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Contains fulltext : 251784.pdf (Publisher’s version ) (Closed access) BACKGROUND: Concerns have been raised regarding the risks of SARS-CoV-2 breakthrough infections in vaccinated patients with immune-mediated inflammatory diseases treated with immunosuppressants, but clinical data on breakthrough infections are still scarce. The primary objective of this study was to compare the incidence and severity of SARS-CoV-2 breakthrough infections between patients with immune-mediated inflammatory diseases using immunosuppressants, and controls (patients with immune-mediated inflammatory diseases not taking immunosuppressants and healthy controls) who had received full COVID-19 vaccinations. The secondary objective was to explore determinants of breakthrough infections of the delta (B.1.617.2) variant of SARS-CoV-2, including humoral immune responses after vaccination. METHODS: In this substudy, we pooled data collected in two large ongoing prospective multicentre cohort studies conducted in the Netherlands (Target to-B! [T2B!] study and Amsterdam Rheumatology Center COVID [ARC-COVID] study). Both studies recruited adult patients (age ≥18 years) with immune-mediated inflammatory diseases and healthy controls. We sourced clinical data from standardised electronic case record forms, digital questionnaires, and medical files. We only included individuals who were vaccinated against SARS-CoV-2. For T2B!, participants were recruited between Feb 2 and Aug 1, 2021, and for ARC-COVID, participants were recruited between April 26, 2020, and March 1, 2021. In this study we assessed data on breakthrough infections collected between July 1 and Dec 15, 2021, a period in which the delta SARS-CoV-2 variant was the dominant variant in the Netherlands. We defined a SARS-CoV-2 breakthrough infection as a PCR-confirmed or antigen test-confirmed SARS-CoV-2 infection that occurred at least 14 days after vaccination. All breakthrough infections during this period were assumed to be due to the delta variant due to its dominance during the study period. We analysed post-vaccination serum samples for anti-receptor binding domain (RBD) antibodies to assess the humoral vaccination response (T2B! study only) and anti-nucleocapsid antibodies to identify asymptomatic breakthrough infections (ARC-COVID study only). We used multivariable logistic regression analyses to explore potential clinical and humoral determinants associated with the odds of breakthrough infections. The T2B! study is registered with the Dutch Trial Register, Trial ID NL8900, and the ARC-COVID study is registered with Dutch Trial Register, trial ID NL8513. FINDINGS: We included 3207 patients with immune-mediated inflammatory diseases who receive immunosuppressants, and 1807 controls (985 patients with immune-mediated inflammatory disease not on immunosuppressants and 822 healthy controls). Among patients receiving immunosuppressants, mean age was 53 years (SD 14), 2042 (64%) of 3207 were female and 1165 (36%) were male; among patients not receiving immunosuppressants, mean age was 54 years (SD 14), 598 (61%) of 985 were female and 387 (39%) were male; and among healthy controls, mean age was 57 years (SD 13), 549 (67%) of 822 were female and 273 (33%) were male. The cumulative incidence of PCR-test or antigen-test confirmed SARS-CoV-2 breakthrough infections was similar in patients on immunosuppressants (148 of 3207; 4·6% [95% CI 3·9-5·4]), patients not on immunosuppressants (52 of 985; 5·3% [95% CI 4·0-6·9]), and healthy controls (33 of 822; 4·0% [95% CI 2·8-5·6]). There was no difference in the odds of breakthrough infection for patients with immune-mediate inflammatory disease on immunosuppressants versus combined controls (ie, patients not on immunosuppressants and healthy controls; adjusted odds ratio 0·88 [95% CI 0·66-1·18]). Seroconversion after vaccination (odds ratio 0·58 [95% CI 0·34-0·98]; T2B! cohort only) and SARS-CoV-2 infection before vaccination (0·34 [0·18-0·56]) were associated with a lower odds of breakthrough infections. INTERPRETATION: The incidence and severity of SARS-CoV-2 breakthrough infections in patients with immune-mediated inflammatory diseases on immunosuppressants was similar to that in controls. However, caution might still be warranted for those on anti-CD20 therapy and those with traditional risk factors. FUNDING: ZonMw (the Netherlands Organization for Health Research and Development) and Reade foundation.

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.

