Reclassification of diffuse large B cell lymphoma to large B cell lymphoma with IRF4 rearrangement in an adult population.

AIMS: Large B cell lymphoma with IRF4 rearrangement (LBCL-IRF4) is a new entity in the 2017 revised World Health Organisation (WHO) classification that was initially mainly reported in children. After identification of a 79-year-old patient, we assessed how often IRF4 rearrangements can be detected in adult diffuse large B cell lymphomas (DLBCLs) which have to be reclassified to LBCL-IRF4 based on fluorescence in-situ hybridisation (FISH) for IRF4. METHODS AND RESULTS: With FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008. CONCLUSIONS: In addition to the index patient, an IRF4 rearrangement was detected in another five of 237 patients (2%). The immunohistochemical profile of these five IRF4 rearranged lymphomas was consistent with previous reports of LBCL-IRF4. One case was recognised to represent transformation of follicular lymphoma rather than de-novo LBCL-IRF4. BCL6 rearrangements were found in two cases of LBCL-IRF4; BCL2 and MYC rearrangements were excluded. Patients presented with limited stage disease with involvement of the head and neck in three patients, and involvement of the lung and thyroid in two others. This study shows that, although rare, LBCL-IRF4 should also be considered in older patients and at localisations other than the head and neck region.

Introduction of primary screening using high-risk HPV DNA detection in the Dutch cervical cancer screening programme: a population-based cohort study.

BACKGROUND: In January 2017, the Dutch cervical cancer screening programme transitioned from cytomorphological to primary high-risk HPV (hrHPV) DNA screening, including the introduction of self-sampling, for women aged between 30 and 60 years. The Netherlands was the first country to switch to hrHPV screening at the national level. We investigated the health impact of this transition by comparing performance indicators from the new hrHPV-based programme with the previous cytology-based programme. METHODS: We obtained data from the Dutch nationwide network and registry of histo- and cytopathology (PALGA) for 454,573 women eligible for screening in 2017 who participated in the hrHPV-based programme between 1 January 2017 and 30 June 2018 (maximum follow-up of almost 21 months) and for 483,146 women eligible for screening in 2015 who participated in the cytology-based programme between 1 January 2015 and 31 March 2016 (maximum follow-up of 40 months). We compared indicators of participation (participation rate), referral (screen positivity; referral rate) and detection (cervical intraepithelial neoplasia (CIN) detection; number of referrals per detected CIN lesion). RESULTS: Participation in the hrHPV-based programme was significantly lower than that in the cytology-based programme (61% vs 64%). Screen positivity and direct referral rates were significantly higher in the hrHPV-based programme (positivity rate: 5% vs 9%; referral rate: 1% vs 3%). CIN2+ detection increased from 11 to 14 per 1000 women screened. Overall, approximately 2.2 times more clinical irrelevant findings (i.e. ≤CIN1) were found in the hrHPV-based programme, compared with approximately 1·3 times more clinically relevant findings (i.e. CIN2+); this difference was mostly due to a national policy change recommending colposcopy, rather than observation, of hrHPV-positive, ASC-US/LSIL results in the hrHPV-based programme. CONCLUSIONS: This is the first time that comprehensive results of nationwide implementation of hrHPV-based screening have been reported using high-quality data with a long follow-up. We have shown that both benefits and potential harms are higher in one screening round of a well-implemented hrHPV-based screening programme than in an established cytology-based programme. Lower participation in the new hrHPV programme may be due to factors such as invitation policy changes and the phased roll-out of the new programme. Our findings add further to evidence from trials and modelling studies on the effectiveness of hrHPV-based screening.

Sexual inactivity and occurrence of STIs in relation to weight status in women: Two large population-based studies.

