Search

Background: Humoral immune responses have previously been associated with improved outcomes, with B cell infiltrates able to independently predict pathologic complete response to neoadjuvant chemotherapy (NACT). B cells represent a diverse population of cells and the complex interplay between specific B cell subsets in the context of chemotherapy treated breast cancers remains unclear. Here, we investigate the dynamic changes in the B cell immune landscape before and after NACT treatment across different breast cancer subtypes. Methods: Treatment naïve, mid-treatment and post-NACT breast tumour tissue samples were dissociated into single cells, stained with two panels of B cell-specific antibodies recognising a total of 24 target proteins, and analysed by flow cytometry. In addition, PMBC before and after NACT were also profiled. B cell subsets were classified as either naïve (CD27—IgD+), class-switched memory (CD27+IgD—), unswitched memory (CD27+IgD+) or double negative (DN)(CD27—IgD—). DN B cells were further characterised into DN1 (CXCR5+CD21+) and DN2 (CXCR5—CD21—) subsets. In vitro co-cultures of breast cancer cell line spheroid and PBMC were carried out. Results: In both treatment naïve and chemotherapy treated samples, we observed a significant expansion in the DN B cell population within the TME compared to the periphery. DN B cells represented on average 40.96% of B cells in treatment naïve tumours vs 9.48% in PBMCs (p< 0.0001), and 71.80% vs 6.34% of B cells in post-NACT tumour vs PBMC samples respectively (p< 0.05). Interestingly, in treatment naïve PBMC and tumour tissue samples, the largest proportion of the DN subset consisted of DN1 cells, 69.35% and 64.11% respectively. In contrast, following NACT, DN2 cells constituted the majority of the DN population both within the TME (86.30%) and in the periphery (50.44%). Although the specific functions of these B cell subsets remain unclassified, deeper phenotyping suggests DN1 cells more closely resemble the phenotype of class-switched memory cells, whilst DN2 cells are thought to have antibody-secreting properties and more closely resemble the plasmablast phenotype. scRNA sequencing of B cells pre- and post NACT is currently underway. Conclusion: To our knowledge, this work is the first to identify an expanded population of DN B cells in breast tumour tissue and highlights the requirement for further investigation into these cells to decipher their role in the context of chemotherapy treatment and resistance in breast cancer. Citation Format: Esme Carpenter, Thanussuyah Alaguthurai, Farhana Hossain, Rosalind Graham, Helen Kakkassery, Sean Keane, Sheeba Irshad. Enrichment of atypical memory double negative (CD27— IgD—) tumour infiltrating B cells following neoadjuvant chemotherapy for early-stage breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-02.

Purpose: To identify potential immune targets in post-neoadjuvant chemotherapy (NAC)–resistant triple-negative breast cancer (TNBC) and ER+HER2– breast cancer disease. Experimental Design: Following pathology review, 153 patients were identified as having residual cancer burden (RCB) II/III disease (TNBC n = 80; ER+HER2–n = 73). Baseline pre-NAC samples were available for evaluation for 32 of 80 TNBC and 36 of 73 ER+HER2– cases. Bright-field hematoxylin and eosin assessment allowed for tumor-infiltrating lymphocyte (TIL) evaluation in all cases. Multiplexed immunofluorescence was used to identify the abundance and distribution of immune cell subsets. Levels of checkpoints including PD-1/PD-L1 expression were also quantified. Findings were then validated using expression profiling of cancer and immune-related genes. Cytometry by time-of-flight characterized the dynamic changes in circulating immune cells with NAC. Results: RCB II/III TNBC and ER+HER2– breast cancer were immunologically “cold” at baseline and end of NAC. Although the distribution of immune cell subsets across subtypes was similar, the mRNA expression profiles were both subtype- and chemotherapy-specific. TNBC RCB II/III disease was enriched with genes related to neutrophil degranulation, and displayed strong interplay across immune and cancer pathways. We observed similarities in the dynamic changes in B-cell biology following NAC irrespective of subtype. However, NAC induced changes in the local and circulating tumor immune microenvironment (TIME) that varied by subtype and response. Specifically, in TNBC residual disease, we observed downregulation of stimulatory (CD40/OX40L) and inhibitory (PD-L1/PD-1) receptor expression and an increase in NK cell populations (especially non-cytolytic, exhausted CD56dimCD16–) within both the local TIME and peripheral white cell populations. Conclusions: This study identifies several potential immunologic pathways in residual disease, which may be targeted to benefit high-risk patients.

