What is the added value of digital image analysis of HER2 immunohistochemistry in breast cancer in clinical practice? A study with multiple platforms

Aims We aimed to compare digital image analysis (DIA) of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) in breast cancer by two platforms: (i) to validate DIA against standard diagnostics; and (ii) to evaluate the added value of DIA in clinical practice. Methods and results HER2 IHC and in-situ hybridisation (ISH) were performed on 152 consecutive invasive breast carcinomas. IHC scores were determined with DIA using two independent platforms. Manual scoring was performed by two independent observers. HER2 status was considered positive in 3+ and ISH-positive 2+ cases. HER2 status using DIA was compared to HER2 status with standard diagnostics (manual scoring with ISH in 2+ cases). Interplatform agreement of IHC scores was 'moderate' (linear weighted kappa = 0.58), agreement between manual scoring and platform A was 'moderate' (kappa = 0.60) and between manual scoring and platform B 'almost perfect' (kappa = 0.85). Compared to manual scoring, DIA resulted in a reduction of 2+ cases from 17.1 to 1.3% with platform A and from 17.1 to 15.8% with platform B. However, compared to standard diagnostics, there were three false-negative cases with DIA using platform A [81.3% sensitivity, 100% specificity, 100% positive predictive value (PPV), 97.8% negative predictive value (NPV)]. Sensitivity, specificity, PPV and NPV were 100% with DIA using platform B. Conclusions DIA of HER2 IHC is a valid tool in determining HER2 status in breast carcinoma. Algorithms in different platforms can behave differently, and optimal calibration is essential. In clinical practice, DIA offers an objective alternative to manual scoring, but a reduction in 2+ cases could result in loss of sensitivity

Feasibility of endometrial sampling by vaginal tampons in women with Lynch syndrome

Background: Endometrial sampling for the surveillance of women with Lynch syndrome is an invasive and painful procedure. The aim of this study was to evaluate the feasibility of a less invasive procedure of collecting vital cells by vaginal tampons. Methods: This was a prospective feasibility study of women scheduled to undergo annual gynecological surveillance, including endometrial sampling. We included consecutive asymptomatic women with Lynch syndrome or first-degree relatives and asked them to insert a vaginal tampon 2-4 h before attending their outpatient appointment. Feasibility was evaluated by the following metrics: Patient acceptance, pain intensity of each procedure (assessed by visual analog scale; range 0-10), and the presence of vital cells obtained by tampon-based or endometrial sampling methods. Two pathologists independently evaluated all samples. Results: In total, 25 of 32 approached women completed the tampon-based procedure, with 23 of these subsequently undergoing invasive endometrial sampling. The median visual analog scale scores for tampon use and invasive endometrial sampling were 0 (range, 0-10) and 5.5 (range, 1-10) (p < 0.001). None of the tampon samples analyzed by cytology showed endometrial cells, but they did contain vital squamous cells and granulocytes. By contrast, 18 (78%) of the invasive endometrial samples contained enough endometrial tissue for analysis. No endometrial abnormalities were found by endometrial sampling. Conclusions: Tampon-based endometrial surveillance was a well-accepted and non-painful procedure, and although tampons contained vital cells, they did not provide endometrial cells. However, this study was limited to asymptomatic women with Lynch syndrome (no endometrial pathology), indicating that research is needed to evaluate whether the tampon method has any utility for endometrial surveillance in women with Lynch syndrome

Association of homozygous variants of STING1 with outcome in human cervical cancer

DNA-sensing receptor Cyclic GMP-AMP Synthase (cGAS) and its downstream signaling effector STimulator of INterferon Genes (STING) have gained significant interest in the field of tumor immunology, as a dysfunctional cGAS-STING pathway is associated with poor prognosis and worse response to immunotherapy. However, studies so far have not taken into account the polymorphic nature of the STING-encoding STING1 gene. We hypothesized that the presence of allelic variance in STING1 would cause variation between individuals as to their susceptibility to cancer development, cancer progression, and potential response to (immuno)therapy. To start to address this, we defined the genetic landscapes of STING1 in cervical scrapings and investigated their corresponding clinical characteristics across a unique cohort of cervical cancer patients and compared them with independent control cohorts. Although we did not observe an enrichment of particular STING1 allelic variants in cervical cancer patients, we did find that the occurrence of homozygous variants HAQ/HAQ and R232H/R232H of STING1 were associated with both younger age of diagnosis and higher recurrence rate. These findings were accompanied by worse survival, despite comparable mRNA and protein levels of STING and numbers of infiltrated CD8(+) T cells. Our findings suggest that patients with HAQ/HAQ and R232H/R232H genotypes may have a dysfunctional cGAS-STING pathway that fails to promote efficient anticancer immunity. Interestingly, the occurrence of these genotypes coincided with homozygous presence of the V48V variant, which was found to be individually associated with worse outcome. Therefore, we propose V48V to be further evaluated as a novel prognostic marker for cervical cancer