The aim of this study was to examine sexual inactivity and occurrence of selected sexually transmitted infections in relation to body mass index. We used data from two large Danish population-based cross-sectional studies conducted in 1991-1995 (HPV study: 6869 women, aged 22-32 years) and in 2004-2005 (Liva study: 19,484 women, aged 18-45 years). Data were collected using a structured interview and measured weight, height, high-risk human papillomavirus DNA, Chlamydia DNA for the HPV study and a structured questionnaire for the Liva study. Overweight and obese women were more likely to have had no lifetime sexual partner or no sexual partner in the last year, e.g., obese women had a threefold (95 percent CI: 1.95-5.04) odds ratio of having had no sexual partner in the last year compared to normal weight women. Additionally, overweight and obese women had a lower likelihood of genital warts and high-risk human papillomavirus infection. A similar tendency was found for self-reported Chlamydia, but not with presence of Chlamydia DNA. If higher likelihood of no lifetime or recent sexual partners among overweight and obese women reflects unmet sexual needs, it could give rise to concern because quality of sexual life is associated with general quality of life.

Vaginal dysbiosis seems associated with hrHPV infection in women attending the Dutch Cervical Cancer Screening Program.

Human papillomavirus (HPV) is a sexually transmitted virus, which infects approximately 80% of all men and women at some time in their lives. Usually, the infection is resolved successfully by the body's immune system. Persistent infection with high-risk HPV (hrHPV) is necessary but not sufficient for cervical cancer development, and additional factors, such as the vaginal microbiome (vaginome), are thought to be involved. The aim of this study is to investigate whether either vaginal dysbiosis (imbalance in vaginal bacterial composition) or sexually transmitted pathogens, e.g., Chlamydia trachomatis (CT), are possible cofactors for hrHPV infection and HPV-induced cervical dysplasia in asymptomatic women attending the Dutch Cervical Cancer Screening Program. In this study, 492 hrHPV-positive and 500 hrHPV-negative cervical smears from women attending the Screening Program were included. Age and cytology were known for the hrHPV-positive samples. All cervical smears were diluted in Aptima® specimen transfer medium and tested with Aptima® transcription-mediated amplification assays targeting CT, Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Candida spp. (CS), C. glabrata (CG), Trichomonas vaginalis (TV), and bacterial vaginosis (BV). The prevalences of CT, NG, MG, CS, CG, TV, and BV in this cohort were found to be 1.9%, 0.0%, 1.7%, 5.4%, 1.4%, 0.1%, and 27.2%, respectively. When comparing HPV groups, it was found that CT, MG, and BV had a significantly higher prevalence in hrHPV-positive smears as compared with hrHPV-negative samples (for all p < 0.001). No significant differences were found when comparing different age groups and cytology outcomes. In conclusion, vaginal dysbiosis seems associated with hrHPV infection in women attending the Dutch Cervical Cancer Screening Program.

Trends in molecular diagnostics: 12th European Meeting on Molecular Diagnostics, Noordwijk aan Zee, the Netherlands, 12-14 October 2022.

This report provides an overview of the highlights of the 12th European Meeting on Molecular Diagnostics held in Noordwijk aan Zee, The Netherlands, 12-14 October 2022. This 3-day conference covered many relevant topics in the field of molecular diagnostics in humans i.e. oncology, infectious diseases, laboratory medicine, pharmacogenetics, pathology, and preventive medicine. Other relevant topics included quality management, laboratory automation, diagnostic preparedness, and lessons learned from the COVID pandemic. More than 400 participants, the majority coming from European countries, attended the meeting. Besides high-quality scientific presentations, more than 40 diagnostic companies presented their latest innovations, altogether in an informal and inspiring ambiance.

Sociodemographic Characteristics and Screening Outcomes of Women Preferring Self-Sampling in the Dutch Cervical Cancer Screening Programme: A Population-Based Study.