S3. Boosted serological responses and persistent T cell responses following SARS-CoV-2 vaccination dose

This study offers longitudinal insight into the impact of three SARS-CoV-2 vaccinations on humoral and cellular immunity in patients with solid cancers, patients with hematologic malignancies, and persons without cancer. For all cohorts, virus-neutralizing immunity was significantly depleted over a period of up to 9 months following the second vaccine dose, the one striking exception being IL2 production by SARS-CoV-2 antigen-specific T cells. Immunity was restored by the third vaccine dose, except in a substantial number of patients with hematologic malignancy, for whom both cancer type and treatment schedule were associated with nonresponse. Thus, whereas most patients with myelodysplastic syndrome were conspicuously good responders, some patients with other hematologic malignancies receiving cancer therapies within 2 weeks of vaccination showed no seroconversion despite three vaccine doses. Moreover, SARS-CoV-2 exposure during the course of the study neither prevented immunity waning, even in healthy controls, nor guaranteed vaccine responsiveness. These data offer real-world human immunologic insights that can inform health policy for patients with cancer.Significance:Global health policy reliant on SARS-CoV-2 vaccine effectiveness is underpinned by our understanding of the durability of protection offered by sequential vaccinations and the efficacy of boosting, especially in immunocompromised patient populations who might constitute virus reservoirs. Here, we have: (i) clarified in patients with cancer the degree of waning of antibodies, serum neutralization titres against parental virus and variants of concern, and T-cell responses; (ii) evaluated the immune response among patients with cancer to a third dose of COVID-19 vaccine; and (iii) provided safety data following the third dose of the BNT162b2 COVID-19 vaccine in patients with cancer.

Supplementary Table & Figure Legends

Supplementary Data from Dynamic Changes in the NK-, Neutrophil-, and B-cell Immunophenotypes Relevant in High Metastatic Risk Post Neoadjuvant Chemotherapy–Resistant Early Breast Cancers

Supplementary Figure from Dynamic Changes in the NK-, Neutrophil-, and B-cell Immunophenotypes Relevant in High Metastatic Risk Post Neoadjuvant Chemotherapy–Resistant Early Breast Cancers

Purpose:To identify potential immune targets in post-neoadjuvant chemotherapy (NAC)–resistant triple-negative breast cancer (TNBC) and ER+HER2– breast cancer disease.Experimental Design:Following pathology review, 153 patients were identified as having residual cancer burden (RCB) II/III disease (TNBC n = 80; ER+HER2–n = 73). Baseline pre-NAC samples were available for evaluation for 32 of 80 TNBC and 36 of 73 ER+HER2– cases. Bright-field hematoxylin and eosin assessment allowed for tumor-infiltrating lymphocyte (TIL) evaluation in all cases. Multiplexed immunofluorescence was used to identify the abundance and distribution of immune cell subsets. Levels of checkpoints including PD-1/PD-L1 expression were also quantified. Findings were then validated using expression profiling of cancer and immune-related genes. Cytometry by time-of-flight characterized the dynamic changes in circulating immune cells with NAC.Results:RCB II/III TNBC and ER+HER2– breast cancer were immunologically “cold” at baseline and end of NAC. Although the distribution of immune cell subsets across subtypes was similar, the mRNA expression profiles were both subtype- and chemotherapy-specific. TNBC RCB II/III disease was enriched with genes related to neutrophil degranulation, and displayed strong interplay across immune and cancer pathways. We observed similarities in the dynamic changes in B-cell biology following NAC irrespective of subtype. However, NAC induced changes in the local and circulating tumor immune microenvironment (TIME) that varied by subtype and response. Specifically, in TNBC residual disease, we observed downregulation of stimulatory (CD40/OX40L) and inhibitory (PD-L1/PD-1) receptor expression and an increase in NK cell populations (especially non-cytolytic, exhausted CD56dimCD16–) within both the local TIME and peripheral white cell populations.Conclusions:This study identifies several potential immunologic pathways in residual disease, which may be targeted to benefit high-risk patients.

