ABSTRACT
PURPOSE: For optimal management of ductal carcinoma in situ (DCIS), reproducible histopathological assessment is essential to distinguish low-risk from high-risk DCIS. Therefore, we analyzed interrater reliability of histopathological DCIS features and assessed their associations with subsequent ipsilateral invasive breast cancer (iIBC) risk. METHODS: Using a case-cohort design, reliability was assessed in a population-based, nationwide cohort of 2767 women with screen-detected DCIS diagnosed between 1993 and 2004, treated by breast-conserving surgery with/without radiotherapy (BCS ± RT) using Krippendorff's alpha (KA) and Gwet's AC2 (GAC2). Thirty-eight raters scored histopathological DCIS features including grade (2-tiered and 3-tiered), growth pattern, mitotic activity, periductal fibrosis, and lymphocytic infiltrate in 342 women. Using majority opinion-based scores for each feature, their association with subsequent iIBC risk was assessed using Cox regression. RESULTS: Interrater reliability of grade using various classifications was fair to moderate, and only substantial for grade 1 versus 2 + 3 when using GAC2 (0.78). Reliability for growth pattern (KA 0.44, GAC2 0.78), calcifications (KA 0.49, GAC2 0.70) and necrosis (KA 0.47, GAC2 0.70) was moderate using KA and substantial using GAC2; for (type of) periductal fibrosis and lymphocytic infiltrate fair to moderate estimates were found and for mitotic activity reliability was substantial using GAC2 (0.70). Only in patients treated with BCS-RT, high mitotic activity was associated with a higher iIBC risk in univariable analysis (Hazard Ratio (HR) 2.53, 95% Confidence Interval (95% CI) 1.05-6.11); grade 3 versus 1 + 2 (HR 2.64, 95% CI 1.35-5.14) and a cribriform/solid versus flat epithelial atypia/clinging/(micro)papillary growth pattern (HR 3.70, 95% CI 1.34-10.23) were independently associated with a higher iIBC risk. CONCLUSIONS: Using majority opinion-based scores, DCIS grade, growth pattern, and mitotic activity are associated with iIBC risk in patients treated with BCS-RT, but interrater variability is substantial. Semi-quantitative grading, incorporating and separately evaluating nuclear pleomorphism, growth pattern, and mitotic activity, may improve the reliability and prognostic value of these features.
Subject(s)
Breast Neoplasms , Carcinoma, Ductal, Breast , Carcinoma, Intraductal, Noninfiltrating , Breast Neoplasms/surgery , Carcinoma, Ductal, Breast/surgery , Carcinoma, Intraductal, Noninfiltrating/surgery , Female , Humans , Mastectomy, Segmental , Neoplasm Recurrence, Local , Prognosis , Reproducibility of ResultsABSTRACT
Fibrinogen is a hexameric plasmatic glycoprotein composed of pairs of three chains (Aα, Bß, and γ), which play an essential role in hemostasis. Conversion of fibrinogen to insoluble polymer fibrin gives structural stability, strength, and adhesive surfaces for growing blood clots. Equally important, the exposure of its non-substrate thrombin-binding sites after fibrin clot formation promotes antithrombotic properties. Fibrinogen and fibrin have a major role in multiple biological processes in addition to hemostasis and thrombosis, i.e., fibrinolysis (during which the fibrin clot is broken down), matrix physiology (by interacting with factor XIII, plasminogen, vitronectin, and fibronectin), wound healing, inflammation, infection, cell interaction, angiogenesis, tumour growth, and metastasis. Congenital fibrinogen deficiencies are rare bleeding disorders, characterized by extensive genetic heterogeneity in all the three genes: FGA, FGB, and FGG (enconding the Aα, Bß, and γ chain, respectively). Depending on the type and site of mutations, congenital defects of fibrinogen can result in variable clinical manifestations, which range from asymptomatic conditions to the life-threatening bleeds or even thromboembolic events. In this manuscript, we will briefly review the main pathogenic mechanisms and risk factors leading to thrombosis, and we will specifically focus on molecular mechanisms associated with mutations in the C-terminal end of the beta and gamma chains, which are often responsible for cases of congenital afibrinogenemia and hypofibrinogenemia associated with thrombotic manifestations.
