To Örebro University

OBJECTIVE: To investigate whether self-sampling is as reliable as professional sampling for HPV testing and genotype detection among postmenopausal women.

METHODS: In the present prospective cross-sectional study, women in Örebro County, Sweden, who had high-risk HPV (hrHPV) and normal cytology results in exit screening tests conducted in between January 1, 2012, and December 31, 2014, were invited to follow-up screenings between February 24, 2015 and May 15, 2015, that included professional sampling and self-sampling. HPV genotypes were identified by a DNA-based assay that could detect 35 HPV genotypes. Findings between the different sampling methods were compared.

RESULTS: Of 143 women who participated, 119 returned a self-sample. Completely concordant results were observed in 67 of these samples when both hrHPV and low-risk HPV genotypes were analyzed. Overall, 99 (83.2%) women had the same clinically relevant finding from both sampling methods. Twenty women had discordant hrHPV results (hrHPV detected in 10 self-samples vs 10 professionally collected samples; Cohen κ 0.66, 95% confidence interval 0.53-0.80). There was no significant difference between the two sampling methods for clinically significant infections (P>0.99) or extended genotyping (P=0.827).

CONCLUSION: Postmenopausal women could be offered self-sampling devices to increase screening-program coverage while maintaining test quality.

AIM: Many cervical cancers occurs among women over 65 and prevalence of HPV genotypes in this age cohort is sparingly studied. One aim of this study was to study the prevalence and distribution of HPV genotypes in women 55-59 years, with normal cytology when exiting the screening program. Secondly, HPV clearance as well as the value of HPV genotyping and/or liquid based cytology as triage tests for identifying histological dysplasia among women with persistent HPV was studied.

METHODS: Women that exited the screening program with normal cytology, between the years 2012-2014, in Örebro County, Sweden, were invited to this study. A total of 2946 samples were analyzed with a broad-spectrum assay to detect both hrHPV and lrHPV in order to investigate the distribution of genotypes. In the consent group, women with a positive hrHPV test were offered a follow-up test and a cone biopsy for histological confirmation, and a follow up sample 6 months post cone.

RESULTS: The overall prevalence of hrHPV was 7.4% and 59% of them remained hrHPV positive in a follow-up test after 12 months. A total of 99 women had a cone biopsy done, where 19% showed histological dysplasia. HPV 53 was the most common genotype, and among women with histology confirmed LSIL or HSIL, HPV 31 was most common. A positive hrHPV result showed a PPV of 25% for LSIL+ and 12.5%for HSIL+. Using detection of HPV 16/18 genotypes as a triage test for hrHPV positive tests, indicated FNR for histological LSIL+ and HSIL+ of 94% and 87.5% respectively, whilst triage based on cervical cytology had a FNR of 69% for LSIL+ and 37.5% for HSIL+.

CONCLUSION: The most common hrHPV genotypes among women 55-59 years of age were non HPV16/18 genotypes, and in this population, these genotypes represented most of the histological verified HSIL lesions. This result does not support the proposition of a HPV 16/18 triaging test after a positive hrHPV test as a marker of histological HSIL+ cervical lesions in women over 55 years of age. Similarly, cytological triage after a positive hrHPV showed no additional benefit in this population. Specific triaging tests should be validated to follow post-menopausal women with a positive hrHPV test.

Background & objectives: HPV-positive penile tumours have been associated with higher survival rates. However, HPV analysis is unavailable in many low-income countries. We investigated if histological assessment of penile squamous cell carcinoma subtypes can replace HPV testing in determining HPV-related/non-HPV-related tumour status.

Methods: We reviewed paraffin-embedded tumour tissue from 345 penile cancer patients, surgically treated between 2009 and 2018 at Örebro University Hospital, Sweden. The histological subtype of squamous cell carcinoma was assessed according to the WHO criteria and ISUP recommendations. HPV-DNA genotyping was performed using the PCR method Anyplex II HPV28. Concordance was assessed by calculating Cohen’s kappa (κ).

Results: A good concordance was found between histological subtype of squamous cell carcinoma and HPV tumour-status with a Cohen’s kappa (κ) of 0,72 corresponding to 86,6% agreement. Of the 46 discordant cases, five had HPV-related histology (mixed subtypes) but were HPV-negative. The remaining 41 cases had non-HPV- related histology (85% usual subtype, 15% mixed subtypes) but were HPV-positive. Noteworthy is that in 21 of the cases with non-HPV-related histology, foci of undifferentiated PeIN was found. In addition, four cases with both undifferentiated PeIN and lichen sclerosus et atrophicus in the tumour margin, 14 cases with both differentiated PeIN and lichen sclerosus et atrophicus and two cases without preneoplastic lesion were identified.

Conclusion: Good concordance between histological subtype of penile squamous cell carcinoma and HPV genotyping shows that when necessary, histological assessment is a good alternative, at least in less resourceful settings, to PCR-based HPV analysis in determining if penile tumours are HPV or non-HPV-related. Discordant cases most likely depend on subjectivity in histological assessment but can also suggest a HPV infection in a non-HPV-related tumour.

