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Background: Recent efforts of gene expression profiling analyses recognized at least four different triple-negative breast cancer (TNBC) molecular subtypes. However, little is known regarding their tumor microenvironment (TME) heterogeneity., Methods: Here, we investigated TME heterogeneity within each TNBC molecular subtype, including immune infiltrate localization and composition together with expression of targetable immune pathways, using publicly available transcriptomic and genomic datasets from a large TNBC series totaling 1512 samples. Associations between molecular subtypes and specific features were assessed using logistic regression models. All statistical tests were two-sided., Results: We demonstrated that each TNBC molecular subtype exhibits distinct TME profiles associated with specific immune, vascularization, stroma, and metabolism biological processes together with specific immune composition and localization. The immunomodulatory subtype was associated with the highest expression of adaptive immune-related gene signatures and a fully inflamed spatial pattern appearing to be the optimal candidate for immune check point inhibitors. In contrast, most mesenchymal stem-like and luminal androgen receptor tumors showed an immunosuppressive phenotype as witnessed by high expression levels of stromal signatures. Basal-like, luminal androgen receptor, and mesenchymal subtypes exhibited an immune cold phenotype associated with stromal and metabolism TME signatures and enriched in margin-restricted spatial pattern. Tumors with high chromosomal instability and copy number loss in the chromosome 5q and 15q regions, including genomic loss of major histocompatibility complex related genes, showed reduced cytotoxic activity as a plausible immune escape mechanism., Conclusions: Our results demonstrate that each TNBC subtype is associated with specific TME profiles, setting the ground for a rationale tailoring of immunotherapy in TNBC patients., (© The Author(s) 2019. Published by Oxford University Press.)

Objective: To report outcomes at 2 years corrected age for children of women recruited to a trial comparing alternative policies for timing of cord clamping and immediate neonatal care at very preterm birth., Design: Parallel group randomised (1:1) trial., Setting: Eight UK tertiary maternity units., Participants: Two hundred and seventy-six babies born to 261 women expected to have a live birth before 32 +0 weeks' gestation., Interventions: Deferred cord clamping (≥2 min) and immediate neonatal care with cord intact or immediate (≤20 s) clamping and immediate neonatal care after clamping., Main Outcome Measure: Composite of death or adverse neurodevelopmental outcome at 2 years corrected age., Results: Six babies born after 35 +6 weeks were excluded. At 2 years corrected age, outcome data were not available for a further 52 children, leaving 218 for analysis (115 deferred clamping, 103 immediate clamping). Overall, 24/115 (21%) children allocated deferred clamping died or had an adverse neurodevelopmental outcome compared with 35/103 (34%) allocated immediate clamping; risk ratio (RR) 0.61 (95% CI 0.39 to 0.96); risk difference (RD) -13% (95% CI -25% to -1%). Multiple imputation for missing data gave an RR 0.69 (95% CI 0.44 to 1.09) and RD -9% (95% CI -21% to 2%)., Conclusions: Deferred clamping and immediate neonatal care with cord intact may reduce the risk of death or adverse neurodevelopmental outcome at 2 years of age for children born very premature. Confirmation in larger studies is needed to determine the real benefits and harms., Trial Registration Number: ISRCTN21456601., Competing Interests: Competing interests: JD reports grants from NIHR during the conduct of the trial; LD reports memberships to CTUs funded by NIHR. All other authors have nothing to disclose., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Background: Biomarkers of placental function can potentially aid the diagnosis and prediction of pregnancy complications. This randomised controlled pilot trial assessed whether for women with reduced fetal movement (RFM), intervention directed by the measurement of a placental biomarker in addition to standard care was feasible and improved pregnancy outcome compared with standard care alone., Methods: Women aged 16-50 years presenting at eight UK maternity units with RFM between 36 +0 and 41 +0  weeks' gestation with a viable singleton pregnancy and no indication for immediate delivery were eligible. Participants were randomised 1:1 in an unblinded manner to standard care and a biomarker blood test result revealed and acted on (intervention arm) or standard care where the biomarker result was not available (control arm). The objectives were to determine the feasibility of a main trial by recruiting 175-225 participants over 9 months and to provide proof of concept that informing care by measurement of placental biomarkers may improve outcome. Feasibility was assessed via the number of potentially eligible women, number recruited, reasons for non-recruitment and compliance. Proof of concept outcomes included the rates of the induction of labour and caesarean birth, and a composite adverse pregnancy outcome., Results: Overall, 2917 women presented with RFM ≥ 36 weeks, 352 were approached to participate and 216 (61%) were randomised (intervention n = 109, control n = 107). The main reason for not approaching women was resource/staff issues ( n = 1510). Ninety-seven women declined the trial, mainly due to not liking blood tests ( n = 24) or not wanting to be in a trial ( n = 21). Compliance with the trial interventions was 100% in both arms. Labour was induced in 97 (45%) participants (intervention n = 49, control n = 48), while 17 (9%) had planned caesarean sections (intervention n = 9, control n = 8). Overall, 9 (8%) babies in the intervention arm had the composite adverse pregnancy outcome versus 4 (4%) in the control arm., Conclusions: A main trial using a placental biomarker in combination with delivery, as indicated by the biomarker, in women with RFM is feasible. The frequency of adverse outcomes in this population is low, hence, a large sample size would be required along with consideration of the most appropriate outcome measures., Trial Registration: ISRCTN, ISRCTN12067514; registered 8 September 2017., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2020.)

