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Background: Data are scarce on immunogenicity of COVID-19 vaccines in patients with autoimmune diseases, who are often treated with immunosuppressive drugs. We aimed to investigate the effect of different immunosuppressive drugs on antibody development after COVID-19 vaccination in patients with autoimmune diseases. Methods: In this study, we used serum samples collected from patients with autoimmune diseases and healthy controls who were included in two ongoing prospective cohort studies in the Netherlands. Participants were eligible for inclusion in this substudy if they had been vaccinated with any COVID-19 vaccine via the Dutch national vaccine programme, which at the time was prioritising vaccination of older individuals. Samples were collected after the first or second COVID-19 vaccination. No serial samples were collected. Seroconversion rates and IgG antibody titres against the receptor-binding domain of the SARS-CoV-2 spike protein were measured. Logistic and linear regression analyses were used to investigate the association between medication use at the time of vaccination and at least until sampling, seroconversion rates, and IgG antibody titres. The studies from which data were collected are registered on the Netherlands Trial Register, Trial ID NL8513, and ClinicalTrials.org, NCT04498286.Findings: Between April 26, 2020, and March 1, 2021, 3682 patients with rheumatic diseases, 546 patients with multiple sclerosis, and 1147 healthy controls were recruited to participate in the two prospective cohort studies. Samples were collected from patients with autoimmune diseases (n=632) and healthy controls (n=289) after their first (507 patients and 239 controls) or second (125 patients and 50 controls) COVID-19 vaccination. The mean age of both patients and controls was 63 years (SD 11), and 423 (67%) of 632 patients with autoimmune diseases and 195 (67%) of 289 controls were female. Among participants without previous SARS-CoV-2 infection, seroconversion after first vaccination were significantly lower in patients than in controls (210 [49%] of 432 patients vs 154 [73%] of 210 controls; adjusted odds ratio 0·33 [95% CI 0·23-0·48]; pInterpretation: Our data suggest that seroconversion after a first COVID-19 vaccination is delayed in older patients on specific immunosuppressive drugs, but that second or repeated exposure to SARS-CoV-2, either via infection or vaccination, improves humoral immunity in patients treated with immunosuppressive drugs. Therefore, delayed second dosing of COVID-19 vaccines should be avoided in patients receiving immunosuppressive drugs. Future studies that include younger patients need to be done to confirm the generalisability of our results.FUNDING: ZonMw, Reade Foundation, and MS Center Amsterdam.

OBJECTIVE: The objective of this study was to measure humoral responses after SARS-CoV-2 vaccination in MS patients treated with ocrelizumab (OCR) compared to MS patients without disease modifying therapies (DMTs) in relation to timing of vaccination and B-cell count.METHODS: OCR treated patients were divided into an early and a late group (cut-off time 12 weeks between infusion and first vaccination). Patients were vaccinated with mRNA-1273 (Moderna). B-cells were measured at baseline (time of first vaccination) and SARS-CoV-2 antibodies were measured at baseline, day 28, 42, 52 and 70.RESULTS: 87 patients were included (62 OCR patients, 29 patients without DMTs). At day 70, seroconversion occurred in 39.3% of OCR patients compared to 100% of MS patients without DMTs. In OCR patients, seroconversion varied between 26% (early group) to 50% (late group) and between 27% (low B-cells) to 56% (at least 1 detectable B-cell/µL).CONCLUSIONS: Low B-cell counts prior to vaccination and shorter time between OCR infusion and vaccination may negatively influence humoral response but does not preclude seroconversion. We advise OCR treated patients to get their first vaccination as soon as possible. In case of an additional booster vaccination, timing of vaccination based on B-cell count and time after last infusion may be considered.