Subject(s)
Afibrinogenemia/genetics , Fibrinogen/genetics , Fibrinogen/metabolism , Afibrinogenemia/physiopathology , Blood Coagulation Tests , Factor XIII/genetics , Fibrin/genetics , Fibrinolysis/genetics , Hemorrhage , Hemostasis , Hemostatics , Humans , Phenotype , Thrombosis/genetics , Thrombosis/physiopathologyABSTRACT
BACKGROUND: Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. METHODS: Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3+, CD4+, CD8+, CD20+, CD68+, FOXP3+) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures. RESULTS: The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infiltrates were highly variable (r = 0.01-0.56). Similar observations were made for cell type-specific quantifications (r = 0.001-0.54). We observed a strong inter-method variability between the omics-derived estimations, which is further cell type dependent. Finally, we demonstrated that most methods more accurately identify highly infiltrated (sTIL ≥ 60%; area under the curve, AUC, 0.64-0.99) as compared to lowly infiltrated tumors (sTIL ≤ 10%; AUC 0.52-0.82). CONCLUSIONS: There is a lower inter-pathologist concordance for cell-specific quantification as compared to overall infiltration quantification. Microscopic assessments are underestimated when considering small cores (tissue microarray) instead of whole slides. Results further highlight considerable differences between the microscopic-, transcriptomic-, and methylomic-based methods in the assessment of overall and cell-specific immune infiltration in BC. We therefore call for extreme caution when assessing immune infiltrates using current methods and emphasize the need for standardized immune characterization beyond TIL.
Subject(s)
Breast Neoplasms/etiology , Disease Susceptibility , Lymphocytes, Tumor-Infiltrating/immunology , Biomarkers, Tumor , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Epigenome , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Lymphocytes/immunology , Lymphocytes/metabolism , Lymphocytes/pathology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Tissue Array Analysis , Transcriptome , Tumor Microenvironment/immunologyABSTRACT
Congenital fibrinogen disorders are caused by mutations in one of the three fibrinogen genes that affect the synthesis, assembly, intracellular processing, stability or secretion of fibrinogen. Functional studies of mutant Bß-chains revealed the importance of individual residues as well as three-dimensional structures for fibrinogen assembly and secretion. This study describes two novel homozygous fibrinogen Bß chain mutations in two Slovak families with afibrinogenemia and hypofibrinogenemia. Peripheral blood samples were collected from all subjects with the aim of identifying the causative mutation. Coagulation-related tests and rotational thromboelastometry were performed. All exons and exon-intron boundaries of the fibrinogen genes (FGA, FGB and FGG) were amplified by PCR followed by direct sequencing. Sequence analysis of the three fibrinogen genes allowed us to identify two novel homozygous mutations in the FGB gene. A novel Bß chain truncation (BßGln180Stop) was detected in a 28-year-old afibrinogenemic man with bleeding episodes including repeated haemorrhaging into muscles, joints, and soft tissues, and mucocutaneous bleeding and a novel Bß missense mutation (BßTyr368His) was found in a 62-year-old hypofibrinogenemic man with recurrent deep and superficial venous thromboses of the lower extremities. The novel missense mutation was confirmed by molecular modelling. Both studying the molecular anomalies and the modelling of fibrinogenic mutants help us to understand the extremely complex machinery of fibrinogen biosynthesis and finally better assess its correlation with the patient's clinical course.