Background: Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably., Methods: Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3 + , CD4 + , CD8 + , CD20 + , CD68 + , FOXP3 + ) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures., Results: The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infiltrates were highly variable (r = 0.01-0.56). Similar observations were made for cell type-specific quantifications (r = 0.001-0.54). We observed a strong inter-method variability between the omics-derived estimations, which is further cell type dependent. Finally, we demonstrated that most methods more accurately identify highly infiltrated (sTIL ≥ 60%; area under the curve, AUC, 0.64-0.99) as compared to lowly infiltrated tumors (sTIL ≤ 10%; AUC 0.52-0.82)., Conclusions: There is a lower inter-pathologist concordance for cell-specific quantification as compared to overall infiltration quantification. Microscopic assessments are underestimated when considering small cores (tissue microarray) instead of whole slides. Results further highlight considerable differences between the microscopic-, transcriptomic-, and methylomic-based methods in the assessment of overall and cell-specific immune infiltration in BC. We therefore call for extreme caution when assessing immune infiltrates using current methods and emphasize the need for standardized immune characterization beyond TIL.

Background: Bacterial vaginosis (BV) affects 30-50% of women at some time in their lives and is an embarrassing and distressing condition which can be associated with potentially serious comorbidities. Current antibiotic treatments such as metronidazole are effective but can result in side effects, and recurrence is common. This trial aims to investigate whether lactic acid gel is clinically effective and cost effective in the treatment of recurrent BV compared with metronidazole., Methods: VITA is an open-label, multicentre, parallel group randomised controlled trial for women with a clinical diagnosis of BV and at least one previous BV episode in the past 2 years. Participants will be randomised 1:1 to intravaginal lactic acid gel 5 ml once daily for 7 days or oral metronidazole tablets 400 mg twice daily for 7 days. All participants will be followed up for 6 months to assess health status and healthcare costs. A subgroup will be interviewed to further explore adherence, tolerability and acceptability of treatment. The estimated sample size is 1900 participants to detect a 6% absolute increase in response rate to 86% in those receiving lactic acid gel. The primary outcome is participant-reported resolution of BV at Week 2., Discussion: Results from this trial will help inform UK treatment guidelines for BV and may provide an alternative effective treatment for recurrent episodes of this condition which avoids repeated exposure to antibiotics., Trial Registration: ISRCTN, ISRCTN14161293. Registered on 8 September 2017.