Introduction: Data on immunogenicity of SARS-CoV-2 vaccines in patients with autoimmune diseases are still scarce, because these patients were largely excluded from SARS-CoV-2 vaccine trials. Methods: Serum samples of patients with autoimmune diseases (n = 480) and healthy controls (n = 204) included in two ongoing prospective cohort studies were collected after first or second SARS-CoV-2 vaccinations. Seroconversion rates and IgG antibody titers against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were measured. Multivariable logistic and linear regression analyses were used to investigate associations between medication, seroconversion rates and IgG antibody titers respectively. Findings: Seroconversion rates after a first SARS-CoV-2 vaccination in 111 patients on methotrexate (25%, adjusted OR: 0.1, 95% CI: 0.1 – 0.2, P < 0.001) and 15 patients on anti-CD20 therapies (0%, P < 0.001) were lower compared to 103 healthy controls (71%). For patients on tumor necrosis factor (TNF) inhibitors (adjusted OR: 0.7, 95% CI: 0.4 – 1.4, P = 0.20) and patients on prednisone monotherapy (adjusted OR: 0.6, 95% CI: 0.2 – 1.5, P = 0.25) seroconversion rates were similar compared to controls. After the second SARS-CoV-2 vaccination seroconversion rates exceeded 88% in all patient subgroups, except in patients treated with anti-CD20 therapies (2 (33%) of 6). Seroconversion rates and IgG antibody titers were similar for patients with a prior SARS-CoV-2 infection who had received a single vaccine dose and patients without a prior SARS-CoV-2 who had received two vaccine doses. Interpretation: Treatment regimens other than anti-CD20 therapies of patients with autoimmune diseases do not need to be postponed when these patients receive a SARS-CoV-2 vaccination. Patients receiving anti-CD20 therapies may benefit from a second booster vaccination, as our data suggest that repeated exposure to SARS-CoV-2 vaccines or the virus itself enhances the development of humoral immunity in these patients. Funding: ZonMw, Reade Foundation and MS Center Amsterdam. Declaration of Interest: None to declare. Ethical Approval: The research protocols were approved by the medical ethical committee of the VU University medical center (registration number 2020.169 and 2020.370). All participants gave informed consent.

Background A growing understanding of the molecular biology of cancer and the identification of specific aberrations driving cancer evolution have led to the development of various targeted agents. Tumors can exhibit significant heterogeneity and this may change over time, also as the result of selective pressure. Circulating tumor cells (CTCs), shed from multiple tumor sites, have demonstrated to represent of the overall tumor burden. We report the results of an ongoing comparative study on mutation and copy number profiles of primary and metastatic tissue, CTCs, and synchronously isolated DTCs from metastatic effusions of patients with clinically progressive MBC. Materials & Methods CTCs and DTCs were enriched from 7.5 ml blood or effusion using the CellSearch system and were further purified and sorted with the DEPArray system. For this study we isolated both 70 single and 70 pools of 10-200 CTCs or DTCs, in order have enough power to detect 5% subclones and analyse heterogeneity. Single and pooled WBCs were isolated as technical controls. DNA was isolated and amplified using the Ampli1-kit and subjected to Illumina WGS and Ion Torrent AmpliSeq panel sequencing. Fresh frozen tissue from solid metastases and the primary tumor, and bulk CTC (CellSearch Profile) were sequenced as comparators for mutation and copy number profiles. DNA of buffy coat was sequenced to enable germline variant detection. For mutational analysis, only somatic variants with good quality metrics, >20x coverage, variant allele frequencies >10%, and being non-synonymous or splice site variants, were taken into account. Results AmpliSeq panel sequencing was performed on 153 unique samples of three patients with a mean coverage depth of 1000x. In patient 1, a PIK3CA hotspot mutation was found clonally in all tumor samples at heterozygous level. Furthermore, various private mutations were found in both CTCs and DTCs, however not in WBC, including several TP53 hotspot mutations. In patient 2, another PIK3CA hotspot was present in all CTCs at heterozygous frequencies. In patient 3, an enormous heterogeneity was observed between all CTC and DTC samples. For patient 3, disease evolution was detected during multiple events of progressive disease over 2 years. At the moment, low pass WGS for CN detection for all samples is being performed and results will be present prior to the SABCS. Conclusion Based on the mutational status we conclude that both clonal mutations as well as various private variants are present in single and pools of CTCs and DTCs. In addition to the detection of targetable aberrations, the evaluation of heterogeneity is of clinical importance, as the effect of targeting subclones is currently being explored in clinical trials. Citation Format: Brouwer A, van Dam P-J, Rutten A, Prové A, Peeters M, Van Laere S, Dirix L. Evaluation of subclonality in the CTC and DTC compartment of patients with metastatic breast cancer using low pass whole genome and AmpliSeq panel sequencing [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-06-01.

