MetDecode: methylation-based deconvolution of cell-free DNA for noninvasive multi-cancer typing.

MOTIVATION: Circulating-cell free DNA (cfDNA) is widely explored as a noninvasive biomarker for cancer screening and diagnosis. The ability to decode the cells of origin in cfDNA would provide biological insights into pathophysiological mechanisms, aiding in cancer characterization and directing clinical management and follow-up. RESULTS: We developed a DNA methylation signature-based deconvolution algorithm, MetDecode, for cancer tissue origin identification. We built a reference atlas exploiting de novo and published whole-genome methylation sequencing data for colorectal, breast, ovarian, and cervical cancer, and blood-cell-derived entities. MetDecode models the contributors absent in the atlas with methylation patterns learnt on-the-fly from the input cfDNA methylation profiles. In addition, our model accounts for the coverage of each marker region to alleviate potential sources of noise. In-silico experiments showed a limit of detection down to 2.88% of tumor tissue contribution in cfDNA. MetDecode produced Pearson correlation coefficients above 0.95 and outperformed other methods in simulations (P < 0.001; T-test; one-sided). In plasma cfDNA profiles from cancer patients, MetDecode assigned the correct tissue-of-origin in 84.2% of cases. In conclusion, MetDecode can unravel alterations in the cfDNA pool components by accurately estimating the contribution of multiple tissues, while supplied with an imperfect reference atlas. AVAILABILITY AND IMPLEMENTATION: MetDecode is available at https://github.com/JorisVermeeschLab/MetDecode.

Dissecting the immune infiltrate of primary luminal B-like breast carcinomas in relation to age.

The impact of aging on the immune landscape of luminal breast cancer (Lum-BC) is poorly characterized. Understanding the age-related dynamics of immune editing in Lum-BC is anticipated to improve the therapeutic benefit of immunotherapy in older patients. To this end, here we applied the 'multiple iterative labeling by antibody neo-deposition' (MILAN) technique, a spatially resolved single-cell multiplex immunohistochemistry method. We created tissue microarrays by sampling both the tumor center and invasive front of luminal breast tumors collected from a cohort of treatment-naïve patients enrolled in the prospective monocentric IMAGE (IMmune system and AGEing) study. Patients were subdivided into three nonoverlapping age categories (35-45 = 'young', n = 12; 55-65 = 'middle', n = 15; ≥70 = 'old', n = 26). Additionally, depending on localization and amount of cytotoxic T lymphocytes, the tumor immune types 'desert' (n = 22), 'excluded' (n = 19), and 'inflamed' (n = 12) were identified. For the MILAN technique we used 58 markers comprising phenotypic and functional markers allowing in-depth characterization of T and B lymphocytes (T&B-lym). These were compared between age groups and tumor immune types using Wilcoxon's test and Pearson's correlation. Cytometric analysis revealed a decline of the immune cell compartment with aging. T&B-lym were numerically less abundant in tumors from middle-aged and old compared to young patients, regardless of the geographical tumor zone. Likewise, desert-type tumors showed the smallest immune-cell compartment and were not represented in the group of young patients. Analysis of immune checkpoint molecules revealed a heterogeneous geographical pattern of expression, indicating higher numbers of PD-L1 and OX40-positive T&B-lym in young compared to old patients. Despite the numerical decline of immune infiltration, old patients retained higher expression levels of OX40 in T helper cells located near cancer cells, compared to middle-aged and young patients. Aging is associated with important numerical and functional changes of the immune landscape in Lum-BC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Research autopsy programmes in oncology: shared experience from 14 centres across the world.

