur for >24 months. Expression of the identified genes/proteins in stage II (n=243) and III (n=176) tumours was analysed by immunohistochemistry on tissue microarrays. Correlation of protein levels with stage-specific outcome was assessed by uni- and multivariable analyses. Results: Both gene expression profiling and proteomics identified Maspin to be differentially expressed in colorectal liver metastases with early (24 months) time to recurrence. Immunohistochemical analysis of Maspin expression on tumour sections revealed that it was an independent predictor of time to recurrence (log-rank P=0.004) and CRC-specific survival (P=0.000) in stage III CRC. High Maspin expression was also correlated with mucinous differentiation. In stage II CRC patients, high Maspin expression did not correlate with survival but was correlated with a right-sided tumour location. Conclusion: High Maspin expression correlates with poor outcome in CRC after spread to the local lymph nodes. Therefore, Maspin may have a stage-specific function possibly related to tumour cell dissemination and/or metastatic outgrowth.

Background: We assessed the association of human papillomavirus (HPV) infection and cervical intraepithelial neoplasia (CIN) with various characteristics, CD4 count and use of combination antiretroviral therapy (cART) among HIV-positive women. Methods: Cross-sectional study of 498 HIV-positive women who underwent HPV PCR-based testing, cytology, and systematic cervical biopsy. Results: In all, 68.7% of women were HPV-positive, 52.6% had high-risk (hr) HPV, and 40.2% multiple type infections. High-risk human papillomavirus-positivity did not vary significantly by age but it was negatively associated with education level. The most frequent types in 113 CIN2/3 were HPV16 (26.5%), HPV35 (19.5%), and HPV58 (12.4%). CD4 count was negatively associated with prevalence of hrHPV (P

Cervical cancer is preceded by well-defined premalignant lesions, which can be identified by detecting abnormal cells in Papanicolaou smear. Cervical screening by cytology with adequate treatment have resulted in a decrease in incidence and mortality of cervical carcinoma (Gustafsson et al, 1997; Vizcaino et al, 2000). In the Dutch screening programme, women aged 30–60 years are invited every 5 years for seven times in a lifetime. Modelling, before the introduction of the Dutch cervical screening programme, predicted a decrease in cervical carcinoma by approximately 75%, assuming full coverage (van Ballegooijen, 1998) within the range mentioned in other studies (Sasieni and Adams, 1999; IARC, 2005). Coverage of the screening programme is currently 77% (Rebolj et al, 2007). Approximately 65% of women attend the screening programme after an invitation, referred to as smears made inside the screening programme and 12% reflects smears made outside the screening programme (opportunistic smears). Approximately 23% of the invited women will not be screened at all (Bais et al, 2007). Collectively, the effect on carcinoma incidence through these two modes of screening will be lower than modelled for the programme, as full coverage is not attained. Moreover, the non-participating fraction of women (referred to as non-attendees) has a higher risk for cervical carcinoma than average, thus further decreasing the effectiveness of a programme in reducing carcinoma incidence (van Oortmarssen and Habbema, 1991). Earlier studies have shown that 40–50% of the women diagnosed with cervical cancer are in the non-compliance group (van der Graaf et al, 1986; Bos et al, 2006). Here we analysed 286 women, with cervical carcinoma from the region Noord-Holland/Flevoland in the Netherlands, diagnosed between 2005 and 2007. We analysed the relationship between the FIGO (International Federation of Gynaecology and Obstetrics) stage of the detected carcinoma and the associated screen status. In addition, we analysed whether the smear was made within or outside the screening programme, and the compliance for referral to the gynaecologist in case of an abnormal smear.

BACKGROUND: DNA aneuploidy reflects gross genomic changes. It can be measured by flow cytometry (FCM-DNA) or image cytometry (ICM-DNA). In gastric cancer, the prevalence of DNA aneuploidy has been reported to range from 27 to 100%, with conflicting associations with clinicopathological variables. The aim of our study was to compare the DNA ploidy status measured using FCM-DNA and ICM-DNA in gastric cancer and to evaluate its association with clinicopathological variables. METHODS: Cell nuclei were isolated from 221 formalin-fixed, paraffin-embedded gastric cancer samples. DNA ploidy was assessed using FCM-DNA and ICM-DNA. RESULTS: A total of 178 (80.5%) gastric cancer samples were classified as DNA aneuploid using FCM-DNA, compared with 172 (77.8%) gastric cancer samples when using ICM-DNA. Results obtained from both methods were concordant in 183 (82.8%) cases (kappa = 0.48). Patients with ICM-DNA diploid gastric cancer survived significantly longer than those with ICM-DNA aneuploid gastric cancer (log rank 10.1, P = 0.001). For FCM-DNA data, this difference did not reach statistical significance. The multivariate Cox model showed that ICM-DNA ploidy status predicted patient survival independently of tumour-node-metastasis status. CONCLUSION: ICM-DNA ploidy status is an independent predictor of survival in gastric cancer patients and may therefore be a more clinically relevant read out of gross genomic damage than FCM-DNA. British Journal of Cancer (2009) 101, 1011-1018. doi:10.1038/sj.bjc.6605266 www.bjcancer.com (C) 2009 Cancer Research UK