Background: The enumeration of circulating tumour cells (CTCs) with the EpCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). The aim of this study was to explore potential differences in the detection and prognostic significance of CTCs in MBC according to immunohistochemical subtypes of breast cancer. Methods: CellSearch CTC counts were obtained from 154 MBC patients before first-line systemic treatment between November 2007 and August 2012. Patients were categorised in five subgroups according to immunohistochemical surrogate definitions of intrinsic subtypes in breast cancer based on hormone receptor status, HER2/neu status and histological grade. Differences in progression-free (PFS) and overall survival (OS) were assessed relative to the cut-off value of >= 5 CTCs per 7.5 ml blood. Results: No significant differences were observed in the absolute CTC counts (P = 0.120) or in CTC positivity rates according to >= 1 and >= 5 CTCs per 7.5 ml blood detection thresholds (P = 0.165 and P = 0.651, respectively) between immunohistochemical subtypes. However, very high CTC counts, defined as >= 80 CTCs per 7.5 ml, were observed more frequently in patients with Luminal A and triple negative (TN) breast cancer (P = 0.024). In the total study population, the presence of X5 CTCs was the single most significant prognostic factor for both PFS and OS in multivariate analysis (P < 0.001). A more limited prognostic impact, not reaching statistical significance, was observed in patients with HER2-positive disease as opposed to patients with Luminal A, Luminal B-HER2-negative and TN disease. Conclusion: The detection of EpCAM + CTCs was not clearly associated with any of the immunohistochemical subtypes of breast cancer in patients with MBC before first-line treatment. Potentially clinically relevant differences were however observed at very high CTC counts. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2-positive patients with MBC.

Background: The enumeration of circulating tumour cells (CTC) has prognostic significance in patients with metastatic breast cancer (MBC) and monitoring of CTC levels over time has considerable potential to guide treatment decisions. However, little is known on CTC kinetics in the human bloodstream. Methods: In this study, we compared the number of CTC in both 7.5 ml central venous blood (CVB) and 7.5 ml peripheral venous blood (PVB) from 30 patients with MBC starting with a new line of chemotherapy. Results: The number of CTC was found to be significantly higher in CVB (median: 43.5; range: 04036) than in PVB (median: 33; range: 04013) (P=0.001). When analysing samples pairwise, CTC counts were found to be significantly higher in CVB than in PVB in 12 out of 26 patients with detectable CTC. In contrast, only 2 out of 26 patients had higher CTC counts in PVB as compared with CVB, whereas in 12 remaining patients no significant difference was seen. The pattern of CTC distribution was independent of the sites of metastatic involvement. Conclusion: A substantial difference in the number of CTC was observed between CVB and PVB of patients with MBC. Registration of the site of blood collection is warranted in studies evaluating the role of CTC assessment in these patients.

Phase 3 trials of bevacizumab combined with chemotherapy in metastatic breast cancer have consistently failed to demonstrate a survival benefit for the addition of bevacizumab. When cancers metastasize to highly vascular organs (including the liver), they can utilize vessel co-option, instead of angiogenesis, as a mechanism to obtain a vascular supply (1). We have repeatedly shown by histopathological analyses that almost all (95%, 2 cohorts) breast cancer liver metastases utilize vessel co-option instead of angiogenesis to vascularize (2,3). The prevalence of vessel co-option in breast cancer could explain, at least in part, why anti-angiogenic therapy has been a disappointing therapeutic approach in metastatic breast cancer. Animal models of non-angiogenic liver and lung metastases also displayed resistance to anti-VEGF treatment (3,4). We have now undertaken a gene expression study (mRNA sequencing) of targeted samples at the tumor-liver interface to discover gene expression patterns and signaling pathways that are associated with non-angiogenic growth of metastatic cancer in the liver (n = 70). A network to detect biological themes of non-angiogenic growth was built by gene set enrichment analysis. Key components of this network are: cancer cell motility and invasion, epithelial-to-mesenchymal transition, stemness and proliferation. This contrasts with the network of angiogenic liver metastases of which the most important components are inflammation and ECM remodeling. Semi-automated image analyses of CD8-immunostained section of liver metastases confirms that non-angiogenic liver metastases have a significantly lower density of CD8-positive cytotoxic T-lymphocytes at the tumor-liver interface when compared with angiogenic liver metastases (300 cells/mm2 and 1000 cells/mm2, respectively (p In conclusion, we provide evidence, based on morphology and gene expression, for the almost exclusive non-angiogenic growth of breast cancer liver metastases. In addition, non-angiogenic breast cancer liver metastases are characterized by a desert immune phenotype. Both observations can have an impact on the treatment strategy of patients with metastatic breast cancer. References: 1. 10.1038/nrc.2018.14 – 2. 10.1038/sj.bjc.6601727 – 3. 10.1038/nm.4197 – 4. 10.1002/path.4845 – 5. 10.1097/NEN.0b013e318233afd7 – 6. 10.1038/onc.2010.518 Citation Format: Vermeulen PB, van Dam P-J, Daelemans S, Latacz E, Joye I, Kockx M, Dirix P, Verhoef K, Grunhagen D, Huget P, Van Laere S, Dirix LY. Breast cancer liver metastases vascularize by vessel co-option, not angiogenesis, and have a desert immune phenotype: A histopathological and gene expression study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-08.