Tumor-infiltrating B-cells (TIL-B) in breast cancer (BC) have previously been associated with improved clinical outcomes; however, their role(s) in tumor immunity is not currently well known. This study confirms and extends the correlation between higher TIL-B densities and positive outcomes through an analysis of HER2-positive and triple-negative BC patients from the BIG 02-98 clinical trial (10yr mean follow-up). Fresh tissue analyses identify an increase in TIL-B density in untreated primary BC compared to normal breast tissues, which is associated with global, CD4+ and CD8+ TIL, higher tumor grades, higher proliferation and hormone receptor negativity. All B-cell differentiation stages are detectable but significant increases in memory TIL-B are consistently present. BC with higher infiltrates are specifically characterized by germinal center TIL-B, which in turn are correlated with TFH TIL and antibody-secreting TIL-B principally located in tertiary lymphoid structures. Some TIL-B also interact directly with tumor cells. Functional analyses reveal TIL-B are responsive to BCR stimulation ex vivo, express activation markers and produce cytokines and immunoglobulins despite reduced expression of the antigen-presenting molecules HLA-DR and CD40. Overall, these data support the concept that ongoing humoral immune responses are generated by TIL-B and help to generate effective anti-tumor immunity at the tumor site.

The formation of new lymphatic vessels, or lymphangiogenesis, is a critical step of the tissue repair program. In pathological conditions involving chronic inflammation or tumorigenesis, this process is often dysregulated and can contribute to disease progression. Yet, lymphangiogenesis is still incompletely understood. In this study, we identified A2a adenosinergic signaling as an important regulator of inflammatory and tumor-associated lymphangiogenesis. Using Adora2a (A2a)-deficient mice, we demonstrated that A2a signaling was involved in the formation of new lymphatic vessels in the context of peritoneal inflammation. We also demonstrated that tumor-associated and sentinel lymph node lymphangiogenesis were impaired in A2a-deficient mice, protecting them from lymph node metastasis. Notably, A2a signaling in both hematopoietic and non-hematopoietic cells contributed to sentinel lymph node metastasis. In A2a-deficient tumor-draining lymph nodes, impaired lymphangiogenesis was associated with a reduced accumulation of B cells and decreased VEGF-C levels. Supporting a role for non-hematopoietic A2a signaling, we observed that primary murine lymphatic endothelial cells (LEC) predominantly expressed A2a receptor and that A2a signaling blockade altered LEC capillary tube formation in vitro . Finally, we observed that Adora2a, Nt5e and Entpd1 gene expression positively correlated with Lyve1, Pdpn and Vegfc in several human cancers, thereby supporting the notion that adenosine production and A2a receptor activation might promote lymphangiogenesis in human tumors. In conclusion, our study highlights a novel pathway regulating lymphangiogenesis and further supports the use of A2a or adenosine blocking agents to inhibit pathological lymphangiogenesis in cancers and block the dissemination of tumor cells through the lymphatic system.

Background: The Cord Pilot Trial compared two alternative policies for cord-clamping at very preterm birth at eight UK tertiary maternity units: clamping after at least 2 min and immediate neonatal care with cord intact, or clamping within 20 s and neonatal care after clamping. This paper reports views and experiences of the women who participated in the trial (261 randomised), based on data from two self-completed questionnaires., Methods: Women were given or posted the first questionnaire between 4 and 8 weeks after birth, and posted a second similar questionnaire at 1 year. Both questionnaires included three questions about experiences of participating in the trial: (1) If time suddenly went backwards and you had to do it all over again, would you agree to participate in the Cord Pilot Trial?; (2) Please tell us if there was anything about the Cord Pilot Trial that you think could have been done better; and (3) Please tell us if there was anything about the Cord Pilot Trial, or your experiences of joining the trial, that you think were particularly good., Results: One hundred and eighty-six women completed the first questionnaire and 133 completed the second. At both time points, 90% responded 'probably' or 'definitely' to participating in the trial again. More women randomised to deferred clamping responded 'definitely yes' than those allocated immediate clamping (78% versus 67% first questionnaire). Women were positive about the level of information and explanations, the friendly and caring staff, and the benefits for their baby and others as a result of participating in the trial. Suggestions for how the trial could be done better included being approached earlier, better staff communication about the trial, more information overall, and better timing of follow-up., Conclusions: Women were largely positive about participating in the trial. Nevertheless, they had suggestions for how the study could have been improved. These suggestions have implications for the design of future trials., Trial Registration: ISRCTN21456601 . Registered on 28 February 2013.

Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC, with implications for current therapies including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B+CD8+ T cells (GzmB+CD8+ T cells), a type 1 IFN signature, and elevated expression of multiple immune inhibitory molecules including indoleamine 2,3-dioxygenase (IDO) and programmed cell death ligand 1 (PD-L1), and resulted in good outcomes. An "immune-cold" microenvironment with an absence of tumoral CD8+ T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma, and poor outcomes. A distinct poor-outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8+ T cells, stromal expression of PD-L1, and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes allowed us to stratify TNBCs, predict outcomes, and identify potential therapeutic targets for TNBC.

Background: The Cord Pilot Trial compared two alternative policies for cord clamping at very preterm birth at eight UK maternity units: clamping after at least 2 min and immediate neonatal care (if needed) with cord intact, or clamping within 20 s and neonatal care after clamping. This paper reports follow-up of the women by two self-completed questionnaires up to one year after the birth., Methods: Women were given or posted the first questionnaire between four and eight weeks after birth, usually before their baby was discharged, and were posted a second similar questionnaire at one year. The questionnaire included the Hospital Anxiety and Depression Scale; the Preterm Birth Experience and Satisfaction Scale (P-BESS) and questions about their baby's feeding., Results: Of 261 women randomised (132 clamping ≥2 min, 129 clamping ≤20 s), six were excluded as birth was after 35 + 6 weeks (2, 4 in each group respectively). Six were not sent either questionnaire. The first questionnaire was given/sent to 244 and returned by 186 (76%) (79, 74%). The second, at one year, was sent to 242 and returned by 133 (55%) (66, 43%). On the first questionnaire, 89 (49%) had a score suggestive of an anxiety disorder, and 55 (30%) had a score suggestive of depression. Satisfaction with care at birth was high: median total P-BESS score 77 [interquartile range 68 to 84] (scale 17 to 85). There was no clear difference in anxiety, depression, or satisfaction with care between the two allocated groups. The median number of weeks after birth women breastfed/expressed was 16 (95% confidence interval (CI) 13 to 20, n = 119) for those allocated clamping ≥2 min and 12 (95% CI 11 to 16, n = 103) for those allocated clamping ≤20 s., Conclusions: The response rate was higher for the earlier questionnaire than at one year. A high proportion of women reported symptoms of anxiety or depression, however there were no clear differences between the allocated groups. Most women reported that they had breastfed or expressed milk and those allocated deferred cord clamping reported continuing this for slightly longer., Trial Registration: ISRCTN 21456601, registered 28th February 2013, http://www.isrctn.com/ISRCTN21456601.

An important role for tumor infiltrating B lymphocytes (TIL-B) in the immune response to cancer is emerging; however, very little is known about the antigen specificity of antibodies produced in situ . The presence of IgA antibodies in the tumor microenvironment has been noted although their biological functions and clinical significance are unknown. This study used a 91-antigen microarray to examine the IgG and IgA autoantibody repertoires in breast cancer (BC). Tumor and adjacent breast tissue supernatants and plasma from BC patients together with normal breast tissue supernatants and plasma from healthy controls (patients undergoing mammary reduction and healthy blood donors) were analyzed to investigate relationships between autoantibodies and the clinical, histological and immunological features of tumors. Our data show that >84% of the BC samples tested contain autoantibodies to one or more antigens on the array, with ANKRD30BL, COPS4, and CTAG1B being most frequently reactive. Ex vivo TIL-B responses were uncoupled from systemic humoral responses in the majority of cases. A comparison of autoantibody frequencies in supernatants and plasma from patients and controls identified eight antigens that elicit BC-associated autoantibody responses. The overall prevalence of IgG and IgA autoantibodies was similar and while IgG and IgA responses were not linked they did correlate with distinct clinical, pathological and immunological features. Higher levels of ex vivo IgG responses to BC-associated antigens were associated with shorter recurrence-free survival (RFS), HER2 overexpression and lower tumor-infiltrating CD8 + T cell counts. Higher IgA levels were associated with estrogen and progesterone receptor-negative cancers but were not significantly associated with RFS. Furthermore, ex vivo IgA but not IgG autoantibodies reactive to BC-associated antigens were linked with germinal center and early memory B cell maturation and the presence of tertiary lymphoid structures suggesting that these TIL-B are activated in the tumor microenvironment. Overall, our results extend the current understanding of the antigen specificity, the biological and the clinical significance of IgG and IgA autoantibodies produced by BC TIL-B in situ .