Background: Disease-specific studies have reported impaired humoral responses after SARS-CoV-2 vaccination in patients with immune-mediated inflammatory disorders treated with specific immunosuppressants. Disease-overarching studies, and data on recall responses and third vaccinations are scarce. Our primary objective was to investigate the effects of immunosuppressive monotherapies on the humoral immune response after SARS-CoV-2 vaccination in patients with prevalent immune-mediated inflammatory disorders.Methods: We did a cohort study in participants treated in outpatient clinics in seven university hospitals and one rheumatology treatment centre in the Netherlands as well as participants included in two national cohort studies on COVID-19-related disease severity. We included patients aged older than 18 years, diagnosed with any of the prespecified immune-mediated inflammatory disorders, who were able to understand and complete questionnaires in Dutch. Participants with immune-mediated inflammatory disorders who were not on systemic immunosuppressants and healthy participants were included as controls. Anti-receptor binding domain IgG responses and neutralisation capacity were monitored following standard vaccination regimens and a three-vaccination regimen in subgroups. Hybrid immune responses-ie, vaccination after previous SARS-CoV-2 infection-were studied as a proxy for recall responses.Findings: Between Feb 2 and Aug 1, 2021, we included 3222 participants in our cohort. Sera from 2339 participants, 1869 without and 470 participants with previous SARS-CoV-2 infection were analysed (mean age 49·9 years [SD 13·7]; 1470 [62·8%] females and 869 [37·2%] males). Humoral responses did not differ between disorders. Anti-CD20 therapy, sphingosine 1-phosphate receptor (S1P) modulators, and mycophenolate mofetil combined with corticosteroids were associated with lower relative risks for reaching seroconversion following standard vaccination (0·32 [95% CI 0·19-0·49] for anti-CD20 therapy, 0·35 [0·21-0·55] for S1P modulators, and 0·61 [0·40-0·90] for mycophenolate mofetil combined with corticosteroids). A third vaccination increased seroconversion for mycophenolate mofetil combination treatments (from 52·6% after the second vaccination to 89·5% after the third) but not significantly for anti-CD20 therapies (from 36·8% to 45·6%) and S1P modulators (from 35·5% to 48·4%). Most other immunosuppressant groups showed moderately reduced antibody titres after standard vaccination that did not increase after a third vaccination, although seroconversion rates and neutralisation capacity were unaffected. In participants with previous SARS-CoV-2 infection, SARS-CoV-2 antibodies were boosted after vaccination, regardless of immunosuppressive treatment.Interpretation: Humoral responses following vaccination are impaired by specific immunosuppressants. After standard vaccination regimens, patients with immune-mediated inflammatory disorders taking most immunosuppressants show similar seroconversion to controls, although antibody titres might be moderately reduced. As neutralisation capacity and recall responses are also preserved in these patients, this is not likely to translate to loss of (short-term) protection. In patients on immunosuppressants showing poor humoral responses after standard vaccination regimens, a third vaccination resulted in additional seroconversion in patients taking mycophenolate mofetil combination treatments, whereas the effect of a third vaccination in patients on anti-CD20 therapy and S1P modulators was limited.Funding: ZonMw (The Netherlands Organization for Health Research and Development).

Background:Patients affected by different types of autoimmune diseases, including common conditions such as multiple sclerosis (MS) and rheumatoid arthritis (RA), are often treated with immunosuppressants to suppress disease activity. It is not fully understood how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral and cellular immunity induced by infection and/or upon vaccination is affected by immunosuppressants.Methods:The dynamics of cellular immune reactivation upon vaccination of SARS-CoV-2 experienced MS patients treated with the humanized anti-CD20 monoclonal antibody ocrelizumab (OCR) and RA patients treated with methotrexate (MTX) monotherapy were analyzed at great depth via high-dimensional flow cytometry of whole blood samples upon vaccination with the SARS-CoV-2 mRNA-1273 (Moderna) vaccine. Longitudinal B and T cell immune responses were compared to SARS-CoV-2 experienced healthy controls (HCs) before and 7 days after the first and second vaccination.Results:OCR-treated MS patients exhibit a preserved recall response of CD8+ T central memory cells following first vaccination compared to HCs and a similar CD4+ circulating T follicular helper 1 and T helper 1 dynamics, whereas humoral and B cell responses were strongly impaired resulting in absence of SARS-CoV-2-specific humoral immunity. MTX treatment significantly delayed antibody levels and B reactivation following the first vaccination, including sustained inhibition of overall reactivation marker dynamics of the responding CD4+ and CD8+ T cells.Conclusions:Together, these findings indicate that SARS-CoV-2 experienced MS-OCR patients may still benefit from vaccination by inducing a broad CD8+ T cell response which has been associated with milder disease outcome. The delayed vaccine-induced IgG kinetics in RA-MTX patients indicate an increased risk after the first vaccination, which might require additional shielding or alternative strategies such as treatment interruptions in vulnerable patients.Funding:This research project was supported by ZonMw (The Netherlands Organization for Health Research and Development, #10430072010007), the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#792532 and #860003), the European Commission (SUPPORT-E, #101015756) and by PPOC (#20_21 L2506), the NHMRC Leadership Investigator Grant (#1173871).

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.

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.

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.