This study offers longitudinal insight into the impact of three SARS-CoV-2 vaccinations on humoral and cellular immunity in patients with solid cancers, patients with hematologic malignancies, and persons without cancer. For all cohorts, virus-neutralizing immunity was significantly depleted over a period of up to 9 months following the second vaccine dose, the one striking exception being IL2 production by SARS-CoV-2 antigen-specific T cells. Immunity was restored by the third vaccine dose, except in a substantial number of patients with hematologic malignancy, for whom both cancer type and treatment schedule were associated with nonresponse. Thus, whereas most patients with myelodysplastic syndrome were conspicuously good responders, some patients with other hematologic malignancies receiving cancer therapies within 2 weeks of vaccination showed no seroconversion despite three vaccine doses. Moreover, SARS-CoV-2 exposure during the course of the study neither prevented immunity waning, even in healthy controls, nor guaranteed vaccine responsiveness. These data offer real-world human immunologic insights that can inform health policy for patients with cancer. Significance: Global health policy reliant on SARS-CoV-2 vaccine effectiveness is underpinned by our understanding of the durability of protection offered by sequential vaccinations and the efficacy of boosting, especially in immunocompromised patient populations who might constitute virus reservoirs. Here, we have: (i) clarified in patients with cancer the degree of waning of antibodies, serum neutralization titres against parental virus and variants of concern, and T-cell responses; (ii) evaluated the immune response among patients with cancer to a third dose of COVID-19 vaccine; and (iii) provided safety data following the third dose of the BNT162b2 COVID-19 vaccine in patients with cancer.

Summary Background The efficacy and safety profiles of vaccines against SARS-CoV-2 in patients with cancer is unknown. We aimed to assess the safety and immunogenicity of the BNT162b2 (Pfizer–BioNTech) vaccine in patients with cancer. Methods For this prospective observational study, we recruited patients with cancer and healthy controls (mostly health-care workers) from three London hospitals between Dec 8, 2020, and Feb 18, 2021. Participants who were vaccinated between Dec 8 and Dec 29, 2020, received two 30 μg doses of BNT162b2 administered intramuscularly 21 days apart; patients vaccinated after this date received only one 30 μg dose with a planned follow-up boost at 12 weeks. Blood samples were taken before vaccination and at 3 weeks and 5 weeks after the first vaccination. Where possible, serial nasopharyngeal real-time RT-PCR (rRT-PCR) swab tests were done every 10 days or in cases of symptomatic COVID-19. The coprimary endpoints were seroconversion to SARS-CoV-2 spike (S) protein in patients with cancer following the first vaccination with the BNT162b2 vaccine and the effect of vaccine boosting after 21 days on seroconversion. All participants with available data were included in the safety and immunogenicity analyses. Ongoing follow-up is underway for further blood sampling after the delayed (12-week) vaccine boost. This study is registered with the NHS Health Research Authority and Health and Care Research Wales (REC ID 20/HRA/2031). Findings 151 patients with cancer (95 patients with solid cancer and 56 patients with haematological cancer) and 54 healthy controls were enrolled. For this interim data analysis of the safety and immunogenicity of vaccinated patients with cancer, samples and data obtained up to March 19, 2021, were analysed. After exclusion of 17 patients who had been exposed to SARS-CoV-2 (detected by either antibody seroconversion or a positive rRT-PCR COVID-19 swab test) from the immunogenicity analysis, the proportion of positive anti-S IgG titres at approximately 21 days following a single vaccine inoculum across the three cohorts were 32 (94%; 95% CI 81–98) of 34 healthy controls; 21 (38%; 26–51) of 56 patients with solid cancer, and eight (18%; 10–32) of 44 patients with haematological cancer. 16 healthy controls, 25 patients with solid cancer, and six patients with haematological cancer received a second dose on day 21. Of the patients with available blood samples 2 weeks following a 21-day vaccine boost, and excluding 17 participants with evidence of previous natural SARS-CoV-2 exposure, 18 (95%; 95% CI 75–99) of 19 patients with solid cancer, 12 (100%; 76–100) of 12 healthy controls, and three (60%; 23–88) of five patients with haematological cancers were seropositive, compared with ten (30%; 17–47) of 33, 18 (86%; 65–95) of 21, and four (11%; 4–25) of 36, respectively, who did not receive a boost. The vaccine was well tolerated; no toxicities were reported in 75 (54%) of 140 patients with cancer following the first dose of BNT162b2, and in 22 (71%) of 31 patients with cancer following the second dose. Similarly, no toxicities were reported in 15 (38%) of 40 healthy controls after the first dose and in five (31%) of 16 after the second dose. Injection-site pain within 7 days following the first dose was the most commonly reported local reaction (23 [35%] of 65 patients with cancer; 12 [48%] of 25 healthy controls). No vaccine-related deaths were reported. Interpretation In patients with cancer, one dose of the BNT162b2 vaccine yields poor efficacy. Immunogenicity increased significantly in patients with solid cancer within 2 weeks of a vaccine boost at day 21 after the first dose. These data support prioritisation of patients with cancer for an early (day 21) second dose of the BNT162b2 vaccine. Funding King's College London, Cancer Research UK, Wellcome Trust, Rosetrees Trust, and Francis Crick Institute.