BACKGROUND: In the Netherlands, lower high-risk human papillomavirus (hrHPV) positivity but higher cervical intraepithelial neoplasia (CIN) 2+ detection were found in self-collected compared with clinician-collected samples. To investigate the possible reason for these differences, we compared sociodemographic and screening characteristics of women and related these to screening outcomes. METHODS: We extracted data from PALGA on all primary hrHPV screens and associated follow-up tests for 857,866 screened women, invited in 2017 and 2018. We linked these data with sociodemographic data from Statistics Netherlands. Logistic regression was performed for hrHPV positivity and CIN 2+/3+ detection. RESULTS: Out of the 857,866 women, 6.8% chose to use a self-sampling device. A higher proportion of self-sampling users was ages 30 to 35 years, was not previously screened, was living in a one-person household, or was the breadwinner in the household. After adjustment for these factors self-sampling had lower hrHPV positivity (aOR, 0.65; 95% CI, 0.63-0.68)) as compared with clinician-collected sampling, as well as lower odds of CIN 2+ (aOR, 0.76; 95% CI, 0.70-0.82) and CIN 3+ (aOR, 0.86; 95% CI, 0.78-0.95) detection. CONCLUSIONS: It is likely that the observed differences between the two sampling methods are not only related to sociodemographic differences, but related to differences in screening test accuracy and/or background risk. IMPACT: Self-sampling can be used for targeting underscreened women, as a more convenient screening tool. Further investigation is required to evaluate how to implement self-sampling, when it is used as a primary instrument in routine screening. See related commentary by Arbyn et al., p. 159.

Comparative study using phenotypic, genotypic, and proteomics methods for identification of coagulase-negative staphylococci.

Five methods were compared to determine the most accurate method for identification of coagulase-negative staphylococci (CoNS) (n = 142 strains). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed the best results for rapid and accurate CoNS differentiation (99.3% of strains correctly identified). An alternative to this approach could be Vitek2 combined with partial tuf gene sequencing (100% of strains correctly identified when both methods are performed simultaneously).

Comprehensive analytical and clinical evaluation of a RNA extraction-free saliva-based molecular assay for SARS-CoV-2.

Standard SARS-CoV-2 testing protocols using nasopharyngeal/throat (NP/T) swabs are invasive and require trained medical staff for reliable sampling. In addition, it has been shown that PCR is more sensitive as compared to antigen-based tests. Here we describe the analytical and clinical evaluation of our in-house RNA extraction-free saliva-based molecular assay for the detection of SARS-CoV-2. Analytical sensitivity of the test was equal to the sensitivity obtained in other Dutch diagnostic laboratories that process NP/T swabs. In this study, 955 individuals participated and provided NP/T swabs for routine molecular analysis (with RNA extraction) and saliva for comparison. Our RT-qPCR resulted in a sensitivity of 82,86% and a specificity of 98,94% compared to the gold standard. A false-negative ratio of 1,9% was found. The SARS-CoV-2 detection workflow described here enables easy, economical, and reliable saliva processing, useful for repeated testing of individuals.

Rapid genotyping of human papillomavirus by post-PCR array-based hybridization techniques.

Kinetic hybridization measurements on a microarray are expected to become a valuable tool for genotyping applications. A method has been developed that enables kinetic hybridization measurements of PCR products on a low-density microarray. This is accomplished by pumping a solution containing PCR products up and down through a porous microarray substrate. After every pumping cycle, the fluorescently labeled PCR products hybridized to capture probes immobilized on the solid surface of the porous microarray substrate are measured. By this method, both binding curves and high-resolution melting curves are obtained instead of the single endpoint hybridization intensities as with commonly used post-PCR array-based hybridization techniques. We used 20 subtypes of the human papillomavirus (HPV) as a model system to test our detection method and blindly analyzed 216 clinical samples. We compared our microarray flowthrough method with a reference method, PCR followed by a reverse line blot (RLB). Real-time hybridization measurements followed by high-resolution melting curves of low concentrations of fluorescently labeled HPV targets on a microarray were successfully carried out without any additional chemical signal amplification. The results of our new method were in good agreement (93%, with a kappa coefficient of κ = 0.88 [95% CI, 0.81 to 0.94]) with the RLB results. All discrepant samples were analyzed by a third method, enzyme immunoassay (EIA). Furthermore, in a number of cases, we were able to identify false-positive samples by making use of the information contained in the kinetic binding and melting curves. This clearly demonstrates the added value of the use of kinetic measurements and high-resolution melting curves, especially for highly homologous targets.