While there is a great clinical need to understand the biology of metastatic cancer in order to treat it more effectively, research is hampered by limited sample availability. Research autopsy programmes can crucially advance the field through synchronous, extensive, and high-volume sample collection. However, it remains an underused strategy in translational research. Via an extensive questionnaire, we collected information on the study design, enrolment strategy, study conduct, sample and data management, and challenges and opportunities of research autopsy programmes in oncology worldwide. Fourteen programmes participated in this study. Eight programmes operated 24 h/7 days, resulting in a lower median postmortem interval (time between death and start of the autopsy, 4 h) compared with those operating during working hours (9 h). Most programmes (n = 10) succeeded in collecting all samples within a median of 12 h after death. A large number of tumour sites were sampled during each autopsy (median 15.5 per patient). The median number of samples collected per patient was 58, including different processing methods for tumour samples but also non-tumour tissues and liquid biopsies. Unique biological insights derived from these samples included metastatic progression, treatment resistance, disease heterogeneity, tumour dormancy, interactions with the tumour micro-environment, and tumour representation in liquid biopsies. Tumour patient-derived xenograft (PDX) or organoid (PDO) models were additionally established, allowing for drug discovery and treatment sensitivity assays. Apart from the opportunities and achievements, we also present the challenges related with postmortem sample collections and strategies to overcome them, based on the shared experience of these 14 programmes. Through this work, we hope to increase the transparency of postmortem tissue donation, to encourage and aid the creation of new programmes, and to foster collaborations on these unique sample collections. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Aging-accumulated methylmalonic acid serum levels at breast cancer diagnosis are not associated with distant metastases.

PURPOSE: Recent evidence suggests that age-accumulated methylmalonic acid (MMA) promotes breast cancer progression in mice. This study aims to investigate the association between baseline serum MMA concentrations in patients with breast cancer and the development of subsequent distant metastases. METHODS: We included 32 patients with early Luminal B-like breast cancer (LumB, median age 62.4y) and 52 patients with early triple-negative breast cancer (TNBC, median age 50.5y) who developed distant metastases within 5 years. They were matched to an equal number of early breast cancer patients (median age 62.2y for LumB and 50.5y for TNBC) who did not develop distant metastases with at least 5 years of follow-up. RESULTS: Baseline serum MMA levels at breast cancer diagnosis showed a positive correlation with age (P < 0.001) and a negative correlation with renal function and vitamin B12 (all P < 0.02), but no statistical association was found with BMI or tumor stage (P > 0.6). Between matched pairs, no significant difference was observed in MMA levels, after adjusting for kidney function and age (P = 0.19). Additionally, in a mouse model, a significant decline in MMA levels was observed in the tumor-bearing group compared to the group without tumors before and after tumor establishment or at identical times for the control group (P = 0.03). CONCLUSION: Baseline serum MMA levels in patients with breast cancer are not correlated with secondary distant metastasis. Evidence in the mouse model suggests that the presence of a tumor perturbates MMA levels.

Integration of Pathological Criteria and Immunohistochemical Evaluation for Invasive Lobular Carcinoma Diagnosis: Recommendations From the European Lobular Breast Cancer Consortium.

Invasive lobular carcinoma (ILC) is the second most frequent type of breast cancer (BC) and its peculiar morphology is mainly driven by inactivation of CDH1, the gene coding for E-cadherin cell adhesion protein. ILC-specific therapeutic and disease-monitoring approaches are gaining momentum in the clinic, increasing the importance of accurate ILC diagnosis. Several essential and desirable morphologic diagnostic criteria are currently defined by the World Health Organization, the routine use of immunohistochemistry (IHC) for E-cadherin is not recommended. Disagreement in the diagnosis of ILC has been repeatedly reported, but interpathologist agreement increases with the use of E-cadherin IHC. In this study, we aimed to harmonize the pathological diagnosis of ILC by comparing 5 commonly used E-cadherin antibody clones (NCH-38, EP700Y, Clone 36, NCL-L-E-cad [Clone 36B5], and ECH-6). We determined their biochemical specificity for the E-cadherin protein and IHC staining performance according to type and location of mutation on the CDH1 gene. Western blot analysis on mouse cell lines with conditional E-cadherin expression revealed a reduced specificity of EP700Y and NCL-L-E-cad for E-cadherin, with cross-reactivity of Clone 36 to P-cadherin. The use of IHC improved interpathologist agreement for ILC, lobular carcinoma in situ, and atypical lobular hyperplasia. The E-cadherin IHC staining pattern was associated with variant allele frequency and likelihood of nonsense-mediated RNA decay but not with the type or position of CDH1 mutations. Based on these results, we recommend the indication for E-cadherin staining, choice of antibodies, and their interpretation to standardize ILC diagnosis in current pathology practice.