Background: Forty percent of babies who are stillborn born die after 36 weeks gestation and have no lethal structural abnormality. Maternal perception of reduced fetal movement (RFM) is associated with stillbirth and is related to abnormal placental structure and function. The ultimate objective of this trial is to assess whether for women with RFM, intervention directed by measurement of placental biochemical factors in addition to standard care improves pregnancy outcome compared with standard care alone. This is the protocol for a pilot trial to determine the feasibility of a definitive trial and also provide proof of concept that informing care by measurement of placental factors improves neonatal outcomes., Methods: ReMIT-2 is a multicentre, pilot randomised controlled trial of care informed by results of an additional placental factor blood test versus standard care alone for women presenting with RFM at or after 36 + 0 weeks gestation. Participants will be randomised 1:1 to the intervention arm where the blood test result is revealed and acted on, or to the control arm where the blood sample is not tested immediately and therefore the result cannot be acted on. All participants will be followed up six weeks after delivery to assess their health status and views of the trial, along with healthcare costs. A sub-group will be interviewed within 16 weeks after delivery to further explore their views of the trial. Outcomes to determine feasibility of a definitive trial include number of potentially eligible women, proportion lost to follow-up, clinical characteristics at randomisation, reasons for non-recruitment, compliance with the trial intervention and views of participants and clinicians about the trial. Proof of concept outcomes include: rates of induction of labour; Caesarean birth; and a composite neonatal outcome of stillbirths and deaths before discharge, 5-min Apgar score < 7, umbilical artery pH < 7.05 and admission to neonatal unit for > 48 h., Discussion: Results from this pilot trial will help determine whether a large definitive trial is feasible. Such a study would provide evidence to guide management of women with RFM and reduce stillbirths., Trial Registration: ISRCTN Registry, ISRCTN12067514 . Registered on 8 September 2017.

Background: Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer after invasive ductal cancer (IDC). Here, we aimed at evaluating the prevalence, levels, and composition of tumor-infiltrating lymphocytes (TILs) and their association with clinico-pathological and outcome variables in ILC, and to compare them with IDC., Methods: We considered two patient series with TIL data: a multicentric retrospective series (n = 614) and the BIG 02-98 study (n = 149 ILC and 807 IDC). We compared immune subsets identified by immuno-histochemistry in the ILC (n = 159) and IDC (n = 468) patients from the Nottingham series, as well as the CIBERSORT immune profiling of the ILC (n = 98) and IDC (n = 388) METABRIC and The Cancer Genome Atlas patients. All ILC/IDC comparisons were done in estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative tumors. All statistical tests were two-sided., Results: TIL levels were statistically significantly lower in ILC compared with IDC (fold-change = 0.79, 95% confidence interval = 0.70 to 0.88, P < .001). In ILC, high TIL levels were associated with young age, lymph node involvement, and high proliferative tumors. In the univariate analysis, high TIL levels were associated with worse prognosis in the retrospective and BIG 02-98 lobular series, although they did not reach statistical significance in the latter. The Nottingham series revealed that the levels of intratumoral but not total CD8+ were statistically significantly lower in ILC compared with IDC. Comparison of the CIBERSORT profiles highlighted statistically significant differences in terms of immune composition., Conclusions: This study shows differences between the immune infiltrates of ER-positive/HER2-negative ILC and IDC in terms of prevalence, levels, localization, composition, and clinical associations.