Clinical performance of high-risk HPV testing on self-samples versus clinician samples in routine primary HPV screening in the Netherlands: An observational study.

BACKGROUND: High-risk human papillomavirus (hrHPV) testing on self-collected samples has potential as a primary screening tool in cervical screening, but real-world evidence on its accuracy in hrHPV-based screening programmes is lacking. METHODS: In the Netherlands, women aged 30-60 years invited for cervical screening can choose between sampling at the clinician's office (Cervex Brush) or self-sampling at home (Evalyn Brush). HrHPV testing is performed using Roche Cobas 4800. We collected screening test results between January 2017 and March 2018 and histological follow-up until August 2019. The main outcome measures were mean cycle threshold (Ct) value, cervical intraepithelial neoplasia (CIN) grade 3 or cancer (CIN3+) and CIN grade 2 or worse (CIN2+). FINDINGS: 30,808 women had a self-collected and 456,207 had a clinician-collected sample. In hrHPV-positive women with adequate cytology, Ct values were higher for self-collection than clinician-collection with a mean Ct difference of 1·25 (95% CI 0·98-1·52) in women without CIN2+, 2·73 (1·75-3·72) in CIN2 and 3·59 (3·03-4·15) in CIN3+. The relative sensitivity for detecting CIN3+ was 0·94 (0·90-0·97) for self-collection versus clinician-collection and the relative specificity was 1·02 (1·02-1·02). INTERPRETATION: The clinical accuracy of hrHPV testing on a self-collected sample for detection of CIN3+ is high and supports its use as a primary screening test for all invited women. Because of the slightly lower sensitivity of hrHPV testing on a self-collected compared to a clinician-collected sample, an evaluation of the workflow procedure to optimise clinical performance seems warranted. FUNDING: National Institute for Public Health and the Environment (the Netherlands) and the European Commission.

Performance analysis of high-throughput HPV testing on three automated workflows.

Primary high-risk human papillomavirus (hrHPV) DNA testing has been introduced in several countries worldwide, including The Netherlands. The objective of this study was to compare three automated workflow procedures for hrHPV testing of which the hrHPV detection assays meet the international guidelines for HPV testing. To mimic a realistic screening situation, we aimed to process 15 000 residual PreservCyt cervical samples in a period of 3 months. During a 3 months period, four technicians were involved in processing 5000 specimens per month on three automated platforms, (1) Qiagen Digene® HC2 HPV DNA test (HC2, signal amplification); (2) Roche Cobas® HPV test (DNA amplification), and (3) Hologic Aptima® HPV test (RNA amplification). We measured and scored general aspects (time-to-results, hands-on-time (HOT)), maintenance, pre-run, run and post-run aspects, inventory (orders, storage), and number of errors on a scale from 1 to 10. As determined for one complete workflow each, maximum processing capacity and HOT were 296 samples and 2 h:55 m, 282 samples and 3 h:20 m, and 264 samples and 4 h:15 m for Aptima, Cobas, and HC2, respectively. The mean throughput time per run was 5 h:51 m for Cobas in which 94 samples could be processed. For Aptima, the mean throughput time per run was 6 h:30 m for 60 samples. Mean throughput time for HC2 is longer since results were provided on day 2. In this study, the fully automated Aptima workflow scores best with a 7.2, followed by Cobas with a score of 7.1 and HC2 with a score of 5.8. Although all HPV tests used in this comparison meet the international test guidelines, the performance (workflow) characteristics of the assays vary widely. A specific choice of a laboratory for high-throughput testing can be different based on the laboratory's demands, but also hands-on-time, time-to-results/ # samples, maintenance, pre-run, run and post-run parameters, consumables, technical support, and number of errors are important operational factors for the selection of a fully automated workflow for hrHPV testing.