Breast cancer patients with advanced metastatic disease can exhibit rapid disease progression, disease stabilisation or partial responses of varying duration. However, for reasons that are not fully elucidated, a small fraction of patients will elicit an exceptional durable response to standard anticancer treatments or survive significantly longer than patients with clinically comparable tumours. Here, we investigate the drivers of immune surveillance mechanisms across breast cancer subtypes in stage IV exceptional survivors (n=13) with matched control cohorts of stage IV typical responders (n=6), early breast cancer patients (n=5) and healthy volunteers (n=17). Peripheral blood mononuclear cells (PBMCs) from patients were stained with 8 panels providing 241 non-redundant immune parameters for flow cytometry analysis. Principle Component Analysis (PCA) showed distinct segregation of the exceptional survivors from the other control groups with an immune signature in exceptional survivors constituting of activated NK, CD8 T cells and gamma delta (gd) T cells pointing towards higher innate immunogenicity in these individuals. Specifically, although these metastatic exceptional responder patients possessed comparable NK cell frequencies, the proportion of NKG2D+CD56dimCD16+ NK cells were significantly enriched compared to the typical responders. Additionally, proportions of CD8+ central memory (CD45RA- CD27+) and effector memory (CD45RA- CD27-) gd T cells, were also seen to be significantly increased. Functional in vitro validation of these findings along with scRNA sequencing of lymph node and tumour tissue is currently underway. To our knowledge, this work is the first to explore in depth the immune signatures in the peripheral blood of exceptional survivors with metastatic breast cancer. Elucidating the immunological reasons for favourable atypical responses alongside functional tumour microenvironment analysis offers unique insights for predictive biomarker identification and discovery of axes that could be exploited therapeutically to benefit those with less favourable responses. Written informed consent was obtained from all individuals in accordance with the Declaration of Helsinki under the following research ethics committee; London-Chelsea approved study (REC ID 13/LO/1248). Citation Format: Helen Kakkassery, Thanussuyah Alaguthurai, Rosalind Graham, Esme Carpenter, Farhana Hossain, Sean Keane, Sheeba Irshad. Local and lymphoid immune surveillance mechanisms in “exceptional survivors” of stage 4 cancers following standard of care chemo- and targeted therapies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-01.

BackgroundThe efficacy and safety profile of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have not been definitively established in immunocompromised patient populations. Patients with a known cancer diagnosis were hitherto excluded from trials of the vaccines currently in clinical use.MethodsThis study presents data on the safety and immune efficacy of the BNT162b2 (Pfizer-BioNTech) vaccine in 54 healthy controls and 151 mostly elderly patients with solid and haematological malignancies, respectively, and compares results for patients who were boosted with BNT162b2 at 3 weeks versus those who were not. Immune efficacy was measured as antibody seroconversion, T cell responses, and neutralisation of SARS-CoV-2 Wuhan strain and of a variant of concern (VOC) (B.1.1.7). We also collected safety data for the BNT162b2 vaccine up to 5 weeks following first dose.FindingsThe vaccine was largely well tolerated. However, in contrast to its very high performance in healthy controls (>90% efficacious), immune efficacy of a single inoculum in solid cancer patients was strikingly low (below 40%) and very low in haematological cancer patients (below 15%). Of note, efficacy in solid cancer patients was greatly and rapidly increased by boosting at 21-days (95% within 2 weeks of boost). Too few haematological cancer patients were boosted for clear conclusions to be drawn.ConclusionsDelayed boosting potentially leaves most solid and haematological cancer patients wholly or partially unprotected, with implications for their own health; their environment and the evolution of VOC strains. Prompt boosting of solid cancer patients quickly overcomes the poor efficacy of the primary inoculum in solid cancer patients.RESEARCH IN CONTEXTEvidence before this studySome cancer patients have been shown to exhibit sustained immune dysregulation, inefficient seroconversion and prolonged viral shedding as a consequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Consequently, their exclusion and, in particular, the exclusion of patients receiving systemic anti-cancer therapies, from the registry trials of the 5 approved COVID-19 vaccines raises questions about the efficacy and safety of SARS-CoV-2 vaccination in this patient population. In addition, whilst the change in the UK’s dosing interval to 12-weeks aimed to maximise population coverage, it is unclear whether this strategy is appropriate for cancer patients and those on systemic anti-cancer therapies.Added value of this studyWe report that the RNA-based SARS-CoV-2 BNT162b2 vaccine administered in cancer patients was well tolerated, and we provide first insights into both antibody and T cell responses to the vaccine in an immunocompromised patient population.Implications of all the available evidenceIn cancer patients, one dose of 30ug of BNT162b2 yields poor vaccine efficacy, as measured by seroconversion rates, viral neutralisation capacity and T cell responses, at 3- and 5-weeks following the first inoculum. Patients with solid cancers exhibited a significantly greater response following a booster at 21-days. These data support prioritisation of cancer patients for an early (21-day) second dose of the BNT162b2 vaccine. Given the globally poor responses to vaccination in patients with haematological cancers, post-vaccination serological testing, creation of herd immunity around these patients using a strategy of ‘ring vaccination’, and careful follow-up should be prioritised.

