Aims
Despite increasing interest in the recently established immunoglobulin 4‐related disease (IgG4‐RD), its pathogenesis and aetiology remain largely unclear. Characteristic histopathological ...features are one of the key elements of diagnosis, including ‘storiform’ fibrosis, obliterative phlebitis, increased lymphoplasmacytic infiltration and increased levels of IgG4 in serum and tissue. Histopathological features of IgG4‐RD are striking but not specific, and can pose a pitfall for surgical pathologists. This paper aims to determine the actual amount of IgG4+ plasma cells in nodular‐sclerosing Hodgkin lymphoma (NSHL) and its potential to be misdiagnosed in routine clinical practice.
Methods and results
IgG4+ plasma cells per high‐power field (HPF) and the ratio of IgG4+ versus IgG+ plasma cells (IgG4/IgG ratio) in lymph node biopsies of 24 patients with nodular‐sclerosing Hodgkin lymphoma (NSHL) were determined using immunohistochemistry and consensus scoring criteria as used for IgG4‐RD. Ten lymph node biopsies with reactive follicular hyperplasia were assessed for comparison. Higher numbers of IgG4+ plasma cells (P < 0.001) were observed in NSHL versus follicular hyperplasia (mean 34 versus 8 per HPF) with a mean IgG4/IgG ratio of 0.38 versus 0.18. Five cases (21%) fulfilled the consensus criteria of IgG4‐RD, with >50 IgG4+ plasma cells per HPF and an IgG4/IgG ratio of >0.4. The mean count of IgG4+ plasma cells per HPF in NSHL varied greatly (3–88) with increased numbers of IgG4+ plasma cells seen near areas of fibrosclerosis.
Conclusions
Significantly higher levels of IgG4+ plasma cells are common in NSHL, emphasising the need to exclude Reed–Sternberg cells by morphology and immunohistochemistry in biopsies where IgG4‐RD is suspected.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Breast tumors of the basal-like, hormone receptor-negative subtype remain an unmet clinical challenge, as there is high rate of recurrence and poor survival in patients following treatment. ...Coevolution of the malignant mammary epithelium and its underlying stroma instigates cancer-associated fibroblasts (CAFs) to support most, if not all, hallmarks of cancer progression. Here we delineate a previously unappreciated role for CAFs as determinants of the molecular subtype of breast cancer. We identified paracrine crosstalk between cancer cells expressing platelet-derived growth factor (PDGF)-CC and CAFs expressing the cognate receptors in human basal-like mammary carcinomas. Genetic or pharmacological intervention of PDGF-CC activity in mouse models of cancer resulted in conversion of basal-like breast cancers into a hormone receptor-positive state that enhanced sensitivity to endocrine therapy in previously resistant tumors. We conclude that specification of breast cancer to the basal-like subtype is under microenvironmental control and is therapeutically actionable.
Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to ...the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Molecular diagnostics is a rapidly evolving area of surgical pathology, that is gradually beginning to transform our diagnostical procedures for a variety of tumors. Next to molecular prognostication ...that has begun to complement our histological diagnosis in breast cancer, additional testing to detect targets and to predict therapy response has become common practice in breast and lung cancer. Prostate cancer is a bit slower in this respect, as it is still largely diagnosed and classified on morphological grounds. Our diagnostic immunohistochemical armamentarium of basal cell markers and positive markers of malignancy now allows to clarify the majority of lesions, if applied to the appropriate morphological context (and step sections). Prognostic immunohistochemistry remains a problematic and erratic yet tempting research field that provides information on tumor relevance of proteins, but little hard data to integrate into our diagnostic workflow. Main reasons are various issues of standardization that hamper the reproducibility of cut-off values to delineate risk categories. Molecular testing of DNA-methylation or transcript profiling may be much better standardized and this review discusses a couple of commercially available tests: The ConfirmDX test measures DNA-methylation to estimate the likelihood of cancer detection on a repeat biopsy and may help to reduce unnecessary biopsies. The tests Prolaris, OncotypeDX Prostate, and Decipher all are transcript tests that have shown to provide prognostic data independent of clinico-pathological parameters and that may aid in therapy planning. However, further validation and more comparative studies will be needed to clarify the many open questions concerning sampling bias and tumor heterogeneity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Circulating cell-free DNA methylation testing in blood has recently received regulatory approval for screening of colorectal cancer. Its application in other clinical settings, including staging, ...prognosis, prediction, and recurrence monitoring is highly promising, and of particular interest in head and neck squamous cell carcinomas (HNSCCs) that represent a heterogeneous group of cancers with unsatisfactory treatment guidelines.
Short stature homeobox 2 (
) and septin 9 (
) DNA methylation in plasma from 649 prospectively enrolled patients (training study: 284 HNSCC/122 control patients; testing study: 141 HNSCC/102 control patients) was quantified before treatment and longitudinally during surveillance.
In the training study, 59% of HNSCC patients were methylation-positive at 96% specificity. Methylation levels correlated with tumor and nodal category (
< 0.001). Initially increased methylation levels were associated with a higher risk of death
: hazard ratio (HR) = 5.27,
= 0.001;
: HR = 2.32,
= 0.024. Disease recurrence/metastases were detected in 47% of patients up to 377 days earlier compared to current clinical practice. The onset of second cancers was detected up to 343 days earlier. In the testing study, sensitivity (52%), specificity (95%), prediction of overall survival (
: HR = 2.78,
= 0.022;
: HR = 2.50,
= 0.026), and correlation with tumor and nodal category (
<0.001) were successfully validated.