Histological characteristics of human papilloma-virus-positive and -negative invasive and in situ squamous cell tumours of the penis.

A high prevalence of cervical cancer associated high-risk types of human papillomavirus (hrHPV) has been demonstrated in premalignant and invasive squamous cell lesions of the penis, but large studies correlating histological characteristics with HPV status are few in number. Tumour tissues from 145 patients with invasive (n = 116) or in situ (n = 29) penile squamous cell carcinoma were subjected to systematic histological evaluation and were PCR-tested for 14 hrHPV types and 23 low-risk HPV types. Around half (52%) of invasive and nine-tenths (90%) of in situ lesions were positive for an hrHPV type, of which HPV 16 was by far the predominant type (91% of hrHPV-positive lesions). In relation to histological characteristics, hrHPV positivity was statistically significantly more common in high-grade tumours, lesions dominated by small tumour cells, lesions with a high number of multinucleated cells and mitoses, and lesions with a small amount of parakeratosis. In conclusion, about half of invasive penile squamous carcinomas in this study were hrHPV-positive, most notably to HPV 16, and probably arose through in situ lesions whereas the other half of invasive penile lesions appeared to be unrelated to hrHPV. A number of histological characteristics differed significantly between hrHPV-positive and -negative invasive penile carcinomas.

Soluble mannose receptor levels in blood correlate to disease severity in patients with community-acquired pneumonia.

PURPOSE: Community-acquired pneumonia (CAP) is the most common form of pneumonia and is a leading infectious cause worldwide. Identification of patients that are at risk to develop severe disease has proven to be a major challenge. Soluble mannose receptor (sMR; sCD206) is a new serum marker for macrophage activation. Recent studies showed that sMR levels are increased in patients suffering from severe infections making it a potential biomarker for improved discrimination of disease severity. For measuring sMR, no standardized assay is available. Aim of this study is to develop an assay for standardized measurement of sMR. Next, this assay was used to assess sMR plasma levels for its ability to predict severe disease development in a patient cohort for community-acquired pneumonia. METHODS: We developed a well-validated sandwich ELISA that enables standardized measurement of sMR in plasma and serum samples. Repeatability was tested by calculating the percentage coefficient of variation (%CV) within and between runs and within and between operators. sMR levels were assessed in a cohort of 100 patients with community-acquired pneumonia. RESULTS: All %CV values were <10%, indicating low variation. Higher sMR levels were observed in patients with severe disease when compared to patients without severe disease development (p = 0.004). Patients with sMR levels between 100-430 ng/ml had 22.7% chance to develop severe disease whereas patients with levels between 430-1000 ng/ml had 33.3% chance to develop severe disease. CONCLUSIONS: We suggest that sMR has potential as a new biomarker for the prediction of disease severity in patients with community-acquired pneumonia.

Risk factors for invasive squamous cell carcinoma of the vulva and vagina--population-based case-control study in Denmark.

The etiology of vulvar and vaginal squamous cell carcinoma (VV-SCC) has received little attention. A total of 182 women with invasive VV-SCC (116 with VV-SCC(vulva), 66 with VV-SCC(vagina)), 164 uterine corpus cancer controls and 518 population controls were interviewed in a population-based case-control study in Denmark, and 87 (48%) of the VV-SCC cases had tissue samples examined for human papillomavirus (HPV) DNA using the GP5+/6+ PCR-EIA assay and subsequent reverse line blotting for HPV typing. Logistic regression-derived odds ratios with 95% confidence intervals served as relative risks. Cervical cancer-associated high-risk HPVs (hrHPVs) were detectable in most (89%) examined cases of VV-SCC(vagina) and in half (50%) of cases of VV-SCC(vulva) (p < 0.001). In site-specific multivariate logistic regression analyses, statistically significant risk factors for both VV-SCC(vulva) and VV-SCC(vagina) included measures of hrHPV exposure (anogenital warts for VV-SCC(vulva); cervical neoplasia and poor genital hygiene for VV-SCC(vagina)), tobacco smoking and alcohol consumption. Furthermore, socioeconomic variables (marital status and years at school) were associated with risk of VV-SCC(vulva). Comparing hrHPV-positive and hrHPV-negative VV-SCCs in polytomous logistic regression analysis revealed that tobacco smoking and cervical neoplasia were significant risk factors only for hrHPV-positive VV-SCCs. Our study shows that VV-SCC(vulva) and VV-SCC(vagina) share measures of prior hrHPV exposure, tobacco smoking and alcohol consumption as statistically significant risk factors. HPV vaccination programs aimed at reducing the burden of cervical cancers are likely to also provide considerable protection against VV-SCCs.

