HIV infection is a significant independent risk factor for both severe COVID-19 presentation at hospital admission and in-hospital mortality. Available information has suggested that people living with HIV and AIDS (PLWHA) could benefit from COVID-19 vaccination. However, there is a dearth of evidence on willingness to receive COVID-19 vaccination among PLWHA.The aim of this study was to investigate willingness to receive COVID-19 vaccination among a national sample of PLWHA in China.This cross-sectional online survey investigated factors associated with willingness to receive COVID-19 vaccination among PLWHA aged 18 to 65 years living in eight conveniently selected Chinese metropolitan cities between January and February 2021. Eight community-based organizations (CBOs) providing services to PLWHA facilitated the recruitment. Eligible PLWHA completed an online survey developed using a widely used encrypted web-based survey platform in China. We fitted a single logistic regression model to obtain adjusted odds ratios (aORs), which involved one of the independent variables of interest and all significant background variables. Path analysis was also used in the data analysis.P<.001), negative attitudes , perceived support from significant others , and perceived behavioral control . At the interpersonal level, receiving advice supportive of COVID-19 vaccination from doctors , CBO staff , friends and/or family members , and PLWHA peers  was associated with higher willingness to receive COVID-19 vaccination. The overall opinion supporting COVID-19 vaccination for PLWHA on the internet or social media was also positively associated with willingness to receive COVID-19 vaccination . Path analysis indicated that interpersonal-level variables were indirectly associated with willingness to receive COVID-19 vaccination through perceptions .Out of 10,845 PLWHA approached by the CBOs, 2740 completed the survey, and 170 had received at least one dose of the COVID-19 vaccine. This analysis was performed among 2570 participants who had never received COVID-19 vaccination. Over half of the participants reported willingness to receive COVID-19 vaccination . Perceptions related to COVID-19 vaccination were significantly associated with willingness to receive COVID-19 vaccination, including positive attitudes (aOR 1.11, 95% CI 1.09-1.12; As compared to PLWHA in other countries and the general population in most parts of the world, PLWHA in China reported a relatively low willingness to receive COVID-19 vaccination. The internet and social media as well as interpersonal communications may be major sources of influence on PLWHA’s perceptions and willingness to receive COVID-19 vaccination. The World Health Organization (WHO) confirmed that HIV infection is a significant independent risk factor for both severe COVID-19 presentation at hospital admission and in-hospital mortality [The COVID-19 vaccine offers the best hope for ending the pandemic. Simulation experiments showed that when vaccine efficacy was 80%, 75% coverage could end the COVID-19 pandemic without any other control measures . A relatThe recommendations or guidelines regarding COVID-19 vaccination for PLWHA are inconsistent across countries. The WHO, the United States Department of Health and Human Services, the British HIV Association, and health authorities in Australia recommend that PLWHA receive COVID-19 vaccination regardless of their CD4+ T cell counts -15. PLWHVaccine hesitancy hindered the successful control of the COVID-19 pandemic. Therefore, it is helpful for governments to plan interventions to improve people’s awareness of the safety and benefits of the COVID-19 vaccine and to reduce vaccine hesitancy. In order to promote the COVID-19 vaccination of PLWHA, it is necessary to understand their willingness to receive COVID-19 vaccination and related facilitators and barriers. However, most studies investigating willingness to receive COVID-19 vaccination and its associated factors were conducted among the general population and medical professionals ; therefoWe applied the socioecological model to understand factors associated with willingness to receive COVID-19 vaccination among PLWHA at individual, interpersonal, and sociostructural levels . InterveTo our knowledge, no studies have investigated willingness to receive COVID-19 vaccination among PLWHA in China. To address knowledge gaps, this study investigated willingness to receive COVID-19 vaccination among a national sample of PLWHA. We examined the effects of the following factors: sociodemographics, HIV-related characteristics, individual-level factors , interpersonal-level variables , and sociostructural-level factors . We further tested the hypothesis that perceptions of COVID-19 vaccination would mediate the association between interpersonal-level variables and willingness to receive COVID-19 vaccination.This study makes use of a multicenter cross-sectional online survey conducted in eight conveniently selected large Chinese cities between January and February 2021. These cities included two in the north (Tianjin and Beijing), two in the northeast (Shenyang and Hohhot), one in the east (Nanjing), and three in the south . Beijing is the capital city of China. Shenzhen is a major special economic zone in China bordering Hong Kong in the south. The other six cities are capital cities of the provinces. Reasons for selecting these cities included the following: (1) each city has a CBO providing services to PLWHA, (2) each city has a large number of PLWHA, and (3) COVID-19 vaccination was first scaled up at these sites. At the time of this study, people in Beijing, Guangzhou, and Shenzhen could make an appointment to receive COVID-19 vaccination. The procedures of making an appointment were simple. People first downloaded a smartphone app developed by the health bureau. After logging in, they could choose the time and location to receive COVID-19 vaccination. In the other five cities, vaccination was arranged by employers and did not allow individuals to make appointments. Only two types of inactivated COVID-19 vaccines—Sinovac-CoronaVac and Sinopharm—were available in China during the study period. They were provided by designated community vaccination centers and people could only receive them at sites in these centers. Immunodeficiency, including HIV infection, was listed as a precaution for COVID-19 vaccination in China