The immune tumour microenvironment has been shown to play a crucial role in the development and progression of cancer. Expression of gene signatures, reflecting immune activation, and the presence of tumour-infiltrating lymphocytes were associated with favourable outcomes in HER2-positive and triple-negative breast cancer. Recently, immunotherapy with immune checkpoint blockade induced long-lasting responses and improved survival in hard-to-treat malignancies (ie, melanoma and non-small cell lung cancer) and are changing treatment paradigms in a variety of neoplastic diseases. Immune checkpoint blockade has been evaluated in breast cancer, particularly in the triple-negative subtype, with promising results observed in monotherapy or in combination with chemotherapy in the metastatic and neoadjuvant settings. However, identification of patients who are most likely to benefit from immune checkpoint blockade remains challenging, with many patients not responding to treatments and a significant financial cost. The combination of immune checkpoint blockade with conventional cancer treatments such as chemotherapy, radiotherapy, targeted therapies or with other immunotherapies is a promising strategy to potentiate its efficacy in breast cancer although further research is required to effectively identify who will respond to these immunotherapies. In this review we report the most recent results that emerged from trials testing immune checkpoint blockade and potential predictive biomarkers and emphasise the new strategies that are under clinical development in breast cancer., Competing Interests: Competing interests: None declared.

Expression of the ectonucleotidase CD73 by tumor cells, stromal cells, and immune cells is associated in cancer with immune suppression. In this study, we investigated the role of CD73 on the activity of the anti-HER2/ErbB2 monoclonal antibody (mAb) trastuzumab. In a prospective, randomized phase III clinical trial evaluating the activity of trastuzumab, high levels of CD73 gene expression were associated significantly with poor clinical outcome. In contrast, high levels of PD-1 and PD-L1 were associated with improved clinical outcome. In immunocompetent mouse models of HER2/ErbB2-driven breast cancer, CD73 expression by tumor cells and host cells significantly suppressed immune-mediated responses mediated by anti-ErbB2 mAb. Furthermore, anti-CD73 mAb therapy enhanced the activity of anti-ErbB2 mAb to treat engrafted or spontaneous tumors as well as lung metastases. Gene ontology enrichment analysis from gene-expression data revealed a positive association of CD73 expression with extracellular matrix organization, TGFβ genes, epithelial-to-mesenchymal transition (EMT) transcription factors and hypoxia-inducible-factor (HIF)-1 gene signature. Human mammary cells treated with TGFβ or undergoing EMT upregulated CD73 cell-surface expression, confirming roles for these pathways. In conclusion, our findings establish CD73 in mediating resistance to trastuzumab and provide new insights into how CD73 is regulated in breast cancer. Cancer Res; 77(20); 5652-63. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The presence of tumor-infiltrating lymphocytes (TIL), reflecting host immune activity, is frequently correlated with better clinical outcomes, particularly in HER2-positive and triple-negative breast cancer. Recent findings suggest that organization of immune infiltrates in tertiary lymphoid structures also has a beneficial effect on survival. This study investigated inter- and intra-observer variation in TIL assessment using conventional hematoxylin-eosin versus immunohistochemical staining to identify immune cells. Global, intratumoral, and stromal TIL, as well as tertiary lymphoid structures were scored independently by experienced pathologists on full-face tumor sections (n=124). The fidelity of scoring infiltrates in core biopsies compared to surgical specimens, and pathological assessment compared to quantitative digital analysis was also evaluated. The inter-observer concordance correlation coefficient was 0.80 for global, 0.72 for intratumoral, and 0.71 for stromal TIL, while the intra-observer concordance correlation coefficient was 0.90 for global, 0.77 for intratumoral, and 0.89 for stromal TIL using immunohistochemical stains. Correlations were lower with hematoxylin-eosin stains, particularly for intratumoral TIL, while global scores had the highest concordance correlation coefficients. Our study concluded that tertiary lymphoid structures are accurately and consistently scored using immunohistochemical but not hematoxylin-eosin stains. A strong association was observed between TIL in core biopsies and surgical samples (R 2 =0.74) but this did not extend to tertiary lymphoid structures (R 2 =0.26). TIL scored by pathologists and digital analysis were correlated but our analysis reveals a constant bias between these methods. These data challenge current criteria for TIL and tertiary lymphoid structure assessment in breast cancer and recommend that how pathologists evaluate immune infiltrates be reexamined for future studies.