Inguinal lymph node surgery is a standard treatment for penile cancer patients with intermediate or high risk for lymph node metastasis (LNM) according to European Association of Urology (EAU) risk grading. We are proposing a more objective histological prognostic grading system for inguinal LNM in these patients. We assessed worst pattern of invasion, lymphocytic host response, lymphovascular invasion, and perineural invasion in a population-based cohort of 306 penile cancer patients. Patients were classified into low, intermediate, and high risk for inguinal LNM. There was a significant association both between risk groups and pT stage (p < 0.001) and between risk groups and LNM. Univariate logistic regression showed 25.43 times higher odds of LNM for patients in the intermediate risk group compared with the low risk group (odds ratio (OR) 25.43; 95% confidence interval (CI): 5.94-108.97) and a 177.13 times higher odds in the high risk group compared to the low risk group (OR 177.13; 95% CI: 40.09-782.51). When comparing our histological risk grading with the EAU grading, we found a higher sensitivity, of 51.28% (95% CI: 45.68-56.88) versus 37.09% (95% CI: 31.68-42.50), as well as a higher area under the curve (0.86; 95% CI: 0.81-0.89; versus 0.65; 95% CI: 0.58-0.71) with our grading system. While our grading classified 111 patients as low risk, only 31 were considered low risk for LNM according to the EAU risk classification. The new histological risk grading system shows a higher sensitivity and includes a higher number of patients in the low risk group in whom lymph node surgery could be avoided, reducing morbidity and costs.

Differentiation between penile squamous cell carcinoma patients who can benefit from limited organ-sparing surgery and those at significant risk of lymph node metastasis is based on histopathological prognostic factors including histological grade and tumor histological subtype. We examined levels of interobserver and intraobserver agreement in assessment of histological subtype and grade in 207 patients with penile squamous cell carcinoma. The cases were assessed by seven pathologists from three hospitals located in Sweden and Italy. There was poor to moderate concordance in assessing both histological subtype and grade, with Fleiss kappas of 0.25 (range: 0.02-0.48) and 0.23 (range: 0.07-0.55), respectively. When choosing HPV-associated and non-HPV-associated subtypes, interobserver concordance ranged from poor to good, with a Fleiss kappa value of 0.36 (range: 0.02-0.79). A re-review of the slides by two of the pathologists showed very good intraobserver concordance in assessing histological grade and subtype, with Cohen's kappa values of 0.94 and 0.91 for grade and 0.95 and 0.84 for subtype. Low interobserver concordance could lead to undertreatment and overtreatment of many patients with penile cancer, and brings into question the utility of tumor histological subtype and tumor grade in determining patient treatment in pT1 tumors. 

Background: Human papilloma virus (HPV) is involved in the development of cancer of the cervix, mouth and throat, anus, penis, vulva, or vagina, but it has not been much considered as a cause of breast cancer. Recently, a number of investigations have linked breast cancer to viral infections. High-risk HPV types, predominantly HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, are established as carcinogens in humans. In this study we aimed to detect 19 high-risk and 9 low-risk HPVs from archived breast tumor tissue among Ethiopian women.

Methods: In this study, 75 breast cancer patients from Tikur Anbassa Specialized Hospital in Addis Ababa (Ethiopia) were included. HPV detection and genotyping were done using the novel Anyplex™ II HPV28 Detection Assay at the Orebro University Hospital, Sweden. The Anyplex™ II PCR System detects 19 high-risk HPV types (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82) and 9 low-risk HPV types (6, 11, 40, 42, 43, 44, 54, 61, and 70). IHC for p16 was done using an automated system, the Dako Autostainer Link.

Results: Out of the 75 valid tests, two were found to be positive (2.7%) for HPV. One of the cases was positive for the high-risk HPV16 genotype while the other was positive both for the high-risk HPV39 and the low-risk HPV6. The cell cycle protein p16 was highly expressed in the case positive for the high-risk HPV16, but it was not expressed in the case positive for HPV39.

Conclusion: The prevalence of HPV is low in Ethiopian breast cancer patients, but the role played by HPV in breast carcinogenesis among Ethiopian breast cancer patients cannot be commented based on these observations.

BACKGROUND: With HPV screening the specificity of screening positives has decreased, even with a cytological triage test. Increases in colposcopies and detection of benign or low-grade dysplasia are reported, not least in older women. These results highlight the necessity to find other triage tests in HPV screening strategies, so that women can be more accurately selected for colposcopy, thus minimizing the clinically irrelevant findings.

METHODS: The study included 55- to 59-year-old women who exited the screening with normal cytology, but later in a follow-up test were positive for the HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 and had a cervical cone biopsy done. To model a screening situation with hrHPV-positive women, three different triage strategies, namely, cytology, genotyping and methylation, were performed. The study considered the effect of direct referral to colposcopy for HPV genotypes 16, 18, 31, 33, 45, 52 and 58, and methylation for FAM19A4 and hsa-mir124-2 and/or any form of abnormal cytology.

RESULTS: Seven out of 49 women aged 55-59 years with hrHPV had a cone biopsy with high-grade squamous intraepithelial lesion. No triage method found all cases, and when comparing positive and negative predictive value and false negative rate, cytology showed better results than genotyping and methylation.