Circulating biomarkers at diagnosis correlate with distant metastases of early luminal-like breast cancer.

There is an urgent need for new and better biomarker modalities to estimate the risk of recurrence within the luminal-like breast cancer (BC) population. Molecular diagnostic tests used in the clinic lack accuracy in identifying patients with early luminal BC who are likely to develop metastases. This study provides proof of concept that various liquid biopsy read-outs could serve as valuable candidates to build a multi-modal biomarker model distinguishing, already at diagnosis, between early metastasizing and non-metastasizing patients. All these blood biomarkers (chemokines, microRNAs, leukemia inhibitory factor, osteopontin, and serum-induced functional myeloid signaling responses) can be measured in baseline plasma/serum samples and could be added to the existing prognostic factors to improve risk stratification and more patient-tailored treatment in early luminal BC.

Common variants in breast cancer risk loci predispose to distinct tumor subtypes.

BACKGROUND: Genome-wide association studies (GWAS) have identified multiple common breast cancer susceptibility variants. Many of these variants have differential associations by estrogen receptor (ER) status, but how these variants relate with other tumor features and intrinsic molecular subtypes is unclear. METHODS: Among 106,571 invasive breast cancer cases and 95,762 controls of European ancestry with data on 173 breast cancer variants identified in previous GWAS, we used novel two-stage polytomous logistic regression models to evaluate variants in relation to multiple tumor features (ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and grade) adjusting for each other, and to intrinsic-like subtypes. RESULTS: Eighty-five of 173 variants were associated with at least one tumor feature (false discovery rate < 5%), most commonly ER and grade, followed by PR and HER2. Models for intrinsic-like subtypes found nearly all of these variants (83 of 85) associated at p < 0.05 with risk for at least one luminal-like subtype, and approximately half (41 of 85) of the variants were associated with risk of at least one non-luminal subtype, including 32 variants associated with triple-negative (TN) disease. Ten variants were associated with risk of all subtypes in different magnitude. Five variants were associated with risk of luminal A-like and TN subtypes in opposite directions. CONCLUSION: This report demonstrates a high level of complexity in the etiology heterogeneity of breast cancer susceptibility variants and can inform investigations of subtype-specific risk prediction.

Histopathological growth patterns of liver metastasis: updated consensus guidelines for pattern scoring, perspectives and recent mechanistic insights.

The first consensus guidelines for scoring the histopathological growth patterns (HGPs) of liver metastases were established in 2017. Since then, numerous studies have applied these guidelines, have further substantiated the potential clinical value of the HGPs in patients with liver metastases from various tumour types and are starting to shed light on the biology of the distinct HGPs. In the present guidelines, we give an overview of these studies, discuss novel strategies for predicting the HGPs of liver metastases, such as deep-learning algorithms for whole-slide histopathology images and medical imaging, and highlight liver metastasis animal models that exhibit features of the different HGPs. Based on a pooled analysis of large cohorts of patients with liver-metastatic colorectal cancer, we propose a new cut-off to categorise patients according to the HGPs. An up-to-date standard method for HGP assessment within liver metastases is also presented with the aim of incorporating HGPs into the decision-making processes surrounding the treatment of patients with liver-metastatic cancer. Finally, we propose hypotheses on the cellular and molecular mechanisms that drive the biology of the different HGPs, opening some exciting preclinical and clinical research perspectives.

Results of a worldwide survey on the currently used histopathological diagnostic criteria for invasive lobular breast cancer.

