Axel Niendorf

Background: An accurate status determination of breast cancer biomarkers (ER, PR, HER2, Ki67) is crucial for guiding patient management. The “gold standard” for assessing these biomarkers in FFPE tissue is IHC, which faces challenges in standardization and exhibits substantial variability. In this study, we compare the concordance of a new commercial RT-qPCR kit with IHC in determining BC biomarker status. Methods: The performance was evaluated using 634 FFPE specimens, which underwent histological analysis in accordance with standard of care methods. HER2 2+ tumors were referred to ISH testing. An immunoreactive score of ≥2/12 was considered positive for ER/PR and 20% staining was used as a cut-off for Ki67 high/low score. RT-qPCR and results calling were performed according to the manufacturer’s instructions. Results: High concordance with IHC was seen for all markers (93.2% for ER, 87.1% for PR, 93.9% for HER2, 77.9% for Ki67 and 80.1% for proliferative signature (assessed against Ki67 IHC)). Conclusions: By assessing the concordance with the results obtained through IHC, we sought to demonstrate the reliability and utility of the kit for precise BC subtyping. Our findings suggest that the kit provides a highly precise and accurate quantitative assessment of BC biomarkers.

Glycosylation is a prominent posttranslational modification, and alterations in glycosylation are a hallmark of cancer. Glycan-binding receptors, primarily expressed on immune cells, play a central role in glycan recognition and immune response. Here, we used the recombinant C-type glycan-binding receptors CD301, Langerin, SRCL, LSECtin, and DC-SIGNR to recognize their ligands on tissue microarrays (TMA) of a large cohort (n = 1859) of invasive breast cancer of different histopathological types to systematically determine the relevance of altered glycosylation in breast cancer. Staining frequencies of cancer cells were quantified in an unbiased manner by a computer-based algorithm. CD301 showed the highest overall staining frequency (40%), followed by LSECtin (16%), Langerin (4%) and DC-SIGNR (0.5%). By Kaplan-Meier analyses, we identified LSECtin and CD301 as prognostic markers in different breast cancer subtypes. Positivity for LSECtin was associated with inferior disease-free survival in all cases, particularly in estrogen receptor positive (ER+) breast cancer of higher histological grade. In triple negative breast cancer, positivity for CD301 correlated with a worse prognosis. Based on public RNA single-cell sequencing data of human breast cancer infiltrating immune cells, we found CLEC10A (CD301) and CLEC4G (LSECtin) exclusively expressed in distinct subpopulations, particularly in dendritic cells and macrophages, indicating that specific changes in glycosylation may play a significant role in breast cancer immune response and progression.

Primary squamous cell carcinoma or squamous intraepithelial lesion of the fallopian tube is a very rare finding with only a small number of cases worldwide. We describe the case of a 51-year-old woman, undergoing an abdominal hysterectomy after the diagnosis of an HPV-associated invasive squamous cell carcinoma of the uterine cervix with the unexpected detection of an HPV16-positive high-grade squamous intraepithelial lesion of the fimbria of the right fallopian tube in the resection specimen. The finding of an isolated, HPV-associated squamous intraepithelial lesion in the fallopian tube raises the question of a de novo development in this body compartment (after exclusion of a continuous metastatic spread from the uterine cervix) by taking a virus-associated field effect into account and should encourage the inclusion of this possibility when examining the fallopian tube in a routine setting.

The diagnosis of breast cancer—including determination of prognosis and prediction—has been traditionally based on clinical and pathological characteristics such as tumor size, nodal status, and tumor grade. The decision-making process has been expanded by the recent introduction of molecular signatures. These signatures, however, have not reached the highest levels of evidence thus far. Yet they have been brought to clinical practice based on statistical significance in prospective as well as retrospective studies. Intriguingly, it has also been reported that most random sets of genes are significantly associated with disease outcome. These facts raise two highly relevant questions: What information gain do these signatures procure? How can one find a signature that is substantially better than a random set of genes? Our study addresses these questions. To address the latter question, we present a hybrid signature that joins the traditional approach with the molecular one by combining the Nottingham Prognostic Index with gene expressions in a data-driven fashion. To address the issue of information gain, we perform careful statistical analysis and comparison of the hybrid signature, gene expression lists of two commercially available tests as well as signatures selected at random, and introduce the Signature Skill Score—a simple measure to assess improvement on random signatures. Despite being based on in silico data, our research is designed to be useful for the decision-making process of oncologists and strongly supports association of random signatures with outcome. Although our study shows that none of these signatures can be considered as the main candidate for providing prognostic information, it also demonstrates that both the hybrid signature and the gene expression list of the OncotypeDx signature identify patients who may not require adjuvant chemotherapy. More importantly, we show that combining signatures substantially improves the identification of patients who do not need adjuvant chemotherapy.