CONCLUSION: This study does not support a switch in triage strategies from cytology to hrHPV genotyping and methylation for women above 55 years of age yet, but demonstrates the need for more evidence on molecular triage strategies.

Background: An updated screening algorithm was introduced in Sweden 2015. Primary HPV test for women >30 years old and a prolonged screening with the last test after 64 years of age were some of the changes. In the region of Örebro County, the previous cut-off age was 60 years and with a screening interval of 5 years, women left their last sample when they were 55-59 years old. In the shift between two screening programs, a group of women, 60-64 years old, that left the program 5-10 years ago were now included in the new screening. For re-inclusion, a two year long program was formed to catch-up this group of women and screen them according to the new screening algorithm. At the same time a research project investigating self-sampling was launched. At the same time as the women were invited for a last screening sample they were also asked to participate in a study where they should take a vaginal self-test up to one week after their ordinary screening sample was taken by a midwife.

Method: Postmenopausal women between 64-70 years was included in the study. HPV status in samples from midwife sampling (MS) was compared to self-sampling (SS) samples. HPV was analyzed using HPV Aptima and all HPV positive samples, independent of sampling method, was triaged with cytology and followed-up according to national guidelines.

Results: So far, 585 women with paired samples have been included in the study. In the MS, 4% of the women are positive for hrHPV compared to 11% in the SS group. In 486/585 women, the results of the two samples are concordant. Among the non-concordant samples (13%), 62% were positive in SS and negative in MS. The opposite, negative in SS and positive in MS were seen in 4% of the samples. Among the MS negative samples, 32% were invalid in SS. Cytology was used as a triage test for HPV positive women, both for MS and SS. Of 23 hrHPV positive, 18 had normal cytology, 2 ASCUS, 1 LSIL and 1 HSIL. In the samples with abnormal cytology, 4/5 were hrHPV positive in both SS and MS. One sample was positive in SS but negative in MS.

Discussion: In this age group, more women are hrHPV positive in SS compared to MS. This is in line with what other have seen. Among the very few hrHPV positive samples with abnormal cytology, the majority was hrHPV positive in both MS and SS. But since cytology is a poor triage marker in this age group clinical follow-up is needed before the effectiveness of the both sampling methods can be concluded.

BACKGROUND: The aim of this study was to examine the natural course of HPV infection in women of 60 years and older who were HPV positive at inclusion, and any association between HPV positivity in historical samples and dysplasia outcome. METHODS: Eighty-nine women aged 60-82 years, who tested positive for HPV between 2012 and 2016 were included. Sampling for cytology and/or histology was also performed. HPV genotyping was carried out on archived material back to 1999.

RESULTS: Of the 89 HPV-positive women 16 had HSIL, 34 had LSIL and 39 were benign at inclusion. Of the women with HSIL, 50.0% had the same HPV type in the archive samples, 12.5% had another type, and 37.5% were HPV negative. Among the 34 women with LSIL, 47.1% had the same HPV type in archive samples, 5.8% had another type, and 47.1% were HPV negative. Of the 39 women without dysplasia at inclusion, 25.6% had the same HPV type in archive samples, 5.1% had another HPV type and 69.2% were HPV negative.

CONCLUSION: Surprisingly few of the elderly women thus seem to have a history with the same or any HPV infection the years before being diagnosed with an HPV infection and dysplasia. The significance of an HPV infection for dysplasia development in elderly women is still not fully understood.

PURPOSE: Cervical cancer prevention for older women can be challenging since there are no specific guidelines for this group. This study aimed to determine the incidence of oncogenic HPV and HPV-related dysplasia in elderly women 5 years after being HPV negative.

METHODS: Invited women participated five years earlier in a study where self-sampling for HPV testing was applied, at this time, they were all HPV negative. The women were now, five years later invited to perform self-sampling for HPV testing. Women with a positive result performed a repeat HPV test. Those with a positive repeat HPV test were examined by colposcopy, biopsy and cytology.

RESULTS: Of the 804 invited women, 634 (76.9%) agreed to participate in the study and a self-sampling kit was sent to them. Of these, 99.6% (632/634) sent a sample to the HPV laboratory. The participation rate in each age group was 93.3% at age 65, 74.0% at age 70, 80.7% at age 75 and 64.6% at age 80. Overall 18 women (2.8%, 95% CI 3.2 to 6.0) were HPV positive in the first test and 8 (1.3%, 95% CI 0.6 to 2.6) in the second test. Sampling for the second test was done on average 5.4 months after the first test. Fifty per cent (4/8) of the women with a positive repeat test had dysplasia in histology.

CONCLUSION: The incidence of HPV in previously HPV-negative elderly women was low. Among women who were HPV positive in a repeat test, there was a high prevalence of low grade dysplasia.

BACKGROUND: Cervical cancer is a global disease and it is well established that cervical cancer is caused by human papillomavirus (HPV). In Sweden self-sampling for HPV is now used as a complement to sampling performed by a midwife. However, there is a lack of knowledge on how older women perceive the self-sampling compared to the sampling performed by a midwife. Therefore, the aim of the study was to describe how women, aged 64 years and older, perceived the process of self-sampling and sampling performed by a midwife for HPV-testing.

