Impact of Prosigna test on adjuvant treatment decision in lymph node-negative early breast cancer-a prospective national multicentre study (EMIT-1).

BACKGROUND: EMIT-1 is a national, observational, single-arm trial designed to assess the value of the Prosigna, Prediction Analysis of Microarray using the 50 gene classifier (PAM50)/Risk of Recurrence (ROR), test as a routine diagnostic tool, examining its impact on adjuvant treatment decisions, clinical outcomes, side-effects and cost-effectiveness. Here we present the impact on treatment decisions. PATIENTS AND METHODS: Patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative pT1-pT2 lymph node-negative early breast cancer (EBC) were included. The Prosigna test and standard histopathology assessments were carried out. Clinicians' treatment decisions were recorded before (pre-Prosigna) and after (post-Prosigna) the Prosigna test results were disclosed. RESULTS: Of 2217 patients included, 2178 had conclusive Prosigna results. The pre-Prosigna treatment decisions were: no systemic treatment (NT) in 27% of patients, endocrine treatment alone (ET) in 38% and chemotherapy (CT) followed by ET (CT + ET) in 35%. Post-Prosigna treatment decisions were 25% NT, 51% ET and 24% CT + ET, respectively. Adjuvant treatment changed in 28% of patients, including 21% change in CT use. Among patients assigned to CT + ET pre-Prosigna, 45% were de-escalated to ET post-Prosigna. Of patients assigned to ET, 12% were escalated to CT + ET and 8% were de-escalated to NT; of those assigned to NT, 18% were escalated to ET/CT + ET. CT was more frequently recommended for patients aged ≤50 years. In the subgroup with pT1c-pT2 G2 and intermediate Ki67 (0.5-1.5× local laboratory median Ki67 score), the pre-Prosigna CT treatment decision varied widely across hospitals (3%-51%). Post-Prosigna, the variability of CT use was markedly reduced (8%-24%). The correlation between Ki67 and ROR score within this subgroup was poor (r = 0.25-0.39). The median ROR score increased by increasing histological grade, but the ROR score ranges were wide (for G1 0-79, G2 0-90, G3 16-94). CONCLUSION: The Prosigna test result changed adjuvant treatment decisions in all EBC clinical risk groups, markedly decreased the CT use for patients categorized as higher clinical risk pre-Prosigna and reduced treatment decision discrepancies between hospitals.

[Psychiatric symptoms and COVID-19: results of a national case register]. / Psychiatrische symptomen en COVID-19: resultaten uit een landelijk register.

BACKGROUND: Psychiatric disorders are associated with a more severe course of COVID-19. COVID-19 can also lead to psychiatric symptoms. AIM: To gain insight into vulnerabilities and protective factors for the course of COVID-19 in a Dutch (neuro)psychiatric population. METHOD: Patients were divided into three groups: patients with pre-existent mental disorders without and with new (neuro)psychiatric symptoms (NPS) during COVID-19 and patients without pre-existent mental disorders who developed de novo NPS during COVID-19. We summarize the characteristics of each group and compare the subgroups with inferential statistics. RESULTS: 186 patients were included in the case register. Patients with NPS showed a more severe course of COVID-19. Mortality in patients with NPS was higher in patients with pre-existent mental disorders compared to patients without pre-existent mental disorders. The most frequently reported de novo psychiatric symptoms during COVID-19 were delirium (46-70%), anxiety (53-54%) and insomnia (18-42%). CONCLUSION: NPS might be an expression of a more severe COVID-19 episode. In patients who developed NPS during COVID-19 we found evidence for a higher mortality risk in patients with pre-existent mental disorders. Extra vigilance for neuropsychiatric symptoms during COVID-19 is warranted.

Olaparib monotherapy as primary treatment in unselected triple negative breast cancer.