Risk factors for squamous cell carcinoma of the penis--population-based case-control study in Denmark.

Few etiologic studies of squamous cell carcinoma (SCC) of the penis have been carried out in populations where childhood circumcision is rare. A total of 71 patients with invasive (n=53) or in situ (n=18) penile SCC, 86 prostate cancer controls, and 103 population controls were interviewed in a population-based case-control study in Denmark. For 37 penile SCC patients, tissue samples were PCR examined for human papillomavirus (HPV) DNA. Overall, 65% of PCR-examined penile SCCs were high-risk HPV-positive, most of which (22 of 24; 92%) were due to HPV16. Penile SCC risk was positively associated with measures of early and high sexual activity, including lifetime number of female sex partners, number of female sex partners before age 20, age at first intercourse, penile-oral sex, a history of anogenital warts, and never having used condoms. Histories of phimosis and priapism at least 5 years before diagnosis were also significant risk factors, whereas alcohol abstinence was associated with reduced risk. Our study confirms sexually transmitted HPV16 infection and phimosis as major risk factors for penile SCC and suggests that penile-oral sex may be an important means of viral transmission. The association with priapism was unexpected and needs replication.

Circumferential margin involvement is the crucial prognostic factor after multimodality treatment in patients with locally advanced rectal carcinoma.

PURPOSE: After preoperative (radio)chemotherapy, histologic determinants for prognostication have changed. It is unclear which variables, including assessment of tumor regression, are the best indicators for local recurrence and survival. EXPERIMENTAL DESIGN: A series of 201 patients with locally advanced rectal cancer (cT3/T4, M0) presenting with an involved or at least threatened circumferential margin (CRM) on preoperative imaging (<2 mm) were evaluated using standard histopathologic variables and four different histologic regression systems. All patients received neoadjuvant radiochemotherapy or radiotherapy. The prognostic value of all factors was tested with univariate survival analysis of time to local recurrence and overall survival. RESULTS: Local recurrence occurred in only 8% of the patients with a free CRM compared with 43% in case of CRM involvement (P < 0.0001). None of the four regression systems were associated with prognosis, not even when corrected for CRM status. However, we did observe a higher degree of tumor regression after radiochemotherapy compared with radiotherapy (P < 0.001). Absence of tumor regression was associated with increasing invasion depth and a positive CRM (P = 0.02 and 0.03, respectively). CONCLUSIONS: Assessment of CRM involvement is the most important pathologic variable after radiochemotherapy. Although tumor regression increases the chance on a free CRM, in cases with positive resection margins prognosis is poor irrespective of the degree of therapy-induced regression.

Cyclooxygenase 2 expression in rectal cancer is of prognostic significance in patients receiving preoperative radiotherapy.