T follicular helper cells (TFH cells) are important regulators of antigen-specific B cell responses. The B cell chemoattractant CXCL13 has recently been linked with TFH cell infiltration and improved survival in human cancer. Although human TFH cells can produce CXCL13, their immune functions are currently unknown. This study presents data from human breast cancer, advocating a role for tumor-infiltrating CXCL13-producing (CXCR5-) TFH cells, here named TFHX13 cells, in promoting local memory B cell differentiation. TFHX13 cells potentially trigger tertiary lymphoid structure formation and thereby generate germinal center B cell responses at the tumor site. Follicular DCs are not potent CXCL13 producers in breast tumor tissues. We used the TFH cell markers PD-1 and ICOS to identify distinct effector and regulatory CD4+ T cell subpopulations in breast tumors. TFHX13 cells are an important component of the PD-1hiICOSint effector subpopulation and coexpanded with PD-1intICOShiFOXP3hi Tregs. IL2 deprivation induces CXCL13 expression in vitro with a synergistic effect from TGFβ1, providing insight into TFHX13 cell differentiation in response to Treg accumulation, similar to conventional TFH cell responses. Our data suggest that human TFHX13 cell differentiation may be a key factor in converting Treg-mediated immune suppression to de novo activation of adaptive antitumor humoral responses in the chronic inflammatory breast cancer microenvironment.

Innate and adaptive immune cells play an important role in the therapeutic activity of anti-ErbB2 mAbs, such as trastuzumab. In the clinic, breast tumors poorly infiltrated with immune cells are more resistant to trastuzumab, and patients have a worse prognosis. Because type I and II IFNs are critical to the immune-mediated activity of anti-ErbB2 mAb, we investigated the effect of combining polyI:C and CpG with trastuzumab-like therapy in immunocompetent mouse models of ErbB2 + breast cancer. We demonstrated that in situ delivery of polyI:C and CpG combined to systemic anti-ErbB2 mAb triggered a potent inflammatory response in breast tumors able to induce long-lasting CD8 + T cell-dependent antitumor immunity. Remarkably, polyI:C and CpG was superior to combined PD-1/CTLA-4 blockade in sensitizing tumors to anti-ErbB2 mAb therapy. Local injection of CpG and polyI:C in a primary tumor significantly enhanced the activity of systemic anti-ErbB2 mAb against a distant untreated tumor. Type I and II IFNs, as well as natural killer cells and CD8 + T cells, were indispensible to the synergistic activity of the combination treatment. Because synthetic RNA analogues and CpG oligodeoxynucleotides have been safely used in clinical trials, our study supports combination treatments with anti-ErbB2 mAbs. Cancer Res; 77(2); 312-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

The clinical relevance of tumor-infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by their demonstrated correlation with long-term positive outcomes. Nevertheless, the relationship between protective immunity, observed in some patients, and critical features of the infiltrate remains unresolved. This study examined TIL density, composition and organization together with PD-1 and PD-L1 expression in freshly collected and paraffin-embedded tissues from 125 patients with invasive primary BC. Tumor and normal breast tissues were analyzed using both flow cytometry and immunohistochemistry. TIL density distribution is a continuum with 25% of tumors identified as TIL-negative at a TIL density equivalent to normal breast tissues. TIL-positive tumors (75%) were equally divided into TIL-intermediate and TIL-high. Tumors had higher mean frequencies of CD4 + T cells and CD19 + B cells and a lower mean frequency of CD8 + T cells compare with normal tissues, increasing the CD4 + /CD8 + T-cell ratio. Tertiary lymphoid structures (TLS), principally located in the peri-tumoral stroma, were detected in 60% of tumors and correlated with higher TIL infiltration. PD-1 and PD-L1 expression were also associated with higher TIL densities and TLS. TIL density, TLS and PD-L1 expression were correlated with more aggressive tumor characteristics, including higher proliferation and hormone receptor negativity. Our findings reveal an important relationship between PD-1/PD-L1 expression, increased CD4 + T and B-cell infiltration, TIL density and TLS, suggesting that evaluating not only the extent but also the nature and location of the immune infiltrate should be considered when evaluating antitumor immunity and the potential for benefit from immunotherapies.