BACKGROUND: The antitumor efficacy of PARP inhibitors (PARPi) for breast cancer patients harboring germline BRCA1/2 (gBRCA1/2) mutations is well established. While PARPi monotherapy was ineffective in patients with metastatic triple negative breast cancer (TNBC) wild type for BRCA1/2, we hypothesized that PARPi may be effective in primary TNBCs without previous chemotherapy exposure. PATIENTS AND METHODS: In the phase II PETREMAC trial, patients with primary TNBC >2 cm received olaparib for up to 10 weeks before chemotherapy. Tumor biopsies collected before and after olaparib underwent targeted DNA sequencing (360 genes) and BRCA1 methylation analyses. In addition, BRCAness (multiplex ligation-dependent probe amplification), PAM50 gene expression, RAD51 foci, tumor-infiltrating lymphocytes (TILs) and PD-L1 analyses were performed on pretreatment samples. RESULTS: The median pretreatment tumor diameter was 60 mm (range 25-112 mm). Eighteen out of 32 patients obtained an objective response (OR) to olaparib (56.3%). Somatic or germline mutations affecting homologous recombination (HR) were observed in 10/18 responders [OR 55.6%, 95% confidence interval (CI) 33.7-75.4] contrasting 1/14 non-responders (OR 7.1%; CI 1.3-31.5, P = 0.008). Among tumors without HR mutations, 6/8 responders versus 3/13 non-responders revealed BRCA1 hypermethylation (P = 0.03). Thus, 16/18 responders (88.9%, CI 67.2-96.9), in contrast to 4/14 non-responders (28.6%, CI 11.7-54.7, P = 0.0008), carried HR mutations and/or BRCA1 methylation. Excluding one gPALB2 and four gBRCA1/2 mutation carriers, 12/14 responders (85.7%, CI 60.1-96.0) versus 3/13 non-responders (23.1%, CI 8.2-50.3, P = 0.002) carried somatic HR mutations and/or BRCA1 methylation. In contrast to BRCAness signature or basal-like subtype, low RAD51 scores, high TIL or high PD-L1 expression all correlated to olaparib response. CONCLUSION: Olaparib yielded a high clinical response rate in treatment-naïve TNBCs revealing HR deficiency, beyond germline HR mutations. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02624973.

Low Protein A20 in Minor Salivary Glands is Associated with Lymphoma in Primary Sjögren's Syndrome.

Patients with primary Sjögren's syndrome (pSS) have an increased risk of developing lymphomas, particularly the subtype mucosa-associated lymphoid tissue (MALT) lymphoma. Chronic antigen stimulation and increased activation of nuclear factor-κB (NF-κB) are important factors for the pathogenesis of MALT lymphomas. Protein A20 is an inhibitor of NF-κB. A recent study of pSS-associated MALT lymphomas identified potential functional abnormalities in the TNFAIP3 gene, which encodes protein A20. The present study aimed to assess protein A20 by immunohistochemistry (IHC) in minor salivary glands (MSGs) and lymphoma tissue sections of patients with pSS and investigate a potential association with lymphoma development. Protein A20 staining in lymphocytes was scored in four categories (0 = negative, 1 = weak, 2 = moderate and 3 = strong). For statistical purposes, these scores were simplified into negative (scores 0-1) and positive (scores 2-3). We investigated associations between protein A20-staining, focus scores, germinal centre (GC)-like structures and monoclonal B-cell infiltration in MSGs. MSG protein A20 staining was weaker in pSS patients with lymphomas than in those without lymphomas (P = 0.01). Weak protein A20 staining was also highly associated with a lack of GC formation (P < 0.01). Finally, weaker A20 staining was observed in the majority of pSS-associated MALT lymphoma tissues. In conclusion, we found absent or weak protein A20 immunoreactivity in MSGs of patients with pSS with lymphomas. This finding indicates that protein A20 downregulation in lymphocytes might be a mechanism underlying lymphoma genesis in patients with pSS.

Reduced Number of CD8+ Cells in Tonsillar Germinal Centres in Children with the Periodic Fever, Aphthous Stomatitis, Pharyngitis and Cervical Adenitis Syndrome.

The syndrome of periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) is an autoinflammatory disorder of unknown aetiology. Tonsillectomy may cause a prompt resolution of the syndrome. The aim was to study the histologic and immunological aspects of the palatine tonsils in PFAPA, to help understand the pathophysiology of the syndrome. Tonsils from children with PFAPA (n = 11) and children with tonsillar hypertrophy (n = 16) were evaluated histologically after haematoxylin and eosin staining. The number of different cell types was identified immunohistochemically by cluster of differentiation (CD) markers: CD3 (T cells), CD4 (T helper cells), CD8 (cytotoxic T cells), CD15 (neutrophils), CD20 (B cells), CD45 (all leucocytes), CD57 (NK cells) and CD163 (monocytes and macrophages). Tonsils from children with PFAPA showed reactive lymphoid hyperplasia dominated by well-developed germinal centres with many tingible body macrophages. The histologic findings were unspecific, and a similar morphologic appearance was also found in the tonsils from controls. The number of CD8+ cells in germinal centres differed between children with PFAPA [median 9 cells (quartiles: 5, 15)] and controls [18 cells (12, 33) (P = 0.001)] and between children with PFAPA with (median 14 cells; 9, 16) and without (4 cells; 3, 8) aphthous stomatitis (P = 0.015). For the other cell types, no differences in germinal centres were found between children with PFAPA and controls. In conclusion, a lower number of CD8+ cells were found in germinal centres of tonsils in children with PFAPA compared to controls, which may be a feature linked to the aetiology of the syndrome.