Subject(s)
Afibrinogenemia/genetics , Fibrinogen/genetics , Hemorrhage/genetics , Mutation , Venous Thrombosis/genetics , Adult , Afibrinogenemia/metabolism , Afibrinogenemia/physiopathology , Base Sequence , Blood Coagulation Tests , Family , Fibrinogen/chemistry , Fibrinogen/metabolism , Fibrinogens, Abnormal/genetics , Fibrinogens, Abnormal/metabolism , Gene Expression , Hemorrhage/metabolism , Hemorrhage/physiopathology , Homozygote , Humans , Male , Middle Aged , Models, Molecular , Pedigree , Protein Structure, Secondary , Severity of Illness Index , Venous Thrombosis/metabolism , Venous Thrombosis/physiopathologyABSTRACT
Platelets are required for the recruitment of bone marrow-derived mononuclear cells (BMMNC) into ischemia-induced vasculature, which underlines their key role in angiogenesis. The difference in platelet immunophenotype between healthy controls and patients with critical limb ischemia (CLI) treated with therapeutic angiogenesis (TA) using BMMNC was assessed. The impact of TA on the expression of platelet membrane markers was studied as well. CLI patients (N = 26) and blood donors as controls (N = 21) were enrolled. Bone marrow (600 ± 50 ml) was centrifuged (3200 g, 20 min, 22 °C). BMMNC (100-120 ml) were separated by Optipress I and implanted to the ischemic limb using deep intramuscular injections. Flow cytometry was employed for the peripheral blood platelets immunophenotyping. CD41FITC, CD62PE, CD36FITC, CD29FITC antibodies were used. Patients were followed up prior to the procedure and at months 1, 3 and 6. The expression of CD41 was lower in CLI patients than in the controls. P-selectin (CD62P) was higher in CLI patients than in controls at the baseline and at month 6. It was significantly down-regulated at month 3, however not at months 1 and 6 compared to baseline. Platelet GPIV (CD36) was higher at the baseline, but not during the follow-up compared to the controls. ß1-integrin (CD29) progressively decreased during the follow-up as compared to the baseline value. Platelets in CLI express P-selectin, GPIV and ß1-integrin more abundantly than platelets of healthy subjects. TA down-regulates the expression of the respective markers. Possible mechanism could be higher clearance of the activated platelets in the ischemic tissues during angiogenesis.
Subject(s)
Blood Platelets/metabolism , Bone Marrow Transplantation , Extremities/blood supply , Ischemia/metabolism , Ischemia/therapy , Platelet Activation , Adult , Antigens, Surface/metabolism , Female , Follow-Up Studies , Humans , Immunophenotyping , Male , Middle Aged , Phenotype , Treatment OutcomeABSTRACT
Congenital fibrinogen disorders are diseases associated with a bleeding tendency; however, there are also reports of thrombotic events. Fibrinogen plays a role in the pathogenesis of thrombosis due to altered plasma concentrations or modifications to fibrinogen's structural properties, which affect clot permeability, resistance to lysis, and its stiffness. Several distinct types of genetic change and pathogenetic mechanism have been described in patients with bleeding and a thrombotic phenotype, including mutations affecting synthesis or processing in three fibrinogen genes. In this paper, we focused on familial hypofibrinogenemia, a rare inherited quantitative fibrinogen disorder characterized by decreased fibrinogen levels with a high phenotypic heterogeneity. To begin, we briefly review the basic information regarding fibrinogen's structure, its function, and the clinical consequences of low fibrinogen levels. Thereafter, we introduce 15 case reports with various gene mutations derived from the fibrinogen mutation database GFHT (French Study Group on Hemostasis and Thrombosis), which are associated with congenital hypofibrinogenemia with both bleeding and thrombosis. Predicting clinical presentations based on genotype data is difficult. Genotype-phenotype correlations would be of help to better understand the pathologic properties of this rare disease and to provide a valuable tool for the identification of patients who are not only at risk of bleeding, but also at risk of a thrombotic event.