This is the first article reporting sentinel node identification in a patient with endometrial cancer recurring in the vagina. A 79-year-old woman presented with a midvaginal recurrence of a stage IB, grade II endometroid carcinoma that had been treated 3 years earlier by a total abdominal hysterectomy, bilateral salpingoophorectomy, and pelvic lymph node sampling, followed by adjuvant brachytherapy to the vaginal vault. A staging examination under anesthetic was performed. Preoperatively, 60-MBq technetium-labeled nannocolloid was injected in the mucosa at 3, 6, 9, and 12 o'clock just adjacent to the tumor recurrence. Three sentinel nodes were detected, respectively, in the left obturator fossa (two) and the right external iliac region, using a laparoscopic probe (Navigator) and removed for pathological assessment. As they proved to be negative, the patient underwent a total vaginectomy, parametrectomy with pelvic lymphadenectomy. The tumor was completely removed, and all lymph nodes proved to be negative. The accuracy of sentinel node identification in patients with recurrent gynecological tumors needs further evaluation. This unique case shows that sentinel node detection is possible after previous radiotherapy and surgery and hopes to stimulate further research in this field.

Introduction: In metastatic breast cancer (MBC), discordant expression levels of the human epidermal growth factor receptor 2 (HER2) have been noted between primary tumours (PT) and matched metastatic lesions (Meta). Therefore reassessment of this predictive marker at time of metastatic disease might help to optimize treatment. Circulating tumour cells (CTCs) offer the potential to provide a repeatedly accessible source of tumour cells for the real-time assessment of actual tumour characteristics. Here we report on a retrospective study analysing over two and a half thousand CTCs in order to evaluate the inter-observer variability when using the semi-quantitative scale (0-3+) described by Riethdorf in 2010. Furthermore we designed a digital scoring system using 13 parameters selected by ImageJ. HER2 status in CTCs was compared to PT and/or Meta of patients with MBC. Materials and methods: 65 patients starting first or second line systemic therapy for MBC and harbouring more than 5 CTC/7.5 mL blood were selected. HER2 status of 2721 CTCs was determined by immunofluorescence using the CellSearch system. HER2 status of the solid lesions was determined by IHC or FISH. Inter-observer variability was calculated using the Kendalls tau tests. 284 CTCs were analysed with the digital scoring system using ImageJ 'plot profile' and 'analyse particles'. Selected parameters comprise cell size, mean and maximum intensity of the cell and its surrounding, and both ratio's and differences of the aforementioned. Dissimilarity matrix was calculated using Pearson Correlation-Distance. Results: Of 2721 CTCs, 1485 cells (55%) were scored 0+ and 2263 cells (83%) were found to be HER2- (0+ or 1+) by both observers. 458 cells (17%) were scored HER2+ (2+ or 3+) by at least one of the observers, however only 175 (6%) by both observers. Inter-observer variability was 0.703, but when omitting the usually undebatable 0+ cells, this variability showed to be 0.278. HER2 scoring of CTC by two observers. HER2- (0+/1+)HER2+ (2+/3+)HER2- (0+/1+)2263 (83%)131 (4.8%)HER2+ (2+/3+)152 (5.6%)175 (6.4%) 24 of 65 patients had at least 80% 0+ CTCs (≥96% HER2- cells). Of these patients, 5 were HER2+ based on their PT. Oppositely, 10 and 20 patients harboured at least 40% and 10% HER2+ CTCs respectively. From these 20 patients only 10 were diagnosed with HER2+ disease on PT or Meta and 1 was shifted form HER2- PT to HER2+ Meta. The digital scoring system was able to identify four groups with different HER2 expression levels. When comparing the identified clusters with the manually scored cells the two moderately related clusters showed to contain almost only 2+ and 3+ CTCs. A very isolated cluster contained almost solely 0+ CTCs. Discussion: The manual scoring system showed to be feasible, however we noticed that there are some discrepancies regarding the scoring of 1+ to 3+ cells. The digital scoring is able to predict the outcome and can by itself cluster CTCs into 4 groups. It strongly distinguishes between the HER2+ and HER2- cells. HER2+ status can change during disease progression, both with gain and loss of HER2 positivity. This can be monitored using CTCs. Citation Format: Brouwer A, van de Wiel M, Peeters B, van Dam P-J, Vermeulen P, Peeters M, Van Laere S, Peeters D, Dirix L. Manual and digital detection of HER2 status of 2721 circulating tumour cells in patients with metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-04-01.