A Critical Appraisal of Circumferential Resection Margins in Esophageal Carcinoma

In esophageal cancer, circumferential resection margins (CRMs) are considered to be of relevant prognostic value, but a reliable definition of tumor-free CRM is still unclear. The aim of this study was to appraise the clinical prognostic value of microscopic CRM involvement and to determine the optimal limit of CRM.To define the optimal tumor-free CRM we included 98 consecutive patients who underwent extended esophagectomy with microscopic tumor-free resection margins (R0) between 1997 and 2006. CRMs were measured in tenths of millimeters with inked lateral margins. Outcome of patients with CRM involvement was compared with a statistically comparable control group of 21 patients with microscopic positive resection margins (R1).A cutoff point of CRM at a parts per thousand currency sign1.0 mm and &gt; 1.0 mm appeared to be an adequate marker for survival and prognosis (both P &lt;0.001). The outcome in patients with CRMs a parts per thousand currency sign1.0 and &gt; 0 mm was equal to that in patients with CRM of 0 mm (P = 0.43). CRM involvement was an independent prognostic factor for both recurrent disease (P = 0.001) and survival (P &lt;0.001). Survival of patients with positive CRMs (a parts per thousand currency sign1 mm) did not significantly differ from patients with an R1 resection (P = 0.12).Involvement of the circumferential resection margins is an independent prognostic factor for recurrent disease and survival in esophageal cancer. The optimal limit for a positive CRM is a parts per thousand currency sign1 mm and for a free CRM is &gt; 1.0 mm. Patients with unfavorable CRM should be approached as patients with R1 resection with corresponding outcome.</p

Serotonin and Dopamine Receptor Expression in Solid Tumours Including Rare Cancers

In preclinical studies serotonin stimulates and dopamine inhibits tumour growth and angiogenesis. Information regarding serotonin and dopamine receptor (5-HTR and DRD) expression in human cancers is limited. Therefore, we screened a large tumour set for receptor mRNA overexpression using functional genomic mRNA (FGmRNA) profiling, and we analysed protein expression and location of 5-HTR1B, 5-HTR2B, DRD1, and DRD2 with immunohistochemistry in different tumour types. With FGmRNA profiling 11,756 samples representing 43 tumour types were compared to 3,520 normal tissue samples to analyse receptor overexpression. 5-HTR2B overexpression was present in many tumour types, most frequently in uveal melanomas (56%). Receptor overexpression in rare cancers included 5-HTR1B in nasopharyngeal carcinoma (17%), DRD1 in ependymoma (30%) and synovial sarcoma (21%), and DRD2 in astrocytoma (13%). Immunohistochemistry demonstrated high 5-HTR2B protein expression on melanoma and gastro-intestinal stromal tumour cells and endothelial cells of colon, ovarian, breast, renal and pancreatic tumours. 5-HTR1B expression was predominantly low. High DRD2 protein expression on tumour cells was observed in 48% of pheochromocytomas, and DRD1 expression ranged from 14% in melanoma to 57% in renal cell carcinoma. In conclusion, 5-HTR1B, 5-HTR2B, DRD1, and DRD2 show mRNA overexpression in a broad spectrum of common and rare cancers. 5-HTR2B protein is frequently highly expressed in human cancers, especially on endothelial cells. These findings support further investigation of especially 5HTR2B as a potential treatment target

Proteomic alterations in early stage cervical cancer

Laser capture microdissection (LCM) allows the capture of cell types or well-defined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, n = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue (n = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by “all-or-nothing” analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer (n = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive “all-or-nothing” way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction Monitoring (PRM). Ingenuity Pathway Analysis using correction for multiple testing by permutation resulted in two networks that were differentially regulated in early stage cervical cancer compared with healthy tissue. From these networks, we learned that specific tumor mechanisms become effective during the early stage of cervical cancer

Lymphadenectomy and Adjuvant Therapy Improve Survival with Uterine Carcinosarcoma:A Large Retrospective Cohort Study

OBJECTIVE: Uterine carcinosarcoma is a rare, aggressive subtype of endometrial cancer. Treatment consists of hysterectomy, bilateral salpingo-oophorectomy, and lymphadenectomy (LND). The survival benefit of LND in relation to adjuvant radio- and/or chemotherapy is unclear. We evaluated the impact of LND on survival in relation to adjuvant therapy in uterine carcinosarcoma. METHODS: Retrospective data on 1,140 cases were combined from the Netherlands Cancer Registry (NCR) and the nationwide network and registry of histo- and cytopathology in the Netherlands (PALGA). LND was defined as the removal of any nodes. Additionally, cases where 10 nodes or less (LND ≤10) or more than 10 nodes (LND > 10) were removed were analyzed separately. Adjuvant therapy was evaluated as radiotherapy, chemotherapy, or radiochemotherapy. Associations were analyzed by χ2 test, log-rank test, and Cox regression analysis. RESULTS: Overall survival (OS) had improved after total abdominal hysterectomy with bilateral salpingo-oophorectomy with LND > 10 (HR 0.62, 95% CI 0.47-0.83). Adjuvant therapy was related to OS with an HR of 0.64 (95% CI 0.54-0.75) for radiotherapy, an HR of 0.65 (95% CI 0.48-0.88) for chemotherapy, and an HR of 0.25 (95% CI 0.13-0.46) for radiochemotherapy. Additionally, adjuvant treatment was related to OS when lymph nodes were positive (HR 0.22, 95% CI 0.11-0.42), but not when they were negative. CONCLUSION: LND is related to improved survival when more than 10 nodes are removed. Adjuvant therapy improves survival when LND is omitted, or when nodes are positive