Background: Cervical cancer incidence in western Africa is among the highest in the world. Methods: To investigate human papillomavirus (HPV) infection in Guinea, we obtained cervical specimens from 831 women aged 18–64 years from the general population of the capital Conakry and from 77 locally diagnosed invasive cervical cancers (ICC). Human papillomavirus was detected using a GP5+/6+ PCR-based assay. Results: Among the general population, the prevalence of cervical abnormalities was 2.6% by visual inspection and 9.5% by liquid-based cytology. Fourteen of 15 high-grade squamous intraepithelial lesions were visual inspection-negative. Human papillomavirus prevalence was 50.8% (32.1% for high-risk types) and relatively constant across all age groups. Being single or reporting ⩾3 sexual partners was significantly associated with HPV positivity. HPV16 was the most common type, both among the general population (7.3%) and, notably in ICC (48.6%). HPV45 (18.6%) and HPV18 (14.3%), the next most common types in ICC, were also more common in ICC than in HPV-positive women with normal cytology from the general population. Conclusion: The heavy burden of HPV infection and severe cervical lesions in Guinean women calls for new effective interventions. Sixty-three per cent of cervical cancers are theoretically preventable by HPV16/18 vaccines in Guinea; perhaps more if some cross-protection exists with HPV45.

Little is known about the dynamics of human papillomavirus (HPV) infection and subsequent development of high-grade cervical intraepithelial neoplasia (CIN2/3), particularly in women >30 years of age. This information is needed to assess the impact of HPV vaccines and consider new screening strategies. A cohort of 1728 women 15-85 years old with normal cytology at baseline was followed every 6 months for an average of 9 years. Women with squamous intraepithelial lesions were referred for biopsy and treatment. The Kaplan-Meier method was used to estimate the median duration of infection and Cox regression analysis was undertaken to assess determinants of clearance and risk of CIN2/3 associated with HPV persistence. No difference in the likelihood of clearance was observed by HPV type or woman's age, with the exception of lower clearance for HPV16 infection in women under 30 years of age. Viral load was inversely associated with clearance. In conclusion, viral load is the main determinant of persistence, and persistence of HPV16 infections carry a higher risk of CIN2/3.

Background:MicroRNAs are small non-coding RNA molecules, which regulate central mechanisms of tumorigenesis. In colorectal tumours, the combination of gain of 8q and 13q is one of the major factors associated with colorectal adenoma to adenocarcinoma progression. Functional studies on the miR-17-92 cluster localised on 13q31 have shown that its transcription is activated by c-myc, located on 8q, and that it has oncogenic activities. We investigated the contribution of the miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression.Methods:Expression levels of the miR-17-92 cluster were determined in 55 colorectal tumours and in 10 controls by real-time RT-PCR. Messenger RNA c-myc expression was also determined by real-time RT-PCR in 48 tumours with array comparative genomic hybridisation (aCGH) data available.Results:From the six members of the miR-17-92 cluster, all except miR-18a, showed significant increased expression in colorectal tumours with miR-17-92 locus gain compared with tumours without miR-17-92 locus gain. Unsupervised cluster analysis clustered the tumours based on the presence of miR-17-92 locus gain. Significant correlation between the expression of c-myc and the six miRNAs was also found.Conclusion:Increased expression of miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression is associated to DNA copy number gain of miR17-92 locus on 13q31 and c-myc expression. © 2009 Cancer Research UK.

Contains fulltext : 69490.pdf (Publisher’s version ) (Closed access) Although the tyrosine kinase inhibitor imatinib has been shown to be an active agent in patients with gastrointestinal stromal tumours (GIST), complete remissions are almost never seen and most patients finally experience disease progression during their course of treatment. An alternative therapeutic option is to target death receptors such as Fas. We showed that a panel of imatinib-sensitive (GIST882) and imatinib-resistant (GIST48, GIST430 and GIST430K-) cell lines expressed Fas. MegaFasL, a recently developed hexameric form of soluble Fas ligand (FasL), appeared to be an active apoptosis-inducing agent in these cell lines. Moreover, MegaFasL potentiated the apoptotic effects of imatinib. Immunohistochemical evaluations, in 45 primary GISTs, underscored the relevance of the Fas pathway: Fas was expressed in all GISTs and was expressed strongly in 93%, whereas FasL was expressed at moderate and strong levels in 35 and 53% of GISTs, respectively. Fas and FasL expression were positively correlated in these primary GISTs, but there was no association between Fas or FasL expression and primary site, histological subtype, tumour size, mitotic index, risk classification, and KIT mutation status. The abundant immunohistochemical Fas and FasL expression were corroborated by western blot analysis. In conclusion, our data implicate Fas as a potential therapeutic target in GIST.