Invasive lobular carcinoma (ILC) represents the second most common subtype of breast cancer (BC), accounting for up to 15% of all invasive BC. Loss of cell adhesion due to functional inactivation of E-cadherin is the hallmark of ILC. Although the current world health organization (WHO) classification for diagnosing ILC requires the recognition of the dispersed or linear non-cohesive growth pattern, it is not mandatory to demonstrate E-cadherin loss by immunohistochemistry (IHC). Recent results of central pathology review of two large randomized clinical trials have demonstrated relative overdiagnosis of ILC, as only ~60% of the locally diagnosed ILCs were confirmed by central pathology. To understand the possible underlying reasons of this discrepancy, we undertook a worldwide survey on the current practice of diagnosing BC as ILC. A survey was drafted by a panel of pathologists and researchers from the European lobular breast cancer consortium (ELBCC) using the online tool SurveyMonkey®. Various parameters such as indications for IHC staining, IHC clones, and IHC staining procedures were questioned. Finally, systematic reporting of non-classical ILC variants were also interrogated. This survey was sent out to pathologists worldwide and circulated from December 14, 2020 until July, 1 2021. The results demonstrate that approximately half of the institutions use E-cadherin expression loss by IHC as an ancillary test to diagnose ILC and that there is a great variability in immunostaining protocols. This might cause different staining results and discordant interpretations. As ILC-specific therapeutic and diagnostic avenues are currently explored in the context of clinical trials, it is of importance to improve standardization of histopathologic diagnosis of ILC diagnosis.

CYP3A7*1C allele: linking premenopausal oestrone and progesterone levels with risk of hormone receptor-positive breast cancers.

BACKGROUND: Epidemiological studies provide strong evidence for a role of endogenous sex hormones in the aetiology of breast cancer. The aim of this analysis was to identify genetic variants that are associated with urinary sex-hormone levels and breast cancer risk. METHODS: We carried out a genome-wide association study of urinary oestrone-3-glucuronide and pregnanediol-3-glucuronide levels in 560 premenopausal women, with additional analysis of progesterone levels in 298 premenopausal women. To test for the association with breast cancer risk, we carried out follow-up genotyping in 90,916 cases and 89,893 controls from the Breast Cancer Association Consortium. All women were of European ancestry. RESULTS: For pregnanediol-3-glucuronide, there were no genome-wide significant associations; for oestrone-3-glucuronide, we identified a single peak mapping to the CYP3A locus, annotated by rs45446698. The minor rs45446698-C allele was associated with lower oestrone-3-glucuronide (-49.2%, 95% CI -56.1% to -41.1%, P = 3.1 × 10-18); in follow-up analyses, rs45446698-C was also associated with lower progesterone (-26.7%, 95% CI -39.4% to -11.6%, P = 0.001) and reduced risk of oestrogen and progesterone receptor-positive breast cancer (OR = 0.86, 95% CI 0.82-0.91, P = 6.9 × 10-8). CONCLUSIONS: The CYP3A7*1C allele is associated with reduced risk of hormone receptor-positive breast cancer possibly mediated via an effect on the metabolism of endogenous sex hormones in premenopausal women.

Concordance between results of inexpensive statistical models and multigene signatures in patients with ER+/HER2- early breast cancer.

Multigene signatures (MGS) are used to guide adjuvant chemotherapy (aCT) decisions in patients diagnosed with estrogen receptor (ER)-positive HER2-negative early breast cancer. We used results from three MGS (Oncotype DX® (ODX), MammaPrint® (MP) or Prosigna®) and assessed the concordance between high or low risk of recurrence and the predicted risk of recurrence based on statistical models. In addition, we looked at the impact of MGS results on final aCT administration during the multidisciplinary meeting (MDM). We retrospectively included 129 patients with ER-positive HER2-negative early breast cancer for which MGS testing was performed after MDM at University Hospitals Leuven between May 2013 and April 2019 in case there was doubt about aCT recommendation. Tumor tissue was analyzed either by ODX (N = 44), MP (N = 28), or Prosigna® (N = 57). Eight statistical models were computed: Magee equations (ME), Memorial Sloan Kettering simplified risk score (MSK-SRS), Breast Cancer Recurrence Score Estimator (BCRSE), OncotypeDXCalculator (ODXC), new Adjuvant! Online (nAOL), Mymammaprint.com (MyMP), PREDICT, and SiNK. Concordance, negative percent agreement, and positive percent agreement were calculated. Of 129 cases, 53% were MGS low and 47% MGS high risk. Concordances of 100.0% were observed between risk results obtained by ODX and ME. For MP, BCRSE demonstrated the best concordance, and for Prosigna® the average of ME. Concordances of <50.0% were observed between risk results obtained by ODX and nAOL, ODX and MyMP, ODX and SiNK, MP and MSK-SRS, MP and nAOL, MP and MyMP, MP and SiNK, and Prosigna® and ODXC. Integration of MGS results during MDM resulted in change of aCT recommendation in 47% of patients and a 15% relative and 9% absolute reduction. In conclusion, statistical models, especially ME and BCRSE, can be useful in selecting ER-positive HER2-negative early breast cancer patients who may need MGS testing resulting in enhanced cost-effectiveness and reduced delay in therapeutic decision-making.