during the study period; it was recommended that PLWHA seek advice from doctors regarding COVID-19 vaccination . ParticiStudy participants included individuals aged 18 to 65 years who have been diagnosed with HIV or AIDS and were living in one of the eight cities. We did not include PLWHA older than 65 years, as COVID-19 vaccination was not approved for this age group in China at the time of the survey. Exclusion criteria included the following: (1) being illiterate and unable to complete the questionnaire survey and (2) being ineligible for COVID-19 vaccination .The eight CBOs that were mainly providing services to marginalized populations , one in each study site, facilitated recruitment through their networks. These CBOs have been working closely with HIV clinical service providers. CBOs in China are the main providers of HIV outreach services to PLWHA, as these routine tasks have been transferred from government agencies to CBOs . A high The survey was carried out through Golden Data, a commonly used, encrypted, web-based survey platform in China. Each individual WeChat account was allowed to access the online questionnaire only once to avoid duplicate responses. The Golden Data tool performed a completeness check before each questionnaire was submitted. Participants could review and change their responses when they completed the questionnaire. The survey took about 13 to 15 minutes to complete. An electronic coupon with a value of 20 Chinese yuan (US $3.1) was sent to each participant upon the completion of the survey. A unique ID was assigned to each participant, which was used to delink the study database from personal identifying data. All data collected by the online surveys were stored in the Golden Data server and protected by a password. Only the designated research team members had access to the database.Signed electronic informed consent forms were obtained from all subjects involved in the study. These were kept separately from the empirical data and stored in a password-protected computer or a locked cabinet in the same locked office. This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Boards of Changzhi Medical College (protocol code RT2021003).A panel consisting of public health researchers, health psychologists, clinicians, CBO staff, and PLWHA was formed to develop the questionnaire used in this study. The panel revised and finalized the questionnaire based on a pilot test among 10 PLWHA. These 10 PLWHA did not participate in the actual survey.Participants reported sociodemographic characteristics, lifestyles , height and weight, and history of other vaccinations in the past 3 years. Participants were also asked whether they had any chronic conditions, such as chronic cardiovascular, respiratory, kidney, and liver diseases; hypertension; diabetes mellitus and its chronic complications; cancers; lymphoma; leukemia; autoimmune diseases; hemorrhagic diseases; and history of severe allergy. The survey also collected some characteristics related to HIV infection .Participants were asked about their likelihood of receiving free COVID-19 vaccination in the future; the response categories were as follows: 1 (very unlikely), 2 (unlikely), 3 , 4 (likely), and 5 (very likely). This study defined willingness to receive COVID-19 vaccination as the responses “likely” or “very likely” .The research team interviewed staff from the Chinese Center for Disease Control and Prevention who were responsible for implementing the COVID-19 vaccination program about whether individuals were allowed to make an appointment to receive COVID-19 vaccination and whether there was a shortage of COVID-19 vaccines during the project period at different study sites. Participants were asked whether they belonged to any of the groups who had priority to receive COVID-19 vaccination as listed by the National Health Commission during the project period.At the individual level, four scales were constructed to measure perceptions related to COVID-19 vaccination. These scales were as follows: (1) the 5-item Positive Attitude Scale, (2) the 5-item Negative Attitude Scale, (3) the 4-item Perceived Subjective Norm Scale , and (4) the 5-item Perceived Behavioral Control Scale . Response categories were as follows: 1 (strongly disagree), 2 (disagree), 3 , 4 (agree), and 5 (strongly agree). Positive and negative attitudes, perceived subjective norm, and perceived behavioral control were significantly associated with willingness to receive COVID-19 vaccination among Chinese people .For interpersonal-level variables, participants were asked whether they received advice given by doctors, CBO staff, friends and/or family members, and other PLWHA regarding COVID-19 vaccination. Participants were also asked about the overall opinion regarding COVID-19 vaccination that they found on the internet or social media; response categories were as follows: 1 (against taking up COVID-19 vaccination), 2 , and 3 (supportive in taking up COVID-19 vaccination).P<.05 in the univariate analysis, associations between independent variables of interest  and the dependent variable were then assessed by adjusted odds ratios (aORs) and 95% CIs. Each aOR was obtained by fitting a single logistic regression model, which involved one of the independent variables of interest and all significant background characteristics.Using willingness to receive COVID-19 vaccination as the dependent variable and background characteristics as independent variables, crude odds ratios were obtained by logistic regression models. After adjusting for variables with P<.05. SPSS for Windows  and Mplus  were used for all analyses.Path analysis was conducted to test the mediation model. The mean scores of the Positive Attitude Scale, the Negative Attitude Scale, the Perceived Subjective Norm Scale, and the Perceived Behavioral Control Scale were used as indicators to represent the latent variable of perceptions related to COVID-19 vaccination. The mean scores of advice given by doctors, CBO staff, friends and/or family members, and other PLWHA, as well as the overall opinion on the internet and social media, were used as indicators to represent the latent variable of interpersonal-level variables. Confirmatory factor analysis was conducted to test goodness of fit of these constructs. The latent variable representing perceptions was used as the independent variable, and willingness to receive COVID-19 vaccination