PURPOSE: To determine the effect of cyclooxygenase (COX)-2 expression on clinical behavior in irradiated and nonirradiated rectal carcinomas. EXPERIMENTAL DESIGN: Tumor samples were collected from 1,231 patients of the Dutch TME trial, in which rectal cancer patients were treated with standardized surgery and randomized for preoperative short-term (5 x 5 Gy) radiotherapy or no preoperative radiotherapy. Tissue microarrays were constructed from primary tumor material, and COX-2 expression was assessed by immunohistochemistry. Tumor cell apoptosis was determined by M30 immunostaining. RESULTS: A high level of COX-2 expression after radiotherapy was associated with low levels of tumor cell apoptosis (P=0.001). COX-2 expression had no significant effect on patient survival or tumor recurrence in nonirradiated tumors. However, in patients receiving preoperative radiotherapy, high level of COX-2 expression was associated with higher incidence of distant recurrences [P=0.003; hazard ratio (HR), 1.7; 95% confidence interval (95% CI), 1.2-2.5] and shorter disease-free survival (P=0.002; HR, 1.8; 95% CI, 1.2-2.5) and overall survival (P=0.009; HR, 1.5; 95% CI, 1.1-2.0), independent of patient age, tumor stage, tumor location, or the presence of tumor cells in the circumferential resection margin. CONCLUSIONS: A high level of COX-2 expression after preoperative radiotherapy in resection specimens is associated with apoptosis resistance, high distant recurrence rates, and a poor prognosis in rectal cancer.

Morphological evidence of an activated cytotoxic T-cell infiltrate in EBV-positive gastric carcinoma preventing lymph node metastases.

Recently, we showed that Epstein-Barr virus (EBV)-positive gastric carcinoma (GC) forms a distinct clinicopathologic entity with a better prognosis due to lower incidence of lymph node metastases (LN+). Here we investigated whether in EBV-positive GC more pronounced activation of cellular immune responses is associated with absence of (micro)metastases. Twenty EBV-positive primary tumors (PT) (9 LN+) were matched with 28 EBV-negative GC (11 LN+) for T- and N-stage, gender, and age. The PT (n = 28) and its LNs were analyzed by EBER RNA in situ hybridization and by immunohistochemistry for MHC class I and II expression, for CD3, CD8, CD4, CD20, CD56, CD83, and Granzyme B (GzB) expression. In LN metastases of EBV-positive GC, the EBV genome is maintained, excluding tumor escape by virus deletion. All GC express MHC class I independently of EBV status. In comparison with EBV-negative GC, EBV-positive GC have higher expression of MHC class II on the tumor cells (P = 0.029) and a more extensive infiltrate (P < 0.0001) of activated GzB+ CD8+ T cells (P = 0.028), which is most abundant in those EBV-positive tumors that do not metastasize (P < 0.0001). In addition, in EBV-positive GC without metastases, the infiltrate contains higher numbers of mature dendritic cells (DC) (P = 0.018). At present, the antigenic target has to be determined. These data support the notion that local triggering of cellular immune responses in EBV-positive GC prevents lymph node metastasis formation.

Detection of HER2 amplification in breast carcinomas: comparison of Multiplex Ligation-dependent Probe Amplification (MLPA) and Fluorescence In Situ Hybridization (FISH) combined with automated spot counting.

In this study the detection of HER2 gene amplification was evaluated using Fluorescence In Situ Hybridization (FISH; PathVysion) in comparison with Multiplex Ligation-dependent Probe Amplification (MLPA), a PCR based technique. These two methods were evaluated on a series of 46 formalin fixed paraffin embedded breast carcinomas, previously tested for protein overexpression by HercepTest (grouped into Hercep 1+, 2+ and 3+). HER2 gene amplification (ratio > or =2.0) by FISH was found in 9/10, 10/30 and 0/6 in IHC 3+, 2+ and 1+/0 cases, respectively. Digitalized automated spot counting performed with recently developed CW4000 CytoFISH software was 100% concordant with manual FISH scoring. Using MLPA 18/46 samples showed a clear HER2 amplification. Comparing MLPA and IHC showed the same results as for FISH and IHC. All but one FISH positive cases (18/19) were confirmed by MLPA for the presence of the gene amplification. The overall concordance of detection of Her2 gene amplification by FISH and MLPA was 98% (45/46). Furthermore, both the level of amplification and equivocal results correlated well between both methods. In conclusion, MLPA is a reliable and reproducible technique and can be used as an either alternative or additional test to determine HER2 status in breast carcinomas.