Interobserver variability in the assessment of stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative invasive breast carcinoma influences the association with pathological complete response: the IVITA study.

High stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC) are associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Histopathological assessment of sTILs in TNBC biopsies is characterized by substantial interobserver variability, but it is unknown whether this affects its association with pCR. Here, we aimed to investigate the degree of interobserver variability in an international study, and its impact on the relationship between sTILs and pCR. Forty pathologists assessed sTILs as a percentage in digitalized biopsy slides, originating from 41 TNBC patients who were treated with NAC followed by surgery. Pathological response was quantified by the MD Anderson Residual Cancer Burden (RCB) score. Intraclass correlation coefficients (ICCs) were calculated per pathologist duo and Bland-Altman plots were constructed. The relation between sTILs and pCR or RCB class was investigated. The ICCs ranged from -0.376 to 0.947 (mean: 0.659), indicating substantial interobserver variability. Nevertheless, high sTILs scores were significantly associated with pCR for 36 participants (90%), and with RCB class for eight participants (20%). Post hoc sTILs cutoffs at 20% and 40% resulted in variable associations with pCR. The sTILs in TNBC with RCB-II and RCB-III were intermediate to those of RCB-0 and RCB-I, with lowest sTILs observed in RCB-I. However, the limited number of RCB-I cases precludes any definite conclusions due to lack of power, and this observation therefore requires further investigation. In conclusion, sTILs are a robust marker for pCR at the group level. However, if sTILs are to be used to guide the NAC scheme for individual patients, the observed interobserver variability might substantially affect the chance of obtaining a pCR. Future studies should determine the 'ideal' sTILs threshold, and attempt to fine-tune the patient selection for sTILs-based de-escalation of NAC regimens. At present, there is insufficient evidence for robust and reproducible sTILs-guided therapeutic decisions.

Behavior of metastatic breast cancer according to subtype.

PURPOSE: To explore the impact of breast cancer subtype on metastatic behavior and long-term outcome defined as breast cancer specific survival (BCSS). METHODS: Retrospective single centre cross-sectional study of 5972 patients with newly diagnosed, unilateral first diagnosis of breast cancer, diagnosed 2000-2010. Patients had either early breast cancer (EBC) treated primarily by surgery (SURG n = 5072), neoadjuvant systemic therapy (NEO n = 592), or upfront metastatic disease (META n = 308). Surrogate breast cancer subtypes were defined according to classical pathological criteria. Analysis was performed using Kaplan-Meier method and logistic/Cox regression. RESULTS: After median follow-up time of 103.6 months (IQR 73.4-139.2 months), 817 patients with EBC at diagnosis (14.4%) developed distant metastases of which 621 (12.2%) SURG and 196 (33.1%) NEO. Metastasis rate after EBC was: LuminalA 8.1%, LuminalB1(HER2-) 20.4%, LuminalB2(HER2+) without (neo)adjuvant trastuzumab 21.7%, LuminalB2(HER2+) with trastuzumab 9.0%, HER2Positive(ER-) without trastuzumab 30.0%, HER2Positive(ER-) with trastuzumab 19.9% and TripleNegative 25.3%. There were major differences in site of first metastases according to subtype. For single site first metastases, median BCSS assessed from time of metastases was worst for brain localization (13.9 months) and best for bone (48.4 months). Multiple sites of first metastases had worse BCSS from date of metastases than single site first metastases (median BCSS for 1 site 40.0, 2 sites 27.1, ≥ 3 sites 20.5 months). Median BCSS from date of metastases is longer in upfront metastases compared to secondary metastases after EBC (43.4 vs. 27.9 months). CONCLUSIONS: Tumor subtype influences the metastatic behavior and survival after development of distant metastases.