In this contribution we describe a high-frequency high-field EPR facility which has been developed at the University of Nijmegen. We present the design of a heterodyne quasi-optical bridge based on a millimeter-wave vector network analyzer as source and detection system. The mm-waves are transported in free-space through Gaussian beam optic elements and through a corrugated guide inside the resonator insert. The Fabri-Perot (TEM00n) resonator is coupled through a metallic mesh and because of its bimodal property it can be operated using orthogonal detection leading to substantial improvement in sensitivity. In the first stage of the project, a multifrequency CW-facility is realized covering the 100–500 GHz range. In our initial explorative experiments we demonstrate the advantages of HF-EPR of high-spin systems: Due to the large microwave quantum, transitions which would be undetectable at X-band due to the large zerofield splitting can now be observed in good sensitivity. As a model for biological high-spin systems a sample of metmyoglobin was measured at D-band (130 GHz). Theg = 5.9 perpendicular line from theS = 5/2 ferric heme was detected and its line-width was compared to data previously obtained at Q-, X-, S- and L-band. As a model for biological integer spin systems theS = 1 signal of Ni(II) in nickel Tutton salt (Ni(NH4)2(SO4)2) was studied at 35 and 130 GHz.

Introduction: The enumeration of circulating tumour cells (CTCs) with the EPCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). However, breast cancer has been shown to be a molecularly heterogeneous disease. The aim of this study was to assess potential differences in the detection and prognostic significance of CTCs according to the immunohistochemically defined molecular subtypes of breast cancer. Methods: CellSearch CTC counts were obtained from 110 patients with MBC prior to first line systemic treatment, treated at GZA Hospitals Sint-Augustinus between november 2007 and december 2011. Clinicopathological variables were prospectively entered in a database. Based on the St-Gallen surrogate definitions of intrinsic breast cancer subtypes (Goldhirsch et al. Ann Oncol 2011), patients were divided in 5 groups: luminal A (ER/PR+, HER2−, Bloom-Richardson histological grade I-II), luminal B – HER2 negative (ER/PR+, Her2−, grade III), luminal B – HER2 positive (ER/PR+, HER2+, any grade), HER2 positive – non luminal (ER/PR−, HER2+), and triple negative (TN) (ER/PR−, HER2−). Differences in progression free survival (PFS) and overall survival (OS) according to the FDA approved prognostic cut-off of ≥5 CTC/7.5 ml blood were estimated using Kaplan Meier and Cox proportional hazard statistics. Results: CTC were detected in 78 of 110 (71%) patients. Higher detection rates and numbers of CTC were observed in patients with luminal A and TN breast cancer as compared to patients with luminal B and HER2 positive disease. However, no differences in positivity rates were observed between molecular subtypes according to the 5 CTC prognostic cut-off point (table 1). After a median FU time of 3.1 years, 39 patients had died. In the total study population, the presence of ≥5 CTC was an independent predictor of PFS and OS in multivariate analysis (PFS: HRCTC≥5=2.236 (1.366–3.658), p = 0.001; OS: HRCTC≥5=3.180 (1.553–6.509), p = 0.002). When analyzing subgroups separately, a lower prognostic power was observed in the HER2 positive and luminal B subgroups. Conclusion: Significant differences were observed in the detection and prognostic significance of EPCAM positive CTC according to the immunohistochemically defined breast cancer subtypes. Interestingly, CTC were detected more frequently in patients with luminal A and TN tumors. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2 positive patients with MBC. Our data independently confirm those reported by Giordano et al. (Ann Oncol 2010) in a large clinically uniform population of patients with MBC before the start of first-line treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-08.