METHODS: Eighteen women were included in a qualitative interview study, and a phenomenographic approach was used for the analysis of the interviews.

RESULTS: Three descriptive categories emerged: Confidence in sampling, Facilitating participation and Being informed. Within the categories, eight conceptions emerged describing the variation relating to how the women perceived the process of self-sampling and sampling performed by a midwife.

CONCLUSIONS: Women in this study describe confidence in self-sampling for HPV-testing and that the self-sampling was saving time and money, both for themselves and for society. Information in relation to an HPV-positive test result is of importance and it must be kept in mind that women affected by HPV may feel guilt and shame, which health care professionals should pay attention to. This knowledge can be used in education of health care staff.

Despite the common perception that the human papilloma virus (HPV) is a requirement for the development of cervical cancer (CC), a considerable number of CCs test HPV negative. Presently, many countries are shifting to HPV primary CC screening, and it is of importance to increase the knowledge about the group of CCs that test HPV negative. The aim of this study was to reinvestigate a proportion of cervical tumors with a primary negative or invalid test result. Reinvestigation with repeated genotyping (targeting L1) was followed by analysis with an alternative target method (targeting E6/E7) on existing or additional tumor material. Consistently negative tumors were histologically evaluated, and cases with low or lacking tumor cell content, consistent invalid test results, or with suspicion of other than cervical origin were excluded. HPV-negative cases were thereafter subjected to immunohistochemistry (Cytokeratin 5, pan cytokeratin, protein 63, P16, and P53). The HPV-negative proportion could after reinvestigation be reduced by one-half (14%-7%). Additional positive samples were often detected in late polymerase chain reaction cycles, with an alternative (E6/E7) or the same (L1) target, or with a method using shorter amplicon lengths. Confirmed HPV negativity was significantly associated with worse prognosis, high patient age, longer storage time, and adenocarcinoma histology. Some of the HPV-negative cases showed strong/diffuse p16 immunoreactivity, indicating some remaining false-negative cases. False HPV negativity in this cohort was mainly linked to methodological limitations in the analysis of stored CC material. The small proportion of presumably true HPV-negative adenocarcinomas is not a reason for hesitation in revision to CC screening with primary HPV testing.

Currently, cervical cancer prevention is undergoing comprehensive development regarding human papillomavirus (HPV) vaccination and cervical cancer screening. In Sweden and many other countries, high coverage vaccinated cohorts are entering screening within the next few years. This entails demands for baseline HPV genotype data across the screening age range for surveillance and a basis for screening program adjustment. In 2016, Örebro County, Sweden, changed to primary HPV screening using HPV mRNA testing followed by cytology triage. An alternative triage method to cytology could allow for a fully molecular screening algorithm and be implemented in a screening program where self-sampling is included. Hypermethylation analysis of the human genes FAM19A4/miR124-2 has been suggested as a promising triage method. HPV mRNA-positive screening samples (n = 529) were included and subjected to genotyping targeting a broad range of both low-risk and high-risk genotypes in addition to hypermethylation analysis of the two human genes FAM19A4/miR124-2. Data were connected to cytological and histological status and age. The most commonly detected genotypes were HPV31, 16, and 52. In addition, HPV18 was one of the most common genotypes in high-grade squamous intraepithelial lesions (HSILs) samples. In relation to available vaccines, 26% of the women with histological HSIL or cancer (≥HSIL) tested positive for only hrHPV included in the quadrivalent vaccine and 77% of the genotypes in the nonavalent vaccine. According to these figures, a relatively large proportion of the HSILs will probably remain, even after age cohorts vaccinated with the quadrivalent vaccine enter the screening program. Hypermethylation positivity was associated with increasing age, but no HPV-related independently predictive factors were found. Accordingly, age needs to be considered in development of future screening algorithms including triage with hypermethylation methodology.

The droplet digital PCR (ddPCR) system enables high-sensitivity detection of nucleic acids and direct absolute quantification of the targets. The aim of this research was to evaluate this system for viral load (VL) analysis of the human papillomavirus (HPV) genotypes HPV31, 35, 39, 51 and 56 measured in number of viral particles per cell. The sample types used for the optimization of the ddPCR assay were formalin-fixed paraffin-embedded (FFPE) tissues and cervical liquid cytology samples. The presently optimized ddPCR assays, together with assays optimized previously for HPV16, 18, 33 and 45, with the same ddPCR method, were used for the VL analysis of cervical tumor samples. Results published previously on the present study cohort showed that women with a cervical tumor containing multiple high-risk HPV genotypes had a worse prognosis compared to women with single-genotype-infected tumors. The VL was therefore analyzed in this study for the same cohort, as a possible explanatory factor to the prognostic differences. The results of the optimization part of the study, with analysis of VL using ddPCR in DNA from varying sample types (FFPE and liquid cytology samples), showed that each of the five assays demonstrated good inter- and intra-assay means with a coefficient of variation (CV) under 8% and 6% respectably. The cohort results showed no difference in VL between tumors with multiple and single HPV infections, and therefore did most likely not constitute a contributing factor for prognostic differences observed previously. However, tumors from women aged 60 years or older or containing certain HPV genotypes and genotype genera were associated with a higher VL.