Breast cancer is a heterogeneous disease representing a number of different histopathologic and molecular types which should be taken into consideration if prognostic or predictive models are to be developed. The aim of the present study was to demonstrate the validity of the long-known Nottingham prognostic index (NPI) in a large retrospective study (n = 6654 women with a first primary unilateral and unifocal invasive breast cancer diagnosed and treated between April 1996 and October 2018; median follow-up time of breast cancer cases was 15.5 years [14.9–16.8]) from a single pathological institution. Furthermore, it was intended to develop an even superior risk stratification model considering an additional variable, namely the patient’s age at the time of diagnosis. Heterogeneity of these cases was addressed by focusing on estrogen receptor-positive as well as Her2-negative cases and taking the WHO-defined different tumor types into account. Calculating progression free survival Cox-regression and CART-analysis revealed significantly superior iAUC as well as concordance values in comparison to the NPI based stratification, leading to an alternative, namely the Altona prognostic index (API). The importance of the histopathological tumor type was corroborated by the fact that when calculated separately and in contrast to the most frequent so-called “No Special Type” (NST) carcinomas, neither NPI nor API could show valid prognostic stratification.

Ligands of the C-type lectin CLEC10A such as Tn and sialyl-Tn representing early intermediates of O-glycosylation are hallmarks of many human malignancies. A variety of regulatory mechanisms underlying their expression are being discussed. CLEC10A ligands were detected in various tissues and cells using the recombinant glycan-binding domain of CLEC10A. In normal breast and endometrium, presence of ligands was correlated to the female cycle. Estrogen- and stress dependent induction of CLEC10A ligands was analyzed in MCF7 and T47D cells exposed to 4-hydroxy-tamoxifen (Tam), zeocin and hydrogen peroxide. The expression and localization of CLEC10A ligands was analyzed by Western blot and immunofluorescence. In breast cancer patients CLEC10A ligand expression and survival was correlated by Kaplan-Meyer analysis. We observed binding of CLEC10A in normal endometrial and breast tissues during the late phase of the female hormonal cycle suggesting a suppressive effect of female sex hormones on CLEC10A ligand expression. Accordingly, CLEC10A ligands were induced in MCF7- and T47D breast cancer cells after Tam treatment and accumulated on the cell surface and in the endosomal/lysosomal compartment. Phagocytosis experiments indicate that macrophages preferentially internalize CLEC10A ligands coated beads and Tam treated MCF7 cells. CLEC10A ligands were also expressed after the addition of zeocin and hydrogen-peroxide. Each substance induced the production of ROS indicating reactive oxygen species as a unifying mechanism of CLEC10A ligand induction. Mechanistically, increased expression of GalNAc-transferase 6 (GalNT6) and translocation of GalNT2 and GalNT6 from cis- towards trans-Golgi compartment was observed, while protein levels of COSMC and T-synthase remained unaffected. In breast cancer patients, positivity for CLEC10A staining in tumor tissues was associated with improved outcome and survival. CLEC10A ligands are inducible by hormone depletion, 4-hydroxy-tamoxifen and agents inducing DNA damage and oxidative stress. Our results indicate that CLEC10A acts as a receptor for damaged and dead cells and may play an important role in the uptake of cell debris by macrophages and dendritic cells.

The receptors for estrogen (ESR1) and progesterone (PGR) are both part of the same signaling pathway and routinely used for breast cancer stratification. We tested the hypothesis if a coordinated analysis could add extra information for prognostic stratification. ESR1 and PGR gene expression was first investigated by quantitative reverse transcription polymerase chain reaction in fresh-frozen invasive ductal breast cancer samples (Hamburg collective, case-control, n=317). Our results were then tested using two datasets generated by different technical approaches: i) a public DNA-chip data set (GSE3494, n=251) and ii) semiquantitative protein expression data based on immunohistochemistry (Stuttgart collective, n=18,528). The PGR/ESR1 gene-expression ratio was a prognostic indicator in those with ESR1/PGR-positive breast cancer (Hamburg collective), with a high PGR/ESR1 expression ratio indicating a favorable outcome. In all three collectives, the PGR/ESR1 mRNA ratio or its protein equivalent was a univariate prognostic factor and also a multivariate prognostic factor in the Hamburg and Stuttgart collectives. Calculation of the PGR/ESR1 gene-expression ratio and its immunohistochemical surrogate could be a useful and simple addition to routine breast cancer diagnostics. A high PGR/ESR1 ratio could be indicative of a favorable clinical outcome.

The well-characterized tubular-type of breast tumors is classified as low-risk breast cancer. We report on the results of a retrospective analysis on clinical and biological features of 248 tubular breast tumors including follow-up and treatment data from two German series of 21,065 breast cancer cases. The majority of tumors were stage I or stage II, ER- and PR-positive and c-erbB2-negative with a 5-year survival-rate of 96.3%. 51.3% of patients received hormonal treatment, 75.5% had post-operative radiotherapy and 11.8% were treated with a chemotherapeutical regimen. Our retrospective analysis showed no treatment benefit for either anti-hormonal or chemotherapeutical regimens. Post-operative radiotherapy, however, improved the survival rate of patients with tubular carcinoma (log-rank=5, p=0.025). Our data suggest that post-operative radiotherapy is an important treatment to prolong survival for patients suffering from tubular breast cancer.