Background: Cancer patients are vulnerable populations for COVID-19 complications and mortality. We previously reported on the poor single-dose immunogenicity of BNT162b2 mRNA vaccine in cancer patients, particularly those with haematological malignancies. Methods: In this prospective, observational study relating to the safety and immunogenicity of BNT162b2 mRNA vaccine, 201 vaccinated cancer patients (solid cancer n=125; haematological cancer n=76) and 54 healthy controls (mostly health-care workers “HCW”) were recruited between December 8th, 2020, and April 23rd, 2021. The previously reported interim results covered a period of 101 days since first patient recruitment, during which time 47 subjects received a second “boost” vaccination on day 21. Because of the change in UK Government policy, all others received a delayed vaccine boost at about 12 weeks after their first vaccination, and had their blood sampled 2 weeks’ later. Here, we describe immunogenicity data following the delayed boost in 31 HCWs, 72 solid cancer and 56 haematological cancer patients. Seroconversion, virus neutralisation, and T cell assays were as described previously, with an additional test for neutralisation of the B.1.617.2 (delta) variant-ofconcern (VOC). The primary endpoint of the study was the impact on seroconversion following delayed (>21days) vaccine boosting in solid and haematological cancer patients. The secondary endpoints were: safety following delayed vaccine boost; T cell responses; and neutralisation of SARS-CoV-2 Wuhan (“wild type” [WT]), B.1.1.7 (alpha), and B.1.617.2 (delta) variants. Findings: Delayed (>21days) boost vaccination of solid cancer patients and haematological cancer patients with the BNT162b2 vaccine was well tolerated, as the primary vaccination had been. There was no vaccine-associated death. Boosting significantly increased solid cancer patients’ seroconversion responses, that had been strikingly poor in response to a single dose: from 38% to 84%. Boosting also significantly improved vaccine immunogenicity for haematological cancer patients, but most (57%) still failed to seroconvert. Seroconversion correlated strongly with the capacity to neutralise SARSCoV- 2 cell entry, although neutralisation of the WT variant was typically greater than of the VOC. Neutralisation was significantly increased by boosting for HCWs but not for cancer patients. In comparison to seroconversion, boosting achieved higher rates of functional T cell responsiveness (de novo responses) but had little impact on the magnitude of T cell responses for those who had responded to first-dose vaccination. When patients were scored as showing both seroconversion and T cell responses, the unfavourable situation of haematological cancer patients was overt with only 36% (12/33) defined as being responders compared to 78% (25/32) of solid cancer patients and 88% (15/17) of HCWs. There was no significant difference in any aspect of immunogenicity for HCWs or solid cancer patients receiving the delayed boost versus the day 21 boost (this comparison could not be made for haematological cancer patients because too few received an early boost). Chemotherapy within 15 days either side of the boost exacerbated the likelihood of non-responsiveness to the vaccine. Interpretation: Boosting at either 3 weeks or longer (up to 12 weeks) post-primary vaccination shows high efficacy in terms of seroconversion of solid cancer patients and increases in their SARS-CoV-2 Spike-specific antibody titres. By contrast, delayed boosting left most haematological cancer patients without serological protection against SARS-CoV-2 infection. These data support the ongoing adjustment of health care measures to limit the evident vulnerability of such individuals to SARS-CoV- 2, and to limit their potential to transmit virus variants that might develop in the context of absent or partial immunoprotection. The absence of any clear improvements in immunogenicity of a delayed boost relative to boosting on day 21 emphasizes the importance of early boosting for cancer patients, and potentially of doing so repeatedly, particularly given how well the vaccine was tolerated. Chemotherapy, if possible should be withheld 15 days before and 15 days after the vaccination date. Trial Registration: The trial is registered with the NHS Health Research Authority (HRA) and Health and Care Research Wales (HCRW) (REC ID: 20/HRA/2031). Funding: KCL, CRUK, Leukemia & Lymphoma Society, Wellcome Trust, Rosetrees Trust, Francis Crick Institute. Declaration of Interest: None to declare. Ethical Approval: The trial was approved by the institutional review boards of the participating institutions (IRAS ID: 282337 REC ID: 20/HRA/2031).