Cervical cancer (CC) is one of the most common cancers in women and virtually all cases of CC are a result of a persistent infection of human papillomavirus (HPV). For disease detected in early stages there is curing treatment but when diagnosed late with recurring disease and metastasis there are limited possibilities. Here we evaluate HPV impact on treatment resistance and metastatic disease progression. Prevalence and distribution of HPV genotypes and HPV16 variants in a Swedish CC patient cohort (n=209) was evaluated, as well as HPV influence on patient prognosis. Tumor samples suitable for analysis (n=204) were genotyped using two different real-time PCR methods. HPV16 variant analysis was made using pyrosequencing. Results showed that HPV prevalence in the total series was 93%. Of the HPV-positive samples, 13% contained multiple infections, typically with two high-risk HPV together. Primary cure rate for the complete series was 95%. Recurrence rate of the complete series was 28% and distant recurrences were most frequent (20%). Patients with tumors containing multiple HPV-strains and particularly HPV genotypes belonging to the alpha 7 and 9 species together had a significantly higher rate of distant tumor recurrences and worse cancer-specific survival rate.

OBJECTIVE To analyse the overall and type-specific human papillomavirus (HPV) prevalence and distribution in penile carcinoma and determine the correlation to histopathological parameters.

PATIENTS AND METHODS In this retrospective study, we analysed HPV status in 241 patients with penile carcinoma, treated at Orebro University Hospital, Orebro, Sweden, between 1984 and 2008. Age and date at diagnosis was recorded. The tumour specimens were categorized according to the UICC 2002 TNM classification. A subset of patients was operatively staged with regard to lymph node status. A commercially available Real Time PCR was used to detect 13 different types of HPV (6,11,16,18,31,33,35,45,51,52,56,58 and 59).

RESULTS We excluded 25 patients due to low DNA quality. Of the remaining 216, 179 (82.9%) tumour specimens were HPV infected. The majority of cases positive for HPV (70.4%) were infected by a single-type. The most frequent type was HPV 16 followed by HPV 18. No significant association between HPV status and pathological tumour stage, grade or lymph node status was found.

CONCLUSION The HPV prevalence found is higher than in most other studies, further strengthening HPV as an etiological agent in penile carcinoma. Furthermore, the high prevalence of HPV 16 and 18 raises the question of what potential impact current HPV vaccines that target these specific HPV types might have on penile carcinoma. No significant association between HPV status and histopathological parameters was found in the present study. Additional investigations are needed to draw final conclusions on the prognostic value of HPV status in penile carcinoma.

The objective of this study was to determine the prevalence and some predictors for vaccine and non-vaccine types of HPV infections among bivalent HPV vaccinated and non-vaccinated young women in Uganda. This was a comparative cross sectional study 5.5 years after a bivalent HPV 16/18 vaccination (Cervarix®, GlaxoSmithKline, Belgium) pilot project in western Uganda. Cervical swabs were collected between July 2014-August 2014 and analyzed with a HPV genotyping test, CLART® HPV2 assay (Genomica, Madrid Spain) which is based on PCR followed by microarray for determination of genotype. Blood samples were also tested for HIV and syphilis infections as well as CD4 and CD8 lymphocyte levels. The age range of the participants was 15-24 years and mean age was 18.6(SD 1.4). Vaccine-type HPV-16/18 strains were significantly less prevalent among vaccinated women compared to non-vaccinated women (0.5% vs 5.6%, p 0.006, OR 95% CI 0.08(0.01-0.64). At type-specific level, significant difference was observed for HPV16 only. Other STIs (HIV/syphilis) were important risk factors for HPV infections including both vaccine types and non-vaccine types. In addition, for non-vaccine HPV types, living in an urban area, having a low BMI, low CD4 count and having had a high number of life time sexual partners were also significant risk factors. Our data concurs with the existing literature from other parts of the world regarding the effectiveness of bivalent HPV-16/18 vaccine in reducing the prevalence of HPV infections particularly vaccine HPV- 16/18 strains among vaccinated women. This study reinforces the recommendation to vaccinate young girls before sexual debut and integrate other STI particularly HIV and syphilis interventions into HPV vaccination packages.

Introduction: The aim of the study was to investigate whether biobanked liquid-based cytology (LBC) vaginal samples could be reanalyzed for the biomarkers HPV DNA and mRNA without loss of sensitivity.

Methods: One hundred LBC samples with ASCUS or CIN1 were tested for HPV DNA and mRNA before and after biobanking. DNA analysis targeted the viral genes E6 and E7, 12 high-risk and 2 low-risk HPV types together with the human control gene HBB, using real-time PCR. The Aptima HPV assay was used for mRNA analysis of 14 high-risk HPV types.