Intravenous cyclophosphamide in patients with chronic systemic inflammatory diseases: morbidity and mortality.

BACKGROUND: Few data exist concerning the development of malignancies and haemorrhagic cystitis in patients with systemic autoimmune diseases previously treated with intravenous (iv) cyclophosphamide (CYC). The use of mesna prophylaxis is also controversial. METHODS: The medical records of all patients with chronic systemic inflammatory diseases treated with iv or oral CYC at Stavanger University Hospital from 1985 to 1999 were reviewed. Eighty-five patients were identified, of whom 75 patients had been treated with iv CYC only and were thus included in this study. Of these 75 patients, 20 (27%) had died and 55 (73%) were alive. Forty-two (76%) out of the 55 patients consented to undergo a comprehensive clinical examination, including a cystoscopy in 33 of them. The medical history of the patients not clinically examined was based solely on medical records. Data from the Cancer Registry of Norway and Statistics Norway were used for comparison with normative data in the general population. RESULTS: Six patients (8%) developed malignant disease compared with an expected number of 4.5, giving a standard incidence ratio of 1.5 [95% confidence interval (CI) 0.7-3.2]. The observed number of deaths was 23 compared to an expected number of 6.3, giving a standard mortality ratio of 3.7 (95% CI 2.4-5.5). CONCLUSIONS: The standard incidence ratio of cancer following iv CYC was increased, although not statistically significantly. No urinary bladder cancer or haemorrhagic cystitis developed even though mesna prophylaxis was not given.

Comparing subjective and digital image analysis HER2/neu expression scores with conventional and modified FISH scores in breast cancer.

BACKGROUND: HER2/neu expression and fluorescence in situ hybridisation (FISH) amplification have therapeutic significance. AIMS: To compare subjective HER2/neu expression scores with digital image analysis (DIA) and conventional and modified FISH scores in breast cancer. METHODS: Sixty HercepTest-immunostained breast carcinomas, prospectively scored as consensus 2+ and 3+ (DAKO protocol) by two observers, were analysed with DIA, and conventional (Vysis) and modified FISH scoring protocols. RESULTS: With consensus scoring, 23 (38%) of the 60 cases were 2+ and 37 (62%) were 3+. Agreement with DIA scores was 100%. With conventional FISH scoring, 4 of the 3+ cases did not show amplification, but all of those negative cases had high HER2/neu copy numbers. With the modified FISH scoring protocol, all HercepTest immunohistochemical 3+ cases were amplified. Of the 2+ cases, 3 were amplified with the modified FISH protocol and 4 with the conventional FISH protocol. CONCLUSIONS: Modified FISH scores were better correlated with HercepTest 3+ consensus and DIA scores than were conventional FISH scores. HER2/neu DIA scoring is a cost-effective supplementary tool in surgical pathology.

Proliferation accurately identifies the high-risk patients among small, low-grade, lymph node-negative invasive breast cancers.

BACKGROUND: The proliferation factor mitotic activity index (MAI) is the strongest prognosticator in lymph node-negative invasive breast cancer patients under age 71. The question remains, whether this also holds for 'favourable prognosis' subgroups. PATIENTS AND METHODS: The study was a multicentre prospective analysis of the MAI for recurrence-free survival and overall cancer-related survival of grade, MAI, and other prognosticators in 853 long-term follow-up, T1-3N0M0 breast cancer patients under 71 years. RESULTS: In all tumours together (N = 853), in grade 3 (n = 269), in tumours <1 cm all grades (n = 84), 1-2 cm, grades 1 + 2 (n = 300), and 2-3 cm, grades 1 + 2 (n = 124), the MAI is prognostically superior. Other features [grade, estrogen receptor (ER), diameter, and age] did not enhance its prognostic value except in grades 1 + 2 tumours 2-3 cm diameter with MAI <10, where ER has an additional prognostic value. CONCLUSIONS: In women <71 years with T1-3N0M0 small or low-grade invasive breast cancer usually not receiving systemic treatment, MAI > or =10 accurately identifies those at high risk. These high-risk patients should be considered for adjuvant systemic therapy.

Dynamic behavioural interpretation of cervical intraepithelial neoplasia with molecular biomarkers.