Vaginal dryness in primary Sjögren's syndrome:a histopathological case control study

The aim was to study clinical, histopathological and immunological changes in the vagina and cervix of women with primary SS, which might explain vaginal dryness. Methods: We included 10 pre-menopausal female primary SS patients with vaginal dryness and 10 pre-menopausal controls undergoing a laparoscopic procedure. The vaginal health index was recorded. Multiplex immunoassays and flow cytometry were performed on endocervical swab and cervicovaginal lavage samples to evaluate cellular and soluble immune markers. Mid-vaginal and endocervical biopsies were taken and stained for various leucocyte markers, caldesmon (smooth muscle cells), avian V-ets erythroblastosis virus E26 oncogene homologue (ERG; endothelial cells) and anti-podoplanin (lymphatic endothelium). The number of positive pixels per square micrometre was calculated. Results: One patient was excluded because of Clamydia trachomatis, and two controls were excluded because of endometriosis observed during their laparoscopy. Vaginal health was impaired in primary SS. CD45+ cells were increased in vaginal biopsies of women with primary SS compared with controls. Infiltrates were predominantly located in the peri-epithelial region, and mostly consisted of CD3+ lymphocytes. In the endocervix, CD45+ infiltrates were present in patients and in controls, but a higher number of B lymphocytes was seen in primary SS. Vascular smooth muscle cells were decreased in the vagina of primary SS patients. No differences were found in leucocyte subsets in the vaginal and endocervical lumen. CXCL10 was increased in endocervical swab samples of primary SS patients. Conclusion: Women with primary SS show impaired vaginal health and increased lymphocytic infiltration in the vagina compared with controls. Vaginal dryness in primary SS might be caused by vascular dysfunction, possibly induced by IFN-mediated pathways

Proteomic alterations in early stage cervical cancer

Laser capture microdissection (LCM) allows the capture of cell types or welldefined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, n = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue (n = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by "all-ornothing" analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer (n = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive "all-or-nothing" way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction

Endometrial cancer survival after breast cancer in relation to tamoxifen treatment:Pooled results from three countries

Introduction: Tamoxifen is an effective treatment for breast cancer but an undesirable side-effect is an increased risk of endometrial cancer, particularly rare tumor types associated with poor prognosis. We investigated whether tamoxifen therapy increases mortality among breast cancer patients subsequently diagnosed with endometrial cancer. Methods: We pooled case-patient data from the three largest case-control studies of tamoxifen in relation to endometrial cancer after breast cancer (1,875 patients: Netherlands, 765; United Kingdom, 786; United States, 324) and collected follow-up information on vital status. Breast cancers were diagnosed in 1972 to 2005 with endometrial cancers diagnosed in 1978 to 2006. We used Cox proportional hazards survival analysis to estimate hazard ratios (HRs) and 95% confidence intervals (CI). Results: A total of 1,104 deaths occurred during, on average, 5.8 years following endometrial cancer (32% attributed to breast cancer, 25% to endometrial cancer). Mortality from endometrial cancer increased significantly with unfavorable non-endometrioid morphologies (P <0.0001), International Federation of Gynaecology and Obstetrics staging system for gynecological malignancy (FIGO) stage (P <0.0001) and age (P <0.0001). No overall association was observed between tamoxifen treatment and endometrial cancer mortality (HR = 1.17 (95% CI: (0.89 to 1.55)). Tamoxifen use for at least five years was associated with increased endometrial cancer mortality (HR = 1.59 (1.13 to 2.25)). This association appeared to be due primarily to the excess of unfavorable histologies and advanced stage in women using tamoxifen for five or more years since the association with mortality was no longer significant after adjustment for morphological type and FIGO stage (HR = 1.37 (0.97 to 1.93)). Those patients with endometrioid tumors, who stopped tamoxifen use at least five years before their endometrial cancer diagnosis, had a greater mortality risk from endometrial cancer than endometrioid patients with no tamoxifen exposure (HR = 2.11 (1.13 to 3.94)). The explanation for this latter observation is not apparent. Conclusions: Patients with endometrial cancer after breast cancer who received tamoxifen treatment for five years for breast cancer have greater endometrial cancer mortality risk than those who did not receive tamoxifen. This can be attributed to non-endometrioid histological subtypes with poorer prognosis among long term tamoxifen users