Aberrant mucin-type O-linked glycosylation is a common occurrence in cancer where the upregulation of sialyltransferases is often seen leading to the early termination of O-glycan chains. Mucin-type O-linked glycosylation is not limited to mucins and occurs on many cell surface glycoproteins including EGFR, where the number of sites can be limited. Upon EGF ligation, EGFR induces a signaling cascade and may also translocate to the nucleus where it directly regulates gene transcription, a process modulated by Galectin-3 and MUC1 in some cancers. Here, we show that upon EGF binding, breast cancer cells carrying different O-glycans respond by transcribing different gene expression signatures. MMP10, the principal gene upregulated when cells carrying sialylated core 1 glycans were stimulated with EGF, is also upregulated in ER-positive breast carcinoma reported to express high levels of ST3Gal1 and hence mainly core 1 sialylated O-glycans. In contrast, isogenic cells engineered to carry core 2 glycans upregulate CX3CL1 and FGFBP1 and these genes are upregulated in ER-negative breast carcinomas, also known to express longer core 2 O-glycans. Changes in O-glycosylation did not significantly alter signal transduction downstream of EGFR in core 1 or core 2 O-glycan expressing cells. However, striking changes were observed in the formation of an EGFR/galectin-3/MUC1/β-catenin complex at the cell surface that is present in cells carrying short core 1-based O-glycans but absent in core 2 carrying cells.

The tumour microenvironment plays a crucial role in the growth and progression of cancer and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies show that TAMs show transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype. These TAMs can recruit and maintain neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This novel macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients.

The tumour microenvironment plays a crucial role in the growth and progression of cancer, and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies have demonstrated that TAMs display transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype that to the best of our knowledge has not previously been described. These TAMs can recruit and prolong the lifespan of neutrophils, inhibit the function of T cells, degrade basement membrane allowing for invasion, are inefficient at phagocytosis, and can induce plasma clotting. This macrophage phenotype is enriched in the stroma at the edge of breast cancer nests and their presence is associated with poor prognosis in breast cancer patients., Beatson et al. show that a sialylated tumour-associated glycoform of the mucin MUC1 induces the differentiation of monocytes into tumour-associated macrophages. These macrophages are found in breast cancer stroma and their presence is associated with poor prognosis.

Myelodysplastic syndromes (MDS) represent a spectrum of clonal bone marrow neoplasms from low risk disease through to those transforming into acute myeloid leukaemia. The COVID-19 pandemic has presented a great risk to those with hematological malignancies who are at higher risk of severe disease and death than the general population. Previous studies looking at the immune response to influenza vaccination in those with MDS had shown promising results, with immune responses not differing from those of healthy family members. Whilst some data exist to reassure the MDS community that majority of patients show seroconversion following Covid-19 vaccination, little data exists on their neutralizing capacity or post vaccination T-cell responses in this cohort. In addition, the majority of patients in these studies received BNT162b2 and there is little published data on vaccine response to the ChAdOx1 nCoV-19 vaccine. We have investigated the humoral and T-cell response of 39 patients with MDS two to four weeks following Covid-19 booster vaccination with BNT162b2 or ChAdOx1 nCoV-19 through the SOAP study (Sars-cov-2 fOr cAncer Patients, IRAS project ID:282337). Plasma and PBMCs from MDS cases and healthy controls have been collected, and are being assessed for both humoral and cellular responses to SARS_CoV_2, the alpha (B.1.1.7) and delta (B.1.617.2) variants. Humoral responses will be assessed using ELISA (peptide binding) and functional viral neutralization assays. Cellular responses will be assessed using IFNy ELISPOT and flow cytometry (CD25 and CD69 expression) after 24h peptide stimulation. All data at time point 1 (2 - 4 weeks following booster vaccination) have been collected and will subsequently be collected at 6 months and 12 months post-vaccination. We also report on the safety data for these vaccines within this patient population. Of this cohort 64% were male with a median age of 65 years (range 21-84). 54% received vaccination with ChAdOx1 nCoV-19 and 44% received BNT162b2 (2% unrecorded). The vaccines were well tolerated with no serious adverse events to date. The mean interval between doses was 70.7 days (range 50 - 90 days). 71% of the cohort were receiving no disease modifying therapy at the time of vaccination, half of whom were receiving supportive therapy and the other half no intervention for their MDS. Of those receiving disease modifying therapy; 5 were receiving azacitidine, (1 in conjunction with low-dose cytarabine) and 3 ciclosporin. We will report the largest study of the humoral and T-cell mediated response to the Covid-19 vaccine in MDS patients to date. This will include cellular response to the delta variant and immunogenicity of both the BNT162b2 and ChAdOx1 nCoV-19 vaccines. Given the vulnerability of these patients to severe disease, investigating the immune response to the vaccines begins to build an evidence base for advising MDS patients on their ongoing risk of infection during the pandemic and going forward. The SOAP study will reassess the immune response at 6 and 12 months post-vaccination to continue to investigate post-vaccine immunity in this cohort. Disclosures Kulasekararaj: F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy; Akari: Consultancy, Honoraria, Speakers Bureau; Biocryst: Consultancy, Honoraria, Speakers Bureau; Achilleon: Consultancy, Honoraria, Speakers Bureau; Alexion: Consultancy, Honoraria, Speakers Bureau; Ra Pharma: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Alexion, AstraZeneca Rare Disease Inc.: Consultancy, Honoraria, Other: Travel support. Patten: JANSSEN: Honoraria; NOVARTIS: Honoraria; GILEAD SCIENCES: Honoraria, Research Funding; ROCHE: Research Funding; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria.