The chemokine CXCL12/SDF-1 and its receptor CXCR4 have been implicated in invasion, survival and proliferation of carcinoma cells. Recently, CXCR7 was identified as a second receptor for CXCL12. We observed that CXCL12 promoted proliferation of CT26 colon and KEP1 mammary carcinoma cells, and this was blocked when CXCR7 was downregulated by 'intrakines' or RNAi, but not by CXCR4 inhibitors. The K1R mutant of CXCL12, which acts as a CXCR4 antagonist, also promoted proliferation through CXCR7 and is therefore a selective CXCR7 agonist. The effect of CXCR7 was not due to reduced apoptosis, and CXCR7 mediated chemotaxis of the carcinoma cells towards CXCL12. These results differ from those in a previous report on other carcinoma cells. We conclude that CXCL12 can be a potent growth factor for carcinoma cells by acting on CXCR7. Nevertheless, we observed no effect of complete and stable CXCR7 suppression on the growth of s.c. tumours or lung metastases of KEP1 and CT26 cells. A CXCR7 inhibitor has been reported to reduce growth of other tumours. Our results indicate that this inhibitor may not be applicable to therapy of all carcinomas.

We determined the prevalence of type-specific hrHPV infections in the Netherlands on cervical scrapes of 45 362 women aged 18-65 years. The overall hrHPV prevalence peaked at the age of 22 with peak prevalence of 24%. Each of the 14 hrHPV types decreased significantly with age (P-values between 0.0009 and 0.03). The proportion of HPV16 in hrHPV-positive infections also decreased with age (OR=0.76 (10-year scale), 95% CI=0.67-0.85), and a similar trend was observed for HPV16 when selecting hrHPV-positive women with cervical intraepithelial neoplasia grade 2 or worse (CIN2+) (OR=0.76, 95% CI=0.56-1.01). In women eligible for routine screening (age 29-61 years) with confirmed CIN2+, 65% was infected with HPV16 and/or HPV18. When HPV16/18-positive infections in women eligible for routine screening were discarded, the positive predictive value of cytology for the detection of CIN2+ decreased from 27 to 15%, the positive predictive value of hrHPV testing decreased from 26 to 15%, and the predictive value of a double-positive test (positive HPV test and a positive cytology) decreased from 54 to 41%. In women vaccinated against HPV16/18, screening remains important to detect cervical lesions caused by non-HPV16/18 types. To maintain a high-positive predictive value, screening algorithms must be carefully re-evaluated with regard to the screening modalities and length of the screening interval.

To investigate the prevalence of, and risk factors for, cervical infection with human papillomavirus (HPV) in Shenyang City, People's Republic of China, we interviewed and obtained cervical cell samples from 685 women aged 15–59 years enumerated from local population lists. Human papillomavirus DNA was detected in cervical cell samples using a GP5+/6+-based PCR assay for 44 HPV types. Human papillomavirus prevalence was 16.8% overall and 13.6% among women without cervical abnormalities (16.6% and 12.4%, respectively, age-standardised to the world standard population), with no significant trends in HPV prevalence by age group. Of the 32 types identified, high-risk HPV types predominated in all age groups, HPV16 being the most common (3.4% of all women), followed by HPV52 (2.5%) and 58 (1.9%). Multiple-type infections accounted for 31.3% of all infected women. Not being married, reporting multiple sexual partners and husband's extramarital sexual relationships were all significantly associated with being HPV-positive. The disclosure of a relatively high HPV prevalence in Shenyang, in comparison with other worldwide populations, raises important questions concerning the prevention of cervical cancer in China, especially given the promising efficacy of prophylactic HPV vaccines.

Gastric cancers express enhanced levels of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Single-nucleotide polymorphisms (SNPs) in MMP and TIMP genes may be associated with disease susceptibility and might also affect their antigen expression. We studied the genotype distribution and allele frequencies of SNPs of MMP-2, -7, -8 and -9 and TIMP-1 and -2 in gastric cancer patients in relation to tumour progression, patient survival and tissue antigen expression. The genotype distribution and allele frequencies were similar in gastric cancer patients and controls, except for MMP-7(-181AG). In addition, the genotype distribution of MMP-7(-181AG) was associated with Helicobacter pylori status (chi(2) 7.8, P=0.005) and tumour-related survival of the patients. Single-nucleotide polymorphism TIMP-2(303CT) correlated significantly with the WHO classification (chi(2) 5.9, P=0.03) and also strongly with tumour-related survival (log rank 11.74, P=0.0006). Single-nucleotide polymorphisms of MMP-2, -8, -9 and TIMP-1 were not associated with tumour-related survival. Only the gene promoter MMP-2(-1306CT) polymorphism correlated significantly with the protein level within the tumours. First-order dendrogram cluster analysis combined with Cox analysis identified the MMP-7(-181AG) and TIMP-2(303CT) polymorphism combination to have a major impact on patients survival outcome. We conclude that MMP-related SNPs, especially MMP-7(-181AG) and TIMP-2(303CT), may be helpful in identifying gastric cancer patients with a poor clinical outcome.