Purpose: Immune checkpoint inhibition has been demonstrated to be an effective anticancer strategy. Several lines of evidence support the study of immunotherapy in triple-negative breast cancer (TNBC). We assessed the safety and antitumor activity of the programmed cell death protein 1 (PD-1) inhibitor pembrolizumab in patients with advanced TNBC., Methods: KEYNOTE-012 (ClinicalTrials.gov identifier: NCT01848834) was a multicenter, nonrandomized phase Ib trial of single-agent pembrolizumab given intravenously at 10 mg/kg every 2 weeks to patients with advanced PD-L1-positive (expression in stroma or ≥ 1% of tumor cells by immunohistochemistry) TNBC, gastric cancer, urothelial cancer, and head and neck cancer. This report focuses on the TNBC cohort., Results: Among 111 patients with TNBC whose tumor samples were screened for PD-L1 expression, 58.6% had PD-L1-positive tumors. Thirty-two women (median age, 50.5 years; range, 29 to 72 years) were enrolled and assessed for safety and antitumor activity. The median number of doses administered was five (range, 1 to 36 doses). Common toxicities were mild and similar to those observed in other tumor cohorts (eg, arthralgia, fatigue, myalgia, and nausea), and included five (15.6%) patients with grade ≥ 3 toxicity and one treatment-related death. Among the 27 patients who were evaluable for antitumor activity, the overall response rate was 18.5%, the median time to response was 17.9 weeks (range, 7.3 to 32.4 weeks), and the median duration of response was not yet reached (range, 15.0 to ≥ 47.3 weeks)., Conclusion: This phase Ib study describes preliminary evidence of clinical activity and a potentially acceptable safety profile of pembrolizumab given every 2 weeks to patients with heavily pretreated, advanced TNBC. A single-agent phase II study examining a 200-mg dose given once every 3 weeks (ClinicalTrials.gov identifier: NCT02447003) is ongoing., (© 2016 by American Society of Clinical Oncology.)

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45(+) cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45(+) cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

Eye disorders are frequently associated with renal diseases, mostly linked to underlying causes such as hypertension, diabetes or autoimmune diseases. Conversely, advanced uraemic states may also lead to progressive vision impairment. The present report concerns a 50-year-old patient who presented with a bilateral, painless, progressive vision loss, a moderate systemic inflammation and chronic renal failure due to hypertension nephrosclerosis. Steroids were given and haemodialysis was initiated, resulting in vision improvement. At 4 months later when the steroids were stopped, the patient developed dyspnoea, cough, fever and fatigue of unclear origin. A lung biopsy showed non-caseating granuloma consistent with pulmonary sarcoidosis. Re-challenge with steroids rapidly improved the respiratory disease. Ophthalmological examinations performed early and later in the course excluded anterior ischaemic optic neuropathy and ocular manifestations of sarcoidosis, leading to a diagnosis of uraemic optic neuropathy. This rare ophthalmological disorder should be promptly recognised since haemodialysis and steroid therapy are highly effective.

Brachyury(T) is a mouse mutation, first described over 70 years ago, that causes defects in mesoderm formation. Recently several related genes, the T-box gene family, that encode a similar N-terminal DNA binding domain, the T-box, and that play critical roles in human embryonic development have been identified. It has been shown that human TBX5 and TBX3, if mutated, cause developmental disorders, Holt-Oram syndrome (OMIM 142900) and ulnar-mammary syndrome (OMIM 181450), respectively. We have identified four new human members of the T-box gene family, EOMES, TBX6, TBX18, and TBX19, and these genes have been mapped to different chromosomal regions by radiation hybrid mapping. The four T-box genes were classified into four different subfamilies and have also been subjected to phylogenomic analysis. Human EOMES maps at 3p21.3-p21.2. This Tbr1-subfamily gene is likely to play a significant role in early embryogenesis similar to that described for Xenopus eomesodermin. Human TBX6 maps at 16p12-q12. This Tbx6-subfamily gene is likely to participate in paraxial mesoderm formation and somitogenesis in human embryo. TBX18 is a novel member of the Tbx1 subfamily that maps at 6q14-q15. Two subgroups, TBX1/10 and TBX15/18 subgroups, could be distinguished within the Tbx1 subfamily. TBX19 is an orthologue of chick TbxT and maps at 1q23-q24. The genomic organization of TBX19 is highly similar to that of human T(Brachyury), another human member of the same subfamily., (Copyright 1999 Academic Press.)