Methylation testing in plasma is a powerful diagnostic tool for molecular disease staging, risk stratification, and disease monitoring. Patients with initially high biomarker levels might benefit from intensified treatment and posttherapeutic surveillance. The early detection of a recurrent/metastatic disease or a second malignancy could lead to an earlier consecutive treatment, thereby improving patients' outcomes.
Purpose: In carcinomas, invasive tumor growth is accompanied by desmoplastic stroma reaction and facilitated by epithelial-mesenchymal
transition (EMT) of cancer cells. We investigated the prognostic ...significance of the EMT indicator proteins periostin and
vimentin in comparison with versican, a putative indicator of the opposite mechanism mesenchymal-epithelial transition (MET),
and to the desmoplasia proteins collagen and elastin in non-small cell lung cancer (NSCLC).
Experimental Design: Tumor of 533 patients with surgically resected NSCLC was used for analysis of stromal and epithelial protein expression by
immunohistochemistry (EMT-MET proteins) and Elastica van Gieson histochemical staining (collagen and elastin). A semiquantitative
sum scoring system was done on three tissue microarrays.
Results: Of the 533 patients, 48% had squamous cell carcinoma, 47% adenocarcinoma, and 5% adenosquamous carcinoma. High expression
of periostin in either stroma or tumor epithelia, independently scored by two pathologists, correlated with male gender, higher
stage, higher pT category, and larger tumor size, and in only stroma with tumor relapse. High expression of versican in either
stroma or epithelia as well as of stromal collagen had fewer but concordant associations with advanced tumor and periostin,
respectively. High expression of elastin was oppositely associated with less advanced disease. Associations of high vimentin
were inconsistent (all P values <0.05). High stromal periostin was found to be a prognostic factor for decreased progression-free survival on univariate
analysis ( P = 0.007).
Conclusions: Because up-regulation is frequently observed in the stromal and epithelial tumor compartment, EMT-MET indicator proteins
may be integrated in progression models of NSCLC.
DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance ...requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives ...endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma‐like TCam‐2 cells to SOX17 in somatic cells and SOX2 in EC‐like 2102EP cells. In seminoma‐like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17‐binding sites overlap in seminoma‐like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma‐like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC‐like cells SOX2 regulates pluripotency‐associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.
What's new?
Seminomas are germ cell tumors of the testis, which are frequent tumors of young men and characterized by features of pluripotency and maintenance of an undifferentiated state. Here the authors examine the genome‐wide binding of the transcription factor SOX17, known as a determinant of endodermal differentiation. They find that in seminomas SOX17 is not controlling cell differentiation but maintains latent pluripotency and germ cell identity. In consequence, loss of SOX17 induces extra‐embryonic differentiation, indicating the importance of SOX17 expression for seminoma cell fate.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Despite advances in combined modality therapy, outcomes in head and neck squamous cell cancer (HNSCC) remain dismal with five-year overall survival rates of less than 50%. Prognostic biomarkers are ...urgently needed to identify patients with a high risk of death after initial curative treatment. Methylation status of the paired-like homeodomain transcription factor 2 (PITX2) has recently emerged as a powerful prognostic biomarker in various cancers. In the present study, the clinical performance of PITX2 methylation was validated in a HNSCC cohort by means of an independent analytical platform (Infinium HumanMethylation450 BeadChip, Illumina, Inc.).
A total of 528 HNSCC patients from The Cancer Genome Atlas (TCGA) were included in the study. Death was defined as primary endpoint. PITX2 methylation was correlated with overall survival and clinicopathological parameters.
PITX2 methylation was significantly associated with sex, tumor site, p16 status, and grade. In univariate Cox proportional hazards analysis, PITX2 hypermethylation analyzed as continuous and dichotomized variable was significantly associated with prolonged overall survival of HNSCC patients (continuous: hazard ratio (HR) = 0.19 95%CI: 0.04-0.88, p = 0.034; dichotomized: HR = 0.52 95%CI: 0.33-0.84, p = 0.007). In multivariate Cox analysis including established clinicopathological parameters, PITX2 promoter methylation was confirmed as prognostic factor (HR = 0.28 95%CI: 0.09-0.84, p = 0.023).
Using an independent analytical platform, PITX2 methylation was validated as a prognostic biomarker in HNSCC patients, identifying patients that potentially benefit from intensified surveillance and/or administration of adjuvant/neodjuvant treatment, i.e. immunotherapy.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The shortage of pathologists in Germany, coupled with an aging workforce, requires innovative approaches to attract medical students to the field. Medical education must address different learning ...styles to ensure that all students are successful.
The pilot project "Practical Pathology" aims to enhance students' understanding of pathology by providing hands-on experience in macroscopic gross analysis through the use of tumor dummies built from scratch.
An evaluation survey, completed by 63 participating students provided positive feedback on the course methodology, its relevance to understanding the pathology workflow, and its improvement over traditional teaching methods. The majority of students recognized the importance of hands-on training in medical education. Students with previous work experience rated the impact of the course on knowledge acquisition even more positively.
The course improved students' understanding of pathological processes and potential sources of clinical-pathological misunderstanding. An increase in motivation for a potential career in the field of pathology was observed in a minority of students, although this exceeded the percentage of pathologists in the total medical workforce.
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