Assessment of stromal tumor infiltrating lymphocytes and immunohistochemical features in invasive micropapillary breast carcinoma with long-term outcomes.

PURPOSE: We studied the long-term outcomes of invasive micropapillary carcinoma (IMPCs) of the breast in relation to stromal tumor infiltrating lymphocytes (sTILs), prognostic biomarkers and clinicopathological features. METHODS: Stage I-III IMPCs treated with upfront surgery at our institution (January 2000 and December 2016) were included. Central pathology review was performed and sTILs (including zonal distribution and hot spot analysis) and tumor-associated plasma cells (TAPC) were evaluated. Expression of P53, BCL2, FOXP3, and WT1, which are variably linked to breast cancer prognosis, was measured by immunohistochemistry using tissue microarrays. Time-to-event endpoints were distant recurrence free interval (DRFI) and breast cancer-specific survival (BCSS). RESULTS: We included 111 patients of whom 59% were pure IMPCs. Standard clinicopathological features were comparable between pure and non-pure IMPCs. Overall, the mean sTILs level was 20% with higher proportion of sTILs present at the invasive front. There were no significant differences between pure- and non-pure IMPCs in sTILs levels, nor in the spatial distribution of the hot spot regions or in the distribution of TAPC. Higher sTILs correlated with worse DRFI (HR = 1.55; p = 0.0172) and BCSS (HR = 2.10; p < 0.001). CONCLUSIONS: Clinicopathological features, geographical distribution of sTILs and TAPC are similar between pure and non-pure IMPCs. Despite a high proportion of grade 3 tumors and lymph node involvement, we observed a low rate of distant recurrences and breast cancer-related death in this cohort of stage I-III IMPCs treated with primary surgery. Caution in interpretation of the observed prognostic correlations is required given the very low number of events, warranting validation in other cohorts.

Prognostic value of histopathological DCIS features in a large-scale international interrater reliability study.

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.

Interobserver variability in upfront dichotomous histopathological assessment of ductal carcinoma in situ of the breast: the DCISion study.

Histopathological assessment of ductal carcinoma in situ, a nonobligate precursor of invasive breast cancer, is characterized by considerable interobserver variability. Previously, post hoc dichotomization of multicategorical variables was used to determine the "ideal" cutoffs for dichotomous assessment. The present international multicenter study evaluated interobserver variability among 39 pathologists who performed upfront dichotomous evaluation of 149 consecutive ductal carcinomas in situ. All pathologists independently assessed nuclear atypia, necrosis, solid ductal carcinoma in situ architecture, calcifications, stromal architecture, and lobular cancerization in one digital slide per lesion. Stromal inflammation was assessed semiquantitatively. Tumor-infiltrating lymphocytes were quantified as percentages and dichotomously assessed with a cutoff at 50%. Krippendorff's alpha (KA), Cohen's kappa and intraclass correlation coefficient were calculated for the appropriate variables. Lobular cancerization (KA = 0.396), nuclear atypia (KA = 0.422), and stromal architecture (KA = 0.450) showed the highest interobserver variability. Stromal inflammation (KA = 0.564), dichotomously assessed tumor-infiltrating lymphocytes (KA = 0.520), and comedonecrosis (KA = 0.539) showed slightly lower interobserver disagreement. Solid ductal carcinoma in situ architecture (KA = 0.602) and calcifications (KA = 0.676) presented with the lowest interobserver variability. Semiquantitative assessment of stromal inflammation resulted in a slightly higher interobserver concordance than upfront dichotomous tumor-infiltrating lymphocytes assessment (KA = 0.564 versus KA = 0.520). High stromal inflammation corresponded best with dichotomously assessed tumor-infiltrating lymphocytes when the cutoff was set at 10% (kappa = 0.881). Nevertheless, a post hoc tumor-infiltrating lymphocytes cutoff set at 20% resulted in the highest interobserver agreement (KA = 0.669). Despite upfront dichotomous evaluation, the interobserver variability remains considerable and is at most acceptable, although it varies among the different histopathological features. Future studies should investigate its impact on ductal carcinoma in situ prognostication. Forthcoming machine learning algorithms may be useful to tackle this substantial diagnostic challenge.