To investigate the prevalence of, and risk factors for, cervical infection with human papillomavirus (HPV) in the rural province of Shanxi, People's Republic of China, which has relatively high cervical cancer mortality rates, we interviewed and obtained cervical cell samples from 662 women aged 15–59 years. A total of 24 different HPV types were identified using a GP5+/6+-based PCR assay able to detect 44 different HPV types. Human papillomavirus prevalence was 14.8% overall and 9.6% among women without cervical abnormalities (14.2 and 8.9%, respectively, age standardised to the world standard population). Multiple-type infections accounted for 30.6% of all infections. By far the most commonly found type was HPV16 (5.7% of all women and 38.8% of HPV-positive women), followed by HPV 58, 52, 33 and 18. Unlike most previous studies published, HPV prevalence was lower among women younger than 35 years (8.7%) than those older than 35 years (17.8%). High-risk HPV types predominated in all age groups. Although low-risk HPV types were rare in young women, they became more common with increasing age. 92.3% of women with cervical intraepithelial neoplasia grade 3 were infected with high-risk HPV types, but none with low-risk types only. No significant difference in HPV positivity was observed by educational level, sexual habits, reproductive history or use of contraceptive methods in this rural low-income Chinese population.

We present the type-distribution of high-risk human papillomavirus (HPV) types in women with normal cytology (n = 1467), adenocarcinoma in situ (ACIS) (n = 61), adenocarcinoma (n = 70), and squamous cell carcinoma (SCC) (n = 83). Cervical adenocarcinoma and ACIS were significantly more frequently associated with HPV18 (ORMH 15.0; 95% CI 8.6-26.1 and 21.8; 95% CI 11.9-39.8, respectively) than normal cytology. Human papillomavirus 16 was only associated with adenocarcinoma and ACIS after exclusion of HPV18-positive cases (OR MH 6.6; 95% CI 2.8-16.0 and 9.4; 95% CI 2.8-31.2, respectively). For SCC, HPV16 prevalence was elevated (ORMH 7.0; 95% CI 3.9-12.4) compared to cases with normal cytology, and HPV18 prevalence was only increased after exclusion of HPV16-positive cases (ORMH 4.3; 95% CI 1.6-11.6). These results suggest that HPV18 is mainly a risk factor for the development of adenocarcinoma whereas HPV16 is associated with both SCC and adenocarcinoma.

The human papillomavirus test (HPV) test could improve the (cost-) effectiveness of cervical screening by selecting women with a very low risk for cervical cancer during a long period. An analysis of a longitudinal study suggests that women with a negative Pap smear and a negative HPV test have a strongly reduced risk of developing cervical abnormalities in the years following the test, and that HPV testing lengthens the detectable stage by 2-5 years, compared to Pap smear detection alone.

Low grade squamous intra-epithelial lesions could be considered as a manifestation of human papillomavirus exposition, however the discrepancy between rates of infection with human papillomavirus and development of low grade squamous intra-epithelial lesions is notable. Here we report a cross-sectional three-armed case–control study in the Colombian population, to compare the risk factors of women with low grade squamous intra-epithelial lesions with that of human papillomavirus DNA-negative and positive women with normal cytology. British Journal of Cancer (2002) 87, 1417–1421. doi:10.1038/sj.bjc.6600650 www.bjcancer.com © 2002 Cancer Research UK

Human papillomavirus is the principal risk factor associated with cervical cancer, the most common malignancy among women in Colombia. We conducted a survey, aiming to report type specific prevalence and determinants of human papillomavirus infection in women with normal cytology. A total of 1859 women from Bogota, Colombia were interviewed and tested for human papillomavirus using a general primer GP5+/GP6+ mediated PCR–EIA. The overall HPV DNA prevalence was 14.8%; 9% of the women were infected by high risk types, 3.1% by low risk types, 2.3% by both high risk/low risk types and 0.4% by uncharacterized types (human papillomavirus X). Thirty-two different human papillomavirus types were detected, being human papillomavirus 16, 58, 56, 81(CP8304) and 18 the most common types. The human papillomavirus prevalence was 26.1% among women younger than 20 years, 2.3% in women aged 45–54 years, and 13.2% in women aged 55 years or more. For low risk types the highest peak of prevalence was observed in women aged 55 years or more. Compared to women aged 35–44 years, women aged less than 20 years had a 10-fold increased risk of having multiple infections. Besides age, there was a positive association between the risk of human papillomavirus infection and number of regular sexual partners and oral contraceptive use. In women aged below 25 years, high educational level and having had casual sexual partners predicted infection risk. In conclusion, there was a broad diversity of human papillomavirus infections with high risk types being the most common types detected. In this population multiplicity of sexual partners and, among young women, high educational level and casual sexual partners seem to determine risk. British Journal of Cancer (2002) 87, 324–333. doi:10.1038/sj.bjc.6600442 www.bjcancer.com © 2002 Cancer Research UK