Background: Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoantibodies in large population cohorts of sera taken before cancer diagnosis. Methods: Serum samples from women who subsequently developed breast cancer, and aged-matched controls, were identified from UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and Guernsey serum banks to formed discovery and validation sets. These were screened on a microarray platform of 60mer MUC1 glycopeptides and recombinant MUC1 containing 16 tandem repeats. Additional case–control sets comprised of women who subsequently developed ovarian, pancreatic and lung cancer were also screened on the arrays. Results: In the discovery (273 cases, 273 controls) and the two validation sets (UKCTOCS 426 cases, 426 controls; Guernsey 303 cases and 606 controls), no differences were found in autoantibody reactivity to MUC1 tandem repeat peptide or glycoforms between cases and controls. Furthermore, no differences were observed between ovarian, pancreatic and lung cancer cases and controls. Conclusion: This robust, validated study shows autoantibodies to MUC1 peptide or glycopeptides cannot be used for breast, ovarian, lung or pancreatic cancer screening. This has significant implications for research on the use of MUC1 in cancer detection.

e15104Background: Many tumors, including breast cancers, contain infiltrating immune cells that are unable to eliminate the tumor and in many cases, promote tumor progression by adding to the permi...

Using a C57Bl/6 mouse model system, where intramuscular (i.m.) injection of full length (FL) MUC1 cDNA protects against subsequent challenge with MUC1-expressing syngeneic tumour cells, we have investigated the importance of the tandem repeat (TR) domain in the induction of T cell-dependent tumour rejection. A MUC1 construct engineered to remove the TR domain (MUC1 0TR) was found to be as effective as the full length MUC1 cDNA in inhibiting the growth of RMA MUC1 cells in C57Bl/6 mice. Protection by i.m. injection of either the FL-MUC1 cDNA or the MUC1 0TR construct depended on the presence of functional CD4+ and CD8+ T cells. Specific CD8+ T cell responses, however, could not be detected in vitro using mouse spleen cells taken after only cDNA injection, but only after challenge in vivo with MUC1-expressing tumour cells. To attempt to enhance the responses of CD4+ T cells, a cDNA construct was developed, where the extracellular domain of MUC1 was fused to the transmembrane and cytoplasmic domain of Lamp1 (MUC1/Lamp1). This construct was equally effective in inducing tumour rejection but did not induce MUC1-specific CTL in mice before challenge with MUC1-expressing tumour cells. Our results indicate that, in this model, T cell responses necessary for protection against MUC1-expressing tumours that are induced by IM injection of MUC1 cDNA are independent of the tandem repeat domain as well as the transmembrane and cytoplasmic domains. A low level of protection was seen with all constructs in BALB/c mice, which show a defect in Th1 responses. C57Bl/6xBALB/c hybrids were, however, well protected against both H2(d) and H2(b) expressing tumour challenge, emphasizing the importance of the host background.