Breast Cancer Detection and Treatment Monitoring Using a Noninvasive Prenatal Testing Platform: Utility in Pregnant and Nonpregnant Populations.

BACKGROUND: Numerous publications have reported the incidental detection of occult malignancies upon routine noninvasive prenatal testing (NIPT). However, these studies were not designed to evaluate the NIPT performance for cancer detection. METHODS: We investigated the sensitivity of a genome-wide NIPT pipeline, called GIPSeq, for detecting cancer-specific copy number alterations (CNAs) in plasma tumor DNA (ctDNA) of patients with breast cancer. To assess whether a pregnancy itself, with fetal cell-free DNA (cfDNA) in the maternal circulation, might influence the detection of ctDNA, results were compared in pregnant (n = 25) and nonpregnant (n = 25) cancer patients. Furthermore, the ability of GIPSeq to monitor treatment response was assessed. RESULTS: Overall GIPSeq sensitivity for detecting cancer-specific CNAs in plasma cfDNA was 26%. Fifteen percent of detected cases were asymptomatic at the time of blood sampling. GIPSeq sensitivity mainly depended on the tumor stage. Also, triple negative breast cancers (TNBC) were more frequently identified compared to hormone-positive or HER2-enriched tumors. This might be due to the presence of high-level gains and losses of cfDNA or high ctDNA loads in plasma of TNBC. Although higher GIPSeq sensitivity was noted in pregnant (36%) than in nonpregnant women (16%), the limited sample size prohibits a definite conclusion. Finally, GIPSeq profiling of cfDNA during therapy allowed monitoring of early treatment response. CONCLUSIONS: The results underscore the potential of NIPT-based tests, analyzing CNAs in plasma cfDNA in a genome-wide and unbiased fashion for breast cancer detection, cancer subtyping and treatment monitoring in a pregnant and nonpregnant target population.

Computerised scoring protocol for identification and quantification of different immune cell populations in breast tumour regions by the use of QuPath software.

AIMS: As important prognostic and predictive information can be obtained from the composition, functionality and spatial arrangement of different immune cell subtypes, this study aims at characterizing the immune infiltrate in breast tumours. METHODS AND RESULTS: Tumour-infiltrating lymphocytes (TILs) in 62 patients with luminal B-like breast cancer were characterised by immunohistochemical staining with standard markers, and were subsequently classified and quantified by the use of QuPath software. In different delineated tumour regions, the proportion and density of CD3+ , CD4+ , CD5+ , CD8+ , CD20+ and FOXP3+ cells were assessed. The results of the software analysis were compared with those of manual counting for CD8 and CD20 staining. The QuPath scoring protocol slightly overestimated positive, negative and total lymphocyte counts and density, while minimally underestimating the proportion of positively stained lymphocytes. However, for density and proportion, no real differences from manual counting were observed. For all markers, the density of positively stained immune cells was higher in the invasive front than in the tumour centre, pointing to an accumulation of immune cells near the tumour boundaries. When we looked at the proportion of immunohistochemically positive immune cells, we observed enrichment of CD5 (P = 0.025) and CD20 (P < 0.001) at the periphery, and FOXP3 enrichment in the centre (P < 0.001). CONCLUSION: The QuPath scoring protocol can adequately identify positively stained immune cells in breast tumours, and allows the evaluation of differences in immune cell proportion and density within different tumour regions. The entire tumour section can be quantitatively assessed quite rapidly, which is a major advantage over manual counting.