We followed 353 women referred with abnormal cervical cytology in a non-intervention cohort study. In 91 pregnant women we compared high-risk human papilloma virus rates in the subsequent trimesters and postpartum in comparison to 262 non-pregnant women. High-risk human papilloma virus clearance was compared with 179 high-risk human papilloma virus positive non-pregnant women. Our main questions were: (1) do high-risk human papilloma virus rates change during pregnancy?; and (2) is there any difference between high-risk human papilloma virus clearance in pregnant and non-pregnant women? Women were monitored 3–4 monthly by cytology, colposcopy, and high-risk human papilloma virus testing. The median follow-up time was 33 months (range 3–74). Non-pregnant women showed prevalence rates of high-risk human papilloma virus of 64, 57, 53, and 50%, respectively, in four subsequent 3-months periods since the start of the study. These high-risk human papilloma virus rates were higher than in the three trimesters of pregnancy, and during the first 3 months postpartum, i.e. 50, 44, 45, and 31%, respectively. Postpartum only, this difference was statistically significant (P=0.004). Paired comparisons of high-risk human papilloma virus prevalence rates of the different trimesters with the postpartum rate showed (McNemar test) decreased rates: first trimester: 18% (P=0.02), second trimester: 13% (P=0.02) and third trimester: 23% (P

During chemo- and radiation therapy, the balance between epithelial cell proliferation, differentiation, and cell death at the villus tip is disrupted by premature death of dividing epithelial cells. This will subsequently lead to the onset of mucosal barrier injury in the whole gastrointestinal tract. Up till now there is no validated method to treat side effects occurring due to therapy. An approach to manage this side effect might be to reversibly arrest growth of epithelial stem cells during therapy using Transforming Growth Factor-β2. A Transforming Growth Factor-β2 enriched fraction prepared from bovine milk was shown to protect small intestinal epithelial cells against cell cycle specific chemotherapeutic agents by arresting the cells in G1-phase. Secondly, in a rat model for induced small intestinal damage, oral supplementation of rats exposed to methotrexate with the Transforming Growth Factor-β2 enriched fraction significantly reduced the chemotherapy-associated weight loss and ileal villus atrophy by reducing cell proliferation in the normal stem cell population. Thus oral supplementation with a bovine milk fraction enriched for Transforming Growth Factor-β2 attenuated the side effects of chemotherapy in the small intestine in rats. British Journal of Cancer (2002) 87, 113–118. doi:10.1038/sj.bjc.6600342 www.bjcancer.com © 2002 Cancer Research UK

In a retrospective case–control study, we investigated high-risk HPV DNA presence by general primer GP5+/6+ PCR in the last normal cervical smear in the patient archives (i.e. baseline smear) of 57 women who later developed cervical cancer. Also, normal cervical smears of 114 age-matched control women were analysed. High-risk HPV DNA was detected in 37 of the 57 (65%) baseline smears of the case women, and 7 (6%) of 114 smears of the control women (OR 28, 95% Cl 11–72). The HPV positive subsequent smears and cervical cancer biopsies of the case women contained the same HPV type as was detected in the baseline smear. After cytological revision, the baseline smears of 48 case women (84%) were reclassified as abnormal, 33 (69%) of which scored high-risk HPV DNA positive. Ultimately, an undisputable normal baseline smear was found in only 10 case women. In 7 (70%) of them this smear was HPV positive, whereas only 7 (7%) of 104 revised, undisputable normal smears of control women were high-risk HPV positive (OR 32, 95% Cl 6.8–153). The results showed that (1) high-risk HPV presence precedes abnormal cytology in women who develop cervical cancer, and (2) high-risk HPV testing signals false-negative smears of women at risk of cervical cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com

We assessed a possible role for high-risk human papillomavirus (HPV) testing in the policy after treatment for cervical intraepithelial neoplasia (CIN) 2 or 3 (moderate to severe dysplasia). According to the Dutch guidelines follow-up after treatment consists of cervical cytology at 6, 12 and 24 months. Colposcopy is only performed in case of abnormal cervical cytology. In this observational study 184 women treated for CIN 2 or 3 were prospectively monitored by cervical cytology and high-risk HPV testing 3, 6, 9, 12 and 24 months after treatment. Post-treatment CIN 2/3 was present in 29 women (15.8%). A positive high-risk HPV test 6 months after treatment was more predictive for post-treatment CIN 2/3 than abnormal cervical cytology (sensitivity 90% and 62% respectively, with similar specificity). At 6 months the negative predictive value of a high-risk HPV negative, normal smear, was 99%. Largely overlapping, partly different groups of women with post-treatment CIN 2/3 were identified by HPV testing and cervical cytology. Based on these results we advocate to include high-risk HPV testing in monitoring women initially treated for CIN 2/3. In case of a high-risk HPV positive test or abnormal cervical cytology, colposcopy is indicated. All women should be tested at 6 and 24 months after treatment and only referred to the population-based cervical cancer screening programme when the tests are negative on both visits. © 2001 Cancer Research Campaign http://www.bjcancer.com