The microscopic phenotype of cervical intraepithelial neoplasia (CIN) reflects a fine balance between factors that promote or reduce CIN development. A shortcoming of the current grading system is its reliance on static morphology and microscopic haematoxylin-eosin features of the epithelium alone. In reality, CIN is a dynamic process, and the epithelium may exhibit differing results over time. Functional biomarkers p16, Ki-67, p53, retinoblastoma protein cytokeratin (CK)14 and CK13, help in the assessment of an individual CIN's lesion's potential for progression and regression. The aggregate information provided by these biomarkers exceeds the value of the classic grading system. Consequently, many more CINs that will either regress or progress can be accurately identified. These findings agree with known molecular interactions between HPV and the host. For accurate interpretation of a CIN, it is essential that these biomarkers be determined quantitatively and separately in the superficial, middle and deep layers of the epithelium. Such geography-specific epithelial evaluations of quantitative biomarkers emphasise the dynamic nature of a particular CIN lesion, thereby changing the art of static morphology grading into dynamic interpretation of the diseased tissue, with a strong prognostic effect.

Microsatellite instability in colorectal cancer.

BACKGROUND: Microsatellite instability (MSI) causes hereditary non-polyposis colorectal cancer (HNPCC), and occurs in about 15 per cent of sporadic colorectal cancers. Although the basic mechanisms are not clear, there is increased understanding of the clinicopathological consequences of MSI. METHODS: Medline was searched for articles with a combination of keywords relating to MSI in colorectal cancer, focusing on molecular mechanisms, clinicopathological implications, and prognostic and predictive value. Emphasis was placed on articles from the past 5 years. RESULTS: The genetic mechanisms differ in hereditary (germline mutation) and sporadic (epigenetic silencing) colorectal cancer. The MSI pathway frequently has altered transforming growth factor beta receptor II and BAX genes, often beta-catenin, and occasionally p16INK4A and PTEN. Changes in K-ras, adenomatous polyposis coli and p53 are rare. Polymerase chain reaction testing for MSI is superior to immunohistochemistry, but complicated by the number and types of nucleotide markers. The Bethesda panel guides HNPCC testing, but guidelines are lacking for general screening. The presence and role of low-frequency MSI remains controversial. Tumours with MSI tend to occur in the proximal colon and be large, but they have a good prognosis. Their reduced response to adjuvant chemotherapy requires confirmation. CONCLUSION: Research on colorectal cancer needs to be stratified according to microsatellite status in order further to explore the molecular mechanisms and clinicopathological consequences of MSI.

Proliferation markers and DNA content analysis in urinary bladder TaT1 urothelial cell carcinomas: identification of subgroups with low and high stage progression risks.

AIMS: To evaluate whether in situ biomarkers Ki67, mitotic activity index (MAI), p53, mean area of the 10 largest nuclei (MNA10), and whole genome DNA ploidy by flow and image cytometry (FCM and ICM, respectively) have independent prognostic value in urinary bladder urothelial cell carcinomas (UCs). METHODS: Ki67 and p53 immunoquantitation was performed in TaT1 consensus diagnosis UCs. MAI and MNA10 were also determined. Single cell suspensions were stained (DAPI for FCM; Feulgen for ICM). There was enough material for all measurements in 171 cases. Kaplan-Meier curves and multivariate survival analysis (Cox) were used to assess the prognostic value of all features (including classic clinicopathological risk factors, such as stage, grade, multicentricity, carcinoma in situ). RESULTS: Thirteen (7.6%) patients progressed. Of the classic factors, grade was strongly prognostic in univariate analysis, as were all the biomarkers. In multivariate analysis, the strongest independent combinations for progression were MNA10 (threshold (T) = 170.0 micro m(2)) plus MAI (T = 30), or MNA10 (T = 170.0 micro m(2)) plus Ki67(T = 25.0%). p53 (T = 35.2%) plus Ki67 (T = 25.0%) also predicted progression well, with high hazard ratios, but p53 measurements were not as reproducible as the other features. The prognostic value of the quantitative biomarkers exceeded that of the classic risk factors and DNA ploidy. The sensitivity, specificity, positive, and negative predictive values of MNA10/MAI or MNA10/Ki67 at the thresholds mentioned were 100%, 79%, 57%, and 100%, respectively. These feature combinations were also strongest prognostically in the high risk treatment subgroup. CONCLUSIONS: The combined biomarkers MNA10/Ki67 or MNA10/MAI are more accurate and reproducible predictors of stage progression in TaT1 UCs than classic prognostic risk factors and DNA ploidy.

DNA cytometric features in biopsies of TaT1 urothelial cell cancer predict recurrence and stage progression more accurately than stage, grade, or treatment modality.