ABSTRACT
Congenital fibrinogen disorders are rare pathologies of the hemostasis, comprising quantitative (afibrinogenemia, hypofibrinogenemia) and qualitative (dysfibrinogenemia and hypodysfibrinogenemia) disorders. The clinical phenotype is highly heterogeneous, being associated with bleeding, thrombosis, or absence of symptoms. Afibrinogenemia and hypofibrinogenemia are the consequence of mutations in the homozygous, heterozygous, or compound heterozygous state in one of three genes encoding the fibrinogen chains, which can affect the synthesis, assembly, intracellular processing, stability, or secretion of fibrinogen. In addition to standard coagulation tests depending on the formation of fibrin, diagnostics also includes global coagulation assays, which are effective in monitoring the management of replacement therapy. Genetic testing is a key point for confirming the clinical diagnosis. The identification of the precise genetic mutations of congenital fibrinogen disorders is of value to permit early testing of other at risk persons and better understand the correlation between clinical phenotype and genotype. Management of patients with afibrinogenemia is particularly challenging since there are no data from evidence-based medicine studies. Fibrinogen concentrate is used to treat bleeding, whereas for the treatment of thrombotic complications, administered low-molecular-weight heparin is most often. This review deals with updated information about afibrinogenemia and hypofibrinogenemia, contributing to the early diagnosis and effective treatment of these disorders.
ABSTRACT
von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder. This disorder develops as a result of defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for VWF-related disorders requires the assessment of both VWF level and VWF activity, the latter requiring multiple assays. As an additional step, an evaluation of VWF structural features by multimer analysis is useful in selective investigations. Multimer analysis is also important for the selection of a suitable VWF therapy preparation (desmopressin, VWF/FVIII concentrate, recombinant VWF) and the determination of the correct dose for the patient. Based on clinical and laboratory findings, including the analysis of VWF multimers, we classified our patients into individual types of VWD. Our study group included 58 patients. The study group consisted of 66% (38 patients) with VWD type 1, 5% (3 patients) with VWD type 2, 7% (4 patients) with VWD type 3, 5% (3 patients) with mixed type 1/2A VWD, and 17% (10 patients) comprising an unclassified group. In this article, we provide an overview of our practical experience using a new complementary method-the analysis of von Willebrand factor multimers with a semi-automatic analyzer Hydrasys 2 scan. We explain the principle, procedure, advantages, and pitfalls associated with the introduction of the VWF multimer analysis methodology into standard VWD diagnostics.
ABSTRACT
The prognostic value of cytonuclear grade in ductal carcinoma in situ (DCIS) is debated, partly due to high interobserver variability and the use of multiple guidelines. The aim of this study was to evaluate interobserver agreement in grading DCIS between Dutch, British, and American pathologists. Haematoxylin and eosin-stained slides of 425 women with primary DCIS were independently reviewed by nine breast pathologists based in the Netherlands, the UK, and the USA. Chance-corrected kappa (κma ) for association between pathologists was calculated based on a generalised linear mixed model using the ordinal package in R. Overall κma for grade of DCIS (low, intermediate, or high) was estimated to be 0.50 (95% confidence interval [CI] 0.44-0.56), indicating a moderate association between pathologists. When the model was adjusted for national guidelines, the association for grade did not change (κma = 0.53; 95% CI 0.48-0.57); subgroup analysis for pathologists using the UK pathology guidelines only had significantly higher association (κma = 0.58; 95% CI 0.56-0.61). To assess if concordance of grading relates to the expression of the oestrogen receptor (ER) and HER2, archived immunohistochemistry was analysed on a subgroup (n = 106). This showed that non-high grade according to the majority opinion was associated with ER positivity and HER2 negativity (100 and 89% of non-high grade cases, respectively). In conclusion, DCIS grade showed only moderate association using whole slide images scored by nine breast pathologists. As therapeutic decisions and inclusion in ongoing clinical trials are guided by DCIS grade, there is a pressing need to reduce interobserver variability in grading. ER and HER2 might be supportive to prevent the accidental and unwanted inclusion of high-grade DCIS in such trials.