Results: With Aptima there was 84% agreement between results before and after biobanking. The sensitivity and specificity were 0.79 (95% CI, 0.68-0.88) and 0.94 (95% CI, 0.80-0.99), respectively. With the DNA-based method, the agreement between results was 87%, the sensitivity 0.85 (95% CI, 0.75-0.92) and the specificity 0.95 (95% CI, 0.77-1.00). Both methods presented a significant difference between positive results before and after biobanking; McNemar test: p = 0.004, p = 0.003, Cohen's kappa: 0.67 (95% CI, 0.53-0.81), 0.68 (95% CI, 0.52-0.84). Cycle threshold values for the DNA method were higher for all genotypes after biobanking, except for HPV-59. Some loss of sensitivity was seen after biobanking but the concordance between HPV detection before and after biobanking was good for both evaluated methods.

Conclusions: Biobanking of LBC vaginal samples offers a good platform for HPV testing and could be extended to further molecular analyses. However, in order to ensure a valid test result a larger portion needs to be analyzed from the biobanked sample.

The underlying cause of cervical cancer is infection with the human papilloma virus (HPV) and HPV testing can be used for cervical cancer screening. The Aptima HPV assay from Hologic is an mRNA HPV test used to identify clinically relevant infections but the method does not discriminate between the different high risk genotypes. The aim of the current study was to evaluate if analyzed Aptima sample transfer tubes could be used as a source for HPV genotyping, using sample DNA. Study samples (n=108); were HPV-tested with mRNA Aptima assay and in parallel DNA was extracted and genotyped with Anyplex II HPV28. Analyzed mRNA Aptima tubes were thereafter used as source for a second DNA extraction and genotyping. Using mRNA Aptima result as reference, 90% of the samples (35/39) were high risk positive with the Anyplex II HPV28. Cohen's kappa 0.78 (95% CI: 0.66-0.90), sensitivity 0.90 (95% CI: 0.76-0.97) and specificity 0.90 (95% CI: 0.80-0.96). Two discordant samples carried low-risk genotypes (HPV 82 and HPV 44) and two were negative. DNA-genotyping results, in parallel to and after mRNA testing, were compared and differed significantly (McNemar test: P=0.021) possibly due to sample extraction volume difference. Cohen's kappa 0.81 (95% CI: 0.70-0.92), sensitivity 0.85 (95% CI: 0.74-0.93) and specificity 0.98 (95% CI: 0.88-1.00). In conclusion, analyzed mRNA Aptima sample tubes could be used as a source for DNA HPV genotyping. The sample volume used for extraction needs to be further explored.

Vulvar carcinoma is the fourth most common gynecological malignancy. Two separate carcinogenic pathways are suggested, where one is associated with the human papillomavirus (HPV) and HPV16 the most common genotype.

The aim of this study was to evaluate HPV-markers in a set of primary tumors, metastases and recurrent lesions of vulvar squamous cell carcinomas (VSCC). Ten HPV16-positive VSCC with metastatic regional lymph nodes, distant lymphoid/hematogenous metastases or local recurrent lesions were investigated for HPV genotype, HPV16 variant, HPV16 viral load, HPV16 integration and HPV16 E2BS3 and 4 methylation.

In all 10 analyzed case series, the same HPV genotype (HPV16), HPV16 variant and level of viral load were detected in all lesions within a patient case. Primary tumors with a high E2/E6 ratio were found to have fewer vulvar recurrences and/or metastases after diagnosis and treatment. Also, a significantly lower viral load was evident in regional lymph nodes compared to primary tumors.

The data presented strengthens the evidence for a clonal HPV-induced pathway for vulvar carcinoma.

Human papillomavirus (HPV) testing and genotyping of FFPE tissue samples is important in epidemiological investigations. Here, we compare four different HPV genotyping methods for use in FFPE clinical samples. Comparative testing was performed on 99 samples with a clinical suspicion of HPV. Specimens were analyzed with Anyplex II HPV28 detecting 28 genotypes using real-time PCR and melting curve analysis, CLART HPV2 detecting 35 genotypes using PCR and microarray detection, and MGP5+/6+ consensus primer system together with pyrosequencing. Results were compared to a real-time PCR reference protocol detecting 14 genotypes. In total, 68% of the samples were positive for an HPV genotype using the reference protocol and MGP5+/6+ primer system. Anyplex II HPV28 analysis and CLART HPV2 had 82% and 72% positive samples, respectively. All four methods showed good agreement when comparing the 14 genotypes included in the reference protocol. When evaluating all genotypes, the Anyplex II HPV28 assay and the CLART assay changed the status of the sample (individually or together) from negative with respect to the reference protocol to positive for either a Group 1 (n = 4) or Group 2 (n = 6) genotype. We conclude from this study that for an extended genotyping approach with a high sensitivity for FFPE specimens, both the Anyplex II HPV28 and CLART HPV2 assays are suitable alternatives despite minor intra-assay differences.

Human papilloma virus (HPV) infection is associated with several anogenital malignancies. Here, we set out to evaluate digital droplet PCR (ddPCR) as a tool for HPV 16, 18, 33 and 45 viral load quantification and, in addition, to compare the efficacy of the ddPCR assay for HPV 16 detection with that of quantitative real-time PCR (qPCR).

Clinical samples, positive for HPV genotypes 16, 18, 33 and 45 were analyzed for viral load using ddPCR. Sample DNA was cleaved before droplet generation and PCR. Droplets positive for VIC and FAM fluorescence were read in a QX200 Droplet reader (TM) (BIO-RAD) after which the viral load was calculated using Quantasoft software.