OBJECTIVES: To compare retrospectively the predictive value for recurrence and stage progression of DNA ploidy and S-phase fraction by flow cytometry and highly automated ultrafast image cytometry (ICM) in biopsies of TaT1 urothelial cell carcinomas (UCCs) of the urinary bladder with stage, grade, other pathologic features, and treatment. METHODS: Three experienced pathologists reviewed the stage and grade of 228 UCCs; 193 (85%) consensus cases were analyzed further. We had enough material for single-cell suspensions for both flow cytometry and ICM in 183 cases (94.8%). The 2001 European Society for Analytical Cellular Pathology standards for DNA ICM were followed. The predictive value of DNA features, classic prognosticators (stage, grade, carcinoma in situ, multicentricity), and treatment modality for recurrence and stage progression were analyzed with univariate (Kaplan-Meier) survival and multivariate (Cox model) regression analysis. Ta and T1 cases were analyzed separately. RESULTS: Of the 228 cases, 88 (51.5%) recurred and 13 (7.6%) progressed. On univariate analysis, most of the DNA features studied were statistically significant. Treatment modality and grade were only prognostic for progression (not for recurrence) and only in Ta cases. On multivariate analysis, DNA ICM features performed best; the strongest recurrence predictor for Ta UCC was a DNA index (DI) of 1.0 versus all others, and for T1 UCC, a DI of less than 1.3 versus 1.3 or greater. The best stage progression predictor for Ta UCCs was a DI of 1.0 plus an S-phase fraction of less than 10%, and for T1 UCCs, a DI of less than 1.3 versus 1.3 or greater. With multivariate analysis, sex, age, grade, carcinoma in situ, multicentricity, and treatment modality were excluded once the DNA ICM features were selected. CONCLUSIONS: DNA image cytometric features predict recurrence and stage progression in TaT1 UCC biopsies more accurately than classic prognostic factors, independent of treatment modality.

Correlation of grade of urothelial cell carcinomas and DNA histogram features assessed by flow cytometry and automated image cytometry.

OBJECTIVE: To analyse how DNA ploidy and S-phase fraction (SPF) by flow cytometry (FCM) and an optimised fully automatic DNA image cytometer (ICM) correlate with grade in TaT1 urothelial cell carcinomas (UC) of the urinary bladder. MATERIALS AND METHODS: Two-hundred-and twenty-eight consensus cases were analysed. Single cell suspensions were stained (DAPI for FCM, Feulgen for ICM). There was enough material for both FCM and ICM in 202 of these cases. FCM and optimised ICM measurements were performed on the 202 UCs. To discriminate between different grades, single- and multivariate analyses was performed on DNA histogram features obtained with the MultiCycle program (using DNA index (DI) and SPF). RESULTS: Overall measurement time of the adapted ICM method was 10.7 minutes per case (range 5.9-29.8 min.) and required little additional interactive object rejection (average 152 objects (84-298) on 3000 objects per case measured, which took 9.9 minutes on average, range 8.3-15.5 minutes). The ICM histograms looked much "cleaner" with less noise than the FCM graphs. The coefficient of variation (CV) of the diploid peak for ICM (5.4%) was significantly lower than for FCM (5.9%) (p<0.0001). ICM features were more strongly correlated to grade than FCM features. In multivariate analysis, the best discriminating set of features was DNA ploidy and SPF (both by ICM). CONCLUSIONS: The adapted fully automated DNA ICM works very well for UCs. Low CV DNA ICM histograms are obtained in a time comparable to FCM. The DNA ICM results have stronger discriminative power than DNA FCM for grade in TaT1 UCs.

Genomics and proteomics in cancer.

Cancer development is driven by the accumulation of DNA changes in the approximately 40000 chromosomal genes. In solid tumours, chromosomal numerical/structural aberrations are common. DNA repair defects may lead to genome-wide genetic instability, which can drive further cancer progression. The genes code the actual players in the cellular processes, the 100000-10 million proteins, which in (pre)malignant cells can also be altered in a variety of ways. Over the past decade, our knowledge of the human genome and Genomics (the study of the human genome) in (pre)malignancies has increased enormously and Proteomics (the analysis of the protein complement of the genome) has taken off as well. Both will play an increasingly important role. In this article, a short description of the essential molecular biological cell processes is given. Important genomic and proteomic research methods are described and illustrated. Applications are still limited, but the evidence so far is exciting. Will genomics replace classical diagnostic or prognostic procedures? In breast cancers, the gene expression array is stronger than classical criteria, but in endometrial hyperplasia, quantitative morphological features are more cost-effective than genetic testing. It is still too early to make strong statements, the more so because it is expected that genomics and proteomics will expand rapidly. However, it is likely that they will take a central place in the understanding, diagnosis, monitoring and treatment of (pre)cancers of many different sites.