Subject(s)
Breast Neoplasms/pathology , Carcinoma, Intraductal, Noninfiltrating/pathology , Pathologists , Biomarkers, Tumor/analysis , Biopsy , Breast Neoplasms/chemistry , Carcinoma, Intraductal, Noninfiltrating/chemistry , Europe , Female , Humans , Immunohistochemistry , Neoplasm Grading , Observer Variation , Predictive Value of Tests , Receptor, ErbB-2/analysis , Receptors, Estrogen/analysis , Reproducibility of Results , Retrospective Studies , United StatesABSTRACT
AKT inhibitors have promising activity in AKT1 E17K-mutant estrogen receptor (ER)-positive metastatic breast cancer, but the natural history of this rare genomic subtype remains unknown. Utilizing AACR Project GENIE, an international clinicogenomic data-sharing consortium, we conducted a comparative analysis of clinical outcomes of patients with matched AKT1 E17K-mutant (n = 153) and AKT1-wild-type (n = 302) metastatic breast cancer. AKT1-mutant cases had similar adjusted overall survival (OS) compared with AKT1-wild-type controls (median OS, 24.1 vs. 29.9, respectively; P = 0.98). AKT1-mutant cases enjoyed longer durations on mTOR inhibitor therapy, an observation previously unrecognized in pivotal clinical trials due to the rarity of this alteration. Other baseline clinicopathologic features, as well as durations on other classes of therapy, were broadly similar. In summary, we demonstrate the feasibility of using a novel and publicly accessible clincogenomic registry to define outcomes in a rare genomically defined cancer subtype, an approach with broad applicability to precision oncology. SIGNIFICANCE: We delineate the natural history of a rare genomically distinct cancer, AKT1 E17K-mutant ER-positive breast cancer, using a publicly accessible registry of real-world patient data, thereby illustrating the potential to inform drug registration through synthetic control data.See related commentary by Castellanos and Baxi, p. 490.
Subject(s)
Breast Neoplasms/genetics , Proto-Oncogene Proteins c-akt/metabolism , Adult , Aged , Aged, 80 and over , Breast Neoplasms/pathology , Female , Humans , Middle Aged , Mutation , Registries , Treatment OutcomeABSTRACT
Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting.
ABSTRACT
Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring.
ABSTRACT
Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls.
ABSTRACT
Cystic form in the spleen was diagnosed accidentally in a 44 years old patient during ultrasonography of the abdomen. It reached 6.0 cms in diameter. The mass was localised superficially, closely beneath the capsule of the spleen. The diagnose was confirmed serologically. Because of the localisation and the size of the cyst, the surgical approach - splenectomy - was indicated and performed.
Subject(s)
Echinococcosis/diagnosis , Echinococcus granulosus , Splenic Diseases/diagnosis , Adult , Animals , Female , HumansABSTRACT
Tumour suppressor protein p53 prevents cancer development through various mechanisms, including the induction of apoptosis. We demonstrated that acute leukaemia, myeloblastic (AML) and lymphoblastic (ALL), is associated with significantly elevated levels of p53 and Bax mRNA in leukaemic cells. Regarding ALL, significantly elevated levels of Bcl-xL mRNA may explain the relative resistance of ALL cells to p53-dependent apoptosis. Altered alternative processing of Bcl-x and myeloid cell leukaemia-1 (MCL1) primary transcripts were observed in the case of AML and AML and ALL, respectively. We assumed that increased glyceraldehyde-3-phosphate dehydrogenase (gapdh) transcription and decreased MCL1s mRNA were not fully responsible for the dysregulation of p53-dependent apoptosis in the case of AML. In addition, transcription of hsp70.1 and Bcl-2 producing anti-apoptotic proteins was not affected in acute leukaemia.