About 5–10% of breast cancers are hereditary; a genetically and clinically heterogeneous disease in which several susceptibility genes, including BRCA1, have been identified. While distinct tumour features can be used to estimate the likelihood that a breast tumour is caused by a BRCA1 germline mutation it is not yet possible to categorize a BRCA1 mutated tumour. The aim of the present study is to molecularly classify BRCA1 mutated breast cancers by resolving gene expression patterns of BRCA1 and matched sporadic surgical breast tumour specimens. The expression profiles of 6 frozen breast tumour tissues with a proven BRCA1 gene mutation were weighed against those from 12 patients without a known family history but who had similar clinico-pathological characteristics. In addition two fibroblast cultures, the breast cancer cell-line HCC1937 and its corresponding B-lymphoblastoid cell line (heterozygous for mutation BRCA1 5382insC) and an epithelial ovarian cancer cell line (A2780) were studied. Using a high density membrane based array for screening of RNA isolated from these samples and standard algorithms and software, we were able to distinguish subgroups of sporadic cases and a group consisting mainly of BRCA1-mutated breast tumours. Furthermore this pilot analysis revealed a gene cluster that differentially expressed genes related to cell substrate formation, adhesion, migration and cell organization in BRCA1-mutated tumours compared to sporadic breast tumours. © 2001 Cancer Research Campaign http://www.bjcancer.com

The bisindole indirubin has been described, more than 30 years ago, as being clinically active in the treatment of human chronic myelocytic leukaemia. However, the underlying mechanism of action has remained unclear. We have reported previously that indirubin and its analogues are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, we investigated the influence of indirubin and derivatives on CDK1/cyclin B kinase in human tumour cells at concentrations known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G1/G0 phase of the cell cycle in the presence of 2 μM indirubin-3′-monoxime. At higher drug concentrations (≥ 5 μM) an increase of the cell population in the G2/M phase is additionally observed. Cells synchronized in G2/M phase by nocodazole remain arrested in the G2/M phase after release, in the presence of indirubin-3′-monoxime (≥5 μM). After 24 h treatment with 10 μM indirubin-3′-monoxime a sub-G2 peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concentrations of indirubin-3′-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a reduction of cyclin B in complex with CDK1 is observed. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumour cells is a major mechanism by which indirubin derivatives exert their potent antitumour efficacy. © 2001 Cancer Research Campaign http://www.bjcancer.com

The efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the β-globin gene appeared to be superior for the GTC-based assays. Using competitive β-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 × 105to 3 × 105genome equivalents in smears containing 5 × 105to 20 × 105nucleated cells, indicating a mean efficiency of 26% (range of 15–44%). In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3–6 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 ± 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears. © 2000 Cancer Research Campaign

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the secretory pathway of perforin/granzymes to kill their target cells. In contrast to NK cells, CTL responses are MHC class I restricted. In this study we analysed the relative activation of CTL and NK cells in relation with MHC class I expression on oral squamous cell carcinomas (OSCCs). MHC class I expression was investigated in 47 OSCCs by immunohistochemistry using HCA2, HC10 and β2-m antibodies. The presence of CTLs, NK cells, and its activation, was investigated in 21 of these OSCCs using respectively, CD8, CD57 and GrB7 antibodies. The Q-Prodit measuring system was used for quantification of cytotoxic cells. All OSCCs showed weak or absent staining of β2-m on the cell surface. The absence of β2-m was significantly associated with absent expression of MHC class I heavy chain as detected by HC10 antibody (P = 0.004). In tumour infiltrates CTLs always outnumbered NK cells, as reflected by the ratio CD57/CD8 being always inferior to one (mean: 0.19; SD: 0.15). The proportion of activated cytotoxic cells as detected by granzyme B expression was generally low (mean: 8.6%; SD 8.9). A clear correlation between MHC class I expression and the relative proportion of NK cells/CTLs was not found. This study shows that the majority of OSCCs show weak or absent expression of MHC class I molecules on the cell surface, possibly due to alterations in the normal β2-m pathway. The low proportion of granzyme B-positive CTLs/NK cells indicates that the secretory pathway of cytotoxicity is poor in these patients. The lack of correlation between MHC class I expression and CTL/NK cell activation as detected by granzyme B expression suggests that, next to poor antigen presentation, also local factors seem to determine the final outcome of the cytotoxic immune response. © 1999 Cancer Research Campaign