We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1–IgG-producing CHO-K1 clones were generated and were found to secrete MUC1–IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galβ1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1–IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1–IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.

The membrane epithelial mucin MUC1 is expressed at the luminal surface of most simple epithelial cells, but expression is greatly increased at lactation and in most breast carcinomas. The increase in level of expression of MUC1 in breast cancer is accompanied by changes in the profile of glycosyl transferases involved in the synthesis of the O-glycans attached to the MUC1 core protein. The cancer-associated mucin is therefore structurally different from the normal mucin, and expresses novel B cell epitopes. MUC1 antibodies are used for in vivo targeting of breast and ovarian tumors, and there is considerable interest in MUC1 as a possible target antigen for the immunotherapy of breast cancer. The different glycoforms can affect cell interactions differently, depending on whether specific interactions with lectins occur. In the absence of such lectin interactions, the long sialylated and negatively charged molecule can inhibit intercellular interactions between other cell surface molecules. The potential role of the different components of the immune system in MUC1 responses are discussed within the framework of how to develop logical strategies for designing clinical studies.

Studies in animal models of mouse mammary carcinoma have shown that ovine submaxillary mucin, which carries multiple sialyl-Tn (STn) epitopes, is effective in stimulating an immune response and inhibiting tumour growth. In similar studies using carbohydrate antigens, pretreatment with low-dose cyclophosphamide has been shown to be important in modulating the immune response to antigen possibly by inhibiting suppresser T-cell activity. In a clinical trial assessing the efficacy and toxicity of synthetic STn, patients with metastatic breast cancer were randomised to receive 100 micrograms STn linked to keyhole limpet haemocyanin (KLH) with DETOX-B adjuvant given by subcutaneous injection at weeks 0, 2, 5 and 9 with or without low-dose cyclophosphamide (CTX, 300 mg m-2) pretreatment, 3 days before the start of immunotherapy. Patients with responding or stable disease after the first four injections were eligible to receive STn-KLH at 4 week intervals. The main toxicity noted was the development of subcutaneous granulomata at injection sites. Of 23 patients randomised, 18 received four injections, 5 patients having developed progressive disease during the initial 12 week period. Two minor responses were noted in the 18 patients who received four active specific immunotherapy (ASI) injections and a further five patients had stable disease. Six patients continued ASI at 4 week intervals and a partial response was noted in a patient who had previously had stable disease. All patients developed IgG and IgM responses to sialyl-Tn and levels of IgM antibodies were significantly higher in those patients who were pretreated with CTX. Measurable tumour responses have been recorded following ASI with STn-KLH plus DETOX and the immunomodulatory properties of low-dose CTX have been confirmed.

The membrane epithelial mucin MUC1 is expressed at the luminal surface of most simple epithelial cells, but expression is greatly increased at lactation and in most breast carcinomas. The increase in level of expression of MUC1 in breast cancer is accompanied by changes in the profile of glycosyl transferases involved in the synthesis of the O-glycans attached to the MUC1 core protein. The cancer-associated mucin is therefore structurally different from the normal mucin, and expresses novel B cell epitopes. MUC1 antibodies are used for in vivo targeting of breast and ovarian tumors, and there is considerable interest in MUC1 as a possible target antigen for the immunotherapy of breast cancer. The different glycoforms can affect cell interactions differently, depending on whether specific interactions with lectins occur. In the absence of such lectin interactions, the long sialylated and negatively charged molecule can inhibit intercellular interactions between other cell surface molecules. The potential role of the different components of the immune system in MUC1 responses are discussed within the framework of how to develop logical strategies for designing clinical studies.

In this study we examined the regulation of expression of the human MUC1 gene in vivo, by developing MUC1 transgenic mice. The data showed that epithelial-specific expression of MUC1 can be directed by just 1.4 kb of 5' flanking sequence using MUC1 cDNA as a reporter gene in vivo. Furthermore, high levels of MUC1 expression were seen in the lactating mammary gland and in spontaneous mammary tumors generated by crossing the MUC1 transgenics with mice transgenic for the polyoma middle T oncogene under the control of the mouse mammary tumor virus promoter. This pattern of expression in epithelial tissues is comparable to the expression of MUC1 in humans and also to the expression pattern in another transgenic mouse line developed with a 10.6-kb genomic MUC1 fragment. This study confirmed that MUC1 is a compact gene and demonstrated that the 1.4-kb 5' sequence not only directs epithelial-specific expression of MUC1 in vivo but also contains the elements governing the up-regulation observed during lactation and in malignancy.