We found that DNAs extracted from formalin fixed paraffin embedded (FFPE) tissue samples yielded lower amplification signals compared to those obtained from liquid based cytology (LBC) samples, but they were clearly distinguishable from negative background signals. The viral limit of detection was 1.6 copies of HPV 16, 2.8 copies of HPV 18, 4.6 copies of HPV 33 and 1.6 copies of HPV 45. The mean inter-assay coefficients of variability (CV) for the assays ranged from 3.4 to 7.0%, and the mean intra-assay CV from 2.6 to 8.2%. The viral load in the different cohorts of tumor samples ranged from 154 to 340,200 copies for HPV 16, 244 to 31,300 copies for HPV 18 and 738 to 69,100 copies for HPV 33. One sample positive for HPV 45 contained 1331 viral copies. When comparing qPCR data with ddPCR copy number data, the qPCR values were found to be 1 to 31 times higher.

Separation of fragments in nanodroplets may facilitate the amplification of fragmented human and viral DNA. The method of digital droplet PCR may, thus, provide a new and promising tool for evaluating the HPV viral load in clinical samples.

Objective: To investigate the human papillomavirus (HPV) and HPV type 16-variant distribution in a series of vulvar squamous cell carcinomas (VSCC) and to evaluate the impact of HPV and HPV 16-variant on prognosis.

Methods: A series of 133 patients who had a diagnosis of VSCC (1983-2008) was selected for the study. Detection of 11 high-risk HPV types (16, 18, 31, 33, 39, 45, 51, 52, 56, 58, and 59) and 2 low-risk HPV types (6 and 11) was performed with real-time polymerase chain reaction. Samples positive for HPV 16 were further analyzed for variant determination of 7 positions in the E6 gene with polymerase chain reaction and pyrosequencing.

Results: Forty (30.8%) of 130 tumors were found to be HPV positive. Human papillomavirus type 16 was found in 31 cases, HPV 18 was found in 2 cases, HPV 33 was found in 5 cases, and HPV 56 and HPV 59 were found in one case each. All but one tumor harboring HPV 16 were of European linage, and the 3 most common variants were E-p (n = 13), E-G350 (n = 7), and E-G131 (n = 5). HPV positivity was associated with the basaloid tumor type and occurred in significantly younger patients. Overall and recurrence-free survival rates were better in HPV-positive cases, but after correction for age and tumor size, HPV status was no longer an independent and significant prognostic factor. The survival rates of the various HPV 16 variants were not significantly different, but there was a trend of worse outcome for the E-G131-variant group.

Conclusions: Human papillomavirus positivity of 30.8% is similar to other reports on VSCC. To our knowledge, this first variant determination of HPV 16 in vulvar carcinoma in a Swedish cohort indicated that the variant E-G131 may have an increased oncogenic potential in patients with VSCC.

Objective

The objectives of this study are to investigate the human papilloma virus (HPV) distribution in vaginal cancer and to evaluate HPV-genotype as well as HPV16-variant impact on prognosis.

Methods

Sixty-nine patients diagnosed with primary vaginal carcinoma (1975-2002) were included in the study. Detection of twelve high-risk HPV (hr HPV) and two low-risk HPV (lr HPV) was performed with realtime-PCR. Samples positive for HPV-16 were analyzed for variants in the E6-gene with PCR and pyrosequencing.

Results

53.6% (37/69) of the tumors were found to be HPV-positive, mostly for HPV-16 (N=26). Other HPV-types were HPV-18 (N=2), HPV-31 (N=2), HPV-33 (N=2), HPV-45 (N=1), HPV-52 (N=2), HPV-56 (N=1) and HPV-58 (N=1). Only European subtypes of HPV-16 were represented and the two most common HPV-16-variants were E-p (N=13) and E-G350 (N=11). Patients with HPV-positive tumors (N=37) had a significantly (log-rank test=3341; p = 0.0008) superior 5-year overall survival rate as well as cancer-specific survival rate and progression-free survival rate (p = 0.0002; p = 0.0004), compared with patients with HPV-negative tumors (N=32). Interestingly, patients with HPV-16-positive tumors had a superior overall survival compared with patients with tumors containing other HPV-genotypes. In a Cox proportional multivariate analysis age, tumor size, and HPV-status were independent and significant prognostic factors with regard to overall survival rate.

Conclusions

HPV-status is of prognostic importance in vaginal carcinoma and varies with viral genotype. In this era of HPV-vaccination, genotypes other than those included in the vaccination program could still lead to vaginal carcinoma with unfavorable prognosis.

Objective: To investigate if viral load, integration and methylation of E2BS3 and 4 represent different ways of tumor transformation in vaginal and vulvar carcinoma and to elucidate its clinical impact.

Methods: Fifty-seven samples, positive for HPV16, were selected for the study. Detection of viral load was made with realtime-PCR using copy numbers of E6 and integration was calculated from comparing E2 to E6-copies. Methylation of E2BS3 and 4 was analysed using bisulphite treatment of tumor DNA, followed by PCR and pyrosequencing.