Prostate-specific antigen (PSA) is a serine protease which may play a role in a variety of cancer types, including breast cancer. In the present study, we evaluated whether the level of PSA in breast tumour cytosol could be associated with prognosis in primary breast cancer, or with response to tamoxifen therapy in recurrent disease. PSA levels were determined by enzyme-linked immunosorbent assay (ELISA) in breast tumour cytosols, and were correlated with prognosis in 1516 patients with primary breast cancer and with response to first-line tamoxifen therapy in 434 patients with recurrent disease. Relating the levels of PSA with classical prognostic factors, low levels were more often found in larger tumours, tumours of older and post-menopausal patients, and in steroid hormone receptor-negative tumours. There was no significant association between the levels of PSA with grade of differentiation or the number of involved lymph nodes. In patients with primary breast cancer, PSA was not significantly related to the rate of relapse, and a positive association of PSA with an improved survival could be attributed to its relationship to age. In patients with recurrent breast cancer, a high level of PSA was significantly related to a poor response to tamoxifen therapy, and a short progression-free and overall survival after start of treatment for recurrent disease. In Cox multivariate analyses for response to therapy and for (progression-free) survival, corrected for age/menopausal status, disease-free interval, site of relapse and steroid hormone receptor status, PSA was an independent variable of poor prognosis. It is concluded that the level of PSA in cytosols of primary breast tumours might be a marker to select breast cancer patients who may benefit from systemic tamoxifen therapy. © 1999 Cancer Research Campaign

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and β-globin DNA. The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines. Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples. We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings. Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach. © 1999 Cancer Research Campaign

There is controversy regarding the prognostic value of cathepsin-D in primary breast cancer. An increased level of cathepsin-D in tumour extracts has been found to be associated with a poor relapse-free and overall survival. Studies performed with immunohistochemistry or Western blotting have produced diverse results. We have analysed 2810 cytosolic extracts obtained from human primary breast tumours for cathepsin-D expression, and have correlated their levels with prognosis. The median follow-up of the patients still alive was 88 months. Patients with high cathepsin-D levels had a significantly worse relapse-free and overall survival, also in multivariate analysis (P < 0.0001). Adjuvant therapy which was associated with an improved prognosis in node-positive patients in univariate analysis, also significantly added to the multivariate models for relapse-free and overall survival. There were no statistically significant interactions between the levels of cathepsin-D and any of the classical prognostic factors in analysis for relapse-free survival, suggesting that the prognostic value of cathepsin-D is not different in the various subgroups of patients. Indeed, multivariate analyses in subgroups of node-negative and -positive patients, pre- and post-menopausal patients, and their combinations, showed that tumours with high cathepsin-D values had a significantly poor relapse-free survival, with relative hazard rates ranging from 1.3 to 1.5, compared with tumours with low cathepsin-D levels. The results presented here on 2810 patients confirm that high cytosolic cathepsin-D values are associated with poor prognosis in human primary breast cancer. © 1999 Cancer Research Campaign

To detect low-level DNA platination, a sensitive immunocyto- and histochemical technique was developed using a polyclonal antibody. The antibody GPt, derived after immunization of rabbits with highly platinated DNA and purified with affinity chromatography, detected the main platinum (Pt)-containing intrastrand and interstrand adducts. Double-fluorescence microscopy image analysis was used to quantify Pt-DNA adducts with Hoechst 33258 fluorescence to locate the nuclei and with fluorescein isothiocyanate fluorescence to measure the immunosignal. A two- to five-fold dose-dependent difference in the level of cisplatin (CDDP)-induced Pt-DNA adducts between a CDDP-sensitive and -resistant human tumour cell line was detected. Large differences in Pt-DNA adduct levels after in vitro CDDP incubation between human buccal cells, lymphocytes and biopsies of different tumour types were observed. Pt-DNA adduct levels were fivefold higher in human testicular tumours than in colon tumours, representing CDDP-sensitive and -resistant tumours, respectively, in the clinic. These data suggest the possibility of predictive testing by measuring Pt-DNA adduct levels. Pt-DNA adducts in patients after treatment with CDDP were shown in normal buccal cells and in imprints of fresh tumour biopsies as well as in paraffin-embedded tumour cells. The analysis of Pt-DNA adducts at a single-cell level in small samples of normal and tumour cells during and/or after treatment is feasible with GPt and will hopefully enable more selective treatment of patients. Images Figure 1 Figure 2 Figure 4 Figure 5