Pictorial Imaging-Histopathology Correlation in a Rabbit with Hepatic VX2 Tumor Treated by Transarterial Vascular Disrupting Agent Administration.

Cancer vasculature is immature, disorganized and hyperpermeable and can serve as a target for anti-cancer therapies. Vascular disrupting agents (VDAs) are tubulin protein binding and depolymerizing agents that induce rapid tumoral vascular shutdown and subsequent cancer necrosis. However, two clinical problems exist with all VDAs, i.e. 1) incomplete anticancer effect and 2) dose-dependent toxicity. To tackle these problems, in our ongoing research, a novel VDA C118P is applied by transarterial administration of half the intravenous dose in rabbits with implanted VX2 liver tumor to assess its therapeutic efficacy. Nearly complete tumor necrosis was achieved by only a single arterial dose of C118P at 5 mg/kg, which was documented in a representative case by in vivo digital subtraction arteriogram (DSA) and magnetic resonance imaging (MRI), and further confirmed by ex vivo microangiogram and histopathology. This convincing and promising preliminary outcome would warrant further comprehensive studies to explore the potentials of VDAs by transarterial administration either in mono-drug or in combination for management of solid cancers.

Prognostic Value of the Progesterone Receptor by Subtype in Patients with Estrogen Receptor-Positive, HER-2 Negative Breast Cancer.

BACKGROUND: In estrogen receptor-positive (ER+), human epidermal growth factor receptor 2 (HER-2) negative breast cancers, the progesterone receptor (PR) is an independent prognostic marker. Little is known about the prognostic value of PR by tumor grade. We assessed this in two independent datasets. PATIENTS AND METHODS: Women with primary operable, invasive ER+ HER-2 negative breast cancer diagnosed between 2000 and 2012, treated at University Hospitals Leuven, were included. We assessed the association of PR status and subtype (grade 1-2 vs. grade 3) with distant recurrence-free interval (DRFI) and breast cancer-specific survival. The interaction between PR status and subtype was investigated, and associations of PR status by subtype were calculated. The BIG 1-98 data set was used for validation. RESULTS: In total, 4,228 patients from Leuven and 5,419 from BIG 1-98 were analyzed. In the Leuven cohort, the adjusted hazard ratio (HR) of PR-positive versus PR-negative tumors for DRFI was 0.66 (95% confidence interval [CI], 0.50-0.89). For the interaction with subtype (p = .34), the HR of PR status was 0.79 (95% CI, 0.61-1.01) in luminal A-like and 0.59 (95% CI, 0.46-0.76) in luminal B-like tumors. In luminal A-like tumors, observed 5-year cumulative incidences of distant recurrence were 4.1% for PR-negative and 2.8% for PR-positive tumors, and in luminal B-like 18.7% and 9.2%, respectively. In the BIG 1-98 cohort, similar results were observed; for the interaction with subtype (p = .12), the adjusted HR of PR status for DRFI was 0.88 (95% CI, 0.57-1.35) in luminal A-like and 0.58 (95% CI, 0.43-0.77) in luminal B-like tumors. Observed 5-year cumulative incidences were similar. CONCLUSION: PR positivity may be more protective against metastatic relapse in luminal B-like versus luminal A-like breast cancer, but no strong conclusions can be made. In absolute risk, results suggest an absent PR is clinically more important in high compared with low proliferative ER+ HER-2 negative tumors. IMPLICATIONS FOR PRACTICE: An absent progesterone receptor (PR) predicts a worse outcome in women treated for an estrogen receptor-positive, human epidermal growth factor receptor 2 negative breast cancer. As low proliferative tumors lacking PR are now also classified high risk, the prognostic value of PR across risk groups was studied. Despite a negative test for interaction of the prognostic value of PR by tumor grade, the magnitude of an absent PR on breast cancer relapse is much larger in high than in low proliferative breast cancers.