Results: Vaginal tumors were found to have a higher viral load (p=0.024) compared to vulvar tumors but a high copy number (> median value, 15 000) as well as high methylation (> 50%) was significantly (p=0.010 and p=0.045) associated with a worse cancer-specific survival rate in vulvar carcinoma, but not in vaginal carcinoma. Four groups could be defined for the complete series using a Cluster Two step analysis; (1) tumors holding episomal viral DNA, viral load below 150 000 copies not highly methylated (n=25, 46.3%); (2) tumors harboring episomal viral DNA and being highly methylated (>50%; n=6, 11.1%); (3) tumors with viral DNA fully integrated (n=11, 20.4%), and (4) tumors harboring episomal viral DNA and being medium-or unmethylated (< 50%) and having a high viral load (> total mean value 150 000; n=12, 22.2%). The completely integrated tumors were found to be distinct group, whilst some overlap between the groups with high methylation and high viral load was observed.

Conclusion: HPV16-related integration, methylation in E2BS3 and 4 and viral load may represent different viral characteristics driving vaginal and vulvar carcinogenesis. HPV16-related parameters were found to be of clinical importance in the vulvar series only.

Biomedical laboratory scientists (BLS) work in many different disciplines but one common denominator for all the fields within the profession is the rapid development in biomedicine and the corresponding increase of advanced technology. A nationally regulated specialist training for BLS is a way for the profession to gain advanced skills and to create career opportunities. From a larger study set, the aims of this sub-study were to investigate BLS student’s and professional’s view on education, choice of workplace, career development, advanced studies and a potential nationally regulated specialist training program. Two surveys were designed using webbenkater.com. The surveys were sent to BLS student members (n=483) and professional members (n=2083) of The Swedish Institute of Biomedical Laboratory Science (IBL), the professional organization of BLS´s in Sweden. Response rate was 57% (276/483) for the student sub-survey and 44% (n=923/2083) for the professional sub-survey. Students from all semesters (1-6, n=272) were represented, with a majority from semester 2, 4 and 6. Top reasons for choosing the BLS education were; easy to get a job (65%), stimulating work tasks (59%) and a good education for further studies (39%). A majority of the students planned for further advanced academic studies (64%) and 54% percent were interested in a potential nationally regulated specialist training. Among professionals, 21% stated there were explicit career paths at their workplace. The individual interest for a potential nationally regulated specialist training was 53% and most responders (93%) stated a need for such an education in Sweden. Among IBL members, there is great interest in a nationally regulated specialized training among both future and present professionals in Sweden. In relation to a future shortage, we also show that in order to attract students to BLS training, we need to be able to offer advanced training as well.

Human papilloma virus (HPV) is considered to be responsible for a large part of vaginal and vulvar carcinomas, and the p53 codon 72 polymorphism has been implicated in susceptibility to cancer induced by this virus, but with contradicting results. In this study, we have investigated the prognostic value of the codon 72 polymorphism by real-time PCR (qPCR) in two cohorts of vaginal (n = 66) and vulvar (n = 123) carcinomas. In vaginal carcinoma, arginine homozygous patients were significantly associated with a higher primary cure rate (p = 0.023) but also associated with a higher recurrence rate (p = 0.073), significant at distant locations (p = 0.009). No significant differences were found in overall survival rate (p = 0.499) or cancer-specific survival rate (p = 0.222). A higher frequency of arginine homozygosity was noted in HPV-positive tumors (p = 0.190) in comparison with HPV-negative tumors. In vulvar carcinoma, the genotype homozygous for arginine was significantly associated with a larger tumor size at diagnosis in the entire cohort (p = 0.015) and a lower cancer-specific survival rate (p = 0.024) compared with heterozygous (arginine/proline) in HPV-negative tumors. Our results indicate that the relation between HPV and the p53 codon 72 polymorphism is complex and the significance and mechanisms responsible for this relationship need to be further elucidated.

Background: Primary vaginal carcinoma (PVC) is a rare malignancy. Established prognostic factors include tumour stage and age at diagnosis. The leucine-rich repeats and immunoglobuline-like domains (LRIG)-1 protein functions as a tumour suppressor, but less is known about the functions of LRIG2 and LRIG3. The present study aimed to evaluate the expression of LRIG proteins and analyse their possible associations with clinical characteristics and survival in a cohort of PVC patients.

Methods: We used immunohistochemistry to investigate LRIG1, LRIG2, and LRIG3 expression in tumour samples from a consecutive cohort of 70 PVC patients. The association between LRIG protein expression and clinical characteristics and cancer-specific survival was investigated using univariate and multivariate analyses.

Results: The majority of PVC patients (72%) had > 50% LRIG1-and LRIG2-positive cells, and no or low LRIG3-positive cells. HPV status was significantly correlated with LRIG1 expression (p = 0.0047). Having high LRIG1 expression was significantly correlated with superior cancer-specific survival in univariate and multivariate analyses. LRIG2 and LRIG3 expression did not significantly correlate with clinical characteristics or survival.

Conclusion: LRIG1 expression might be of interest as a prognostic marker in PVC patients, whereas the role of LRIG2 and LRIG3 expression remains to be clarified.