In this study, the mechanism(s) by which heat increases cis-diamminedichloroplatinum (cisplatin, cDDP) sensitivity in cDDP-sensitive and -resistant cell lines of murine as well as human origin were investigated. Heating cells at 43 degrees C during cDDP exposure was found to increase drug accumulation significantly in the cDDP-resistant cell lines but had little effect on drug accumulation in the cDDP-sensitive cell lines. DNA adduct formation, however, was significantly increased in all cell lines studied. Furthermore, ongoing formation of platinum (Pt)-DNA adducts after the end of cDDP treatment was enhanced and/or adduct removal was decreased in heated cells, resulting in relatively more DNA damage remaining at 24 h after the end of cDDP exposure. Correlation plots with survival revealed weak correlations with cellular Pt accumulation (r2 = 0.59) and initial Pt-DNA adduct formation (r2 = 0.64). Strong correlations, however, were found with Pt-DNA adducts at 6 h (r2 = 0.97) and 24 h (r2 = 0.89) after the incubation with the drug. In conclusion, the mechanism by which heat sensitizes cells for cDDP action seems to be the sum of multiple factors, which comprise heat effects on accumulation, adduct formation and adduct processing. This mechanism did not seem to differ between cDDP-sensitive and -resistant cells, emphasizing the potential of hyperthermia to reduce cDDP resistance. Images Figure 5

Cervical carcinomas are closely associated with high-risk human papillomavirus (HPV) types and are preceded by cervical intraepithelial neoplasia (CIN). Most CIN lesions regress spontaneously and will not evolve to invasive carcinoma. The cellular immune system mediated by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are thought to play an important role in the ultimate decline of CIN lesions. Although TIA-1 is constitutively expressed in the majority of circulating T cells and defines a subpopulation of CD8+ T cells with cytotoxic potential, granzyme B is only expressed in CTLs upon activation. In the present study we have evaluated the expression of these proteins by lymphocytes present in 24 randomly chosen CIN lesions with increasing degree of atypia and in 14 cervical squamous cell carcinomas. As major histocompatibility complex (MHC) class I expression is frequently down-regulated in HPV-induced lesions, thus possibly frustrating tumour cell recognition by infiltrating CTLs, these lesions were also analysed for MHC class I expression. The results indicated that in most CIN lesions only a minority of CTLs are activated, whereas in some carcinomas a massive infiltration of activated, i.e. granzyme B-positive, CTLs were observed. The percentage of activated CTLs was not related to expression of MHC class I on neoplastic cells. These results suggest that in some carcinomas proper activation of CTLs occurs but that most likely local factors or immunoselection of resistant neoplastic cells inhibit a proper response of CTLs to these neoplastic cells. Images Figure 1 Figure 2

We prospectively evaluated the 5-year predictive values of adding high-risk human papillomavirus (hrHPV) testing to cytology for the detection of > or = cervical intraepithelial neoplasia (CIN)3 lesions in a population-based cohort of 2810 women. At baseline, nine (0.3%) women had prevalent lesions > or = CIN3, all being hrHPV positive. After 5 years of follow-up, four (6.5%) of the 62 hrHPV-positive women with normal cytology developed lesions > or = CIN3, vs only one (0.05%) of the 2175 hrHPV-negative women with normal cytology. High-risk human papillomavirus testing or combined screening revealed a much higher sensitivity, at the cost of a small decrease in specificity, and a higher negative predictive value for the detection of lesions > or = CIN3 till the next screening round (5 years) than cytology alone.

The interaction between moderate-dose radiation and cisplatin or carboplatin was studied in a cisplatin-sensitive (GLC4) and -resistant (GLC4-CDDP) human small-cell lung cancer cell line. Cellular toxicity was analysed under oxic conditions with the microculture tetrazolium assay. For the platinum and radiation toxicity with the clinically relevant dose ranges applied, this assay was used to obtain information on cell survival after the treatments. Apart from effects on cell survival effects on DNA were also investigated. Configurational DNA changes could be induced by platinum drugs and thereby these drugs might change the frequency of DNA double-strand breaks (dsbs). DNA fragmentation assayed with the clamped homogeneous electric field (CHEF) technique was used as a measure for dsbs in DNA. The radiosensitising effect of the platinum drugs was expressed as enhancement ratio (ER) calculated directly from survival levels of the initial slope of the curve. The highest ER for cisplatin in GLC4 was 1.39 and in GLC4-CDDP 1.38. These were all at 75% cell survival. Carboplatin showed increased enhancement with prolonged incubation up to 1.21 in GLC4 and was equally effective as cisplatin in GLC4-CDDP. According to isobologram analysis, prolonged incubation with both platinum drugs showed at least additivity with radiation for both cell lines at clinically achievable doses. GLC4-CDDP showed cross-resistance to radiation. The radiosensitising capacity of both lung cancer cell lines was not dependent on their platinum sensitivity. The formation of dsbs in DNA directly after radiation was not influenced by pretreatment of either drug in the sensitive or in the resistant cell line. Drug treatment resulted in decreased DNA extractability in control as well as in irradiated cells. Modest enhancement ratio for radiosensitisation by platinum drugs cannot be explained on the level of dsb formation in DNA in both cell lines. Interaction of radiation with the clinically less toxic carboplatin can be improved by prolonged low-dose carboplatin exposure before irradiation and is as potent as cisplatin in the resistant lung cancer cell line. This suggests an advantage in combining radiation and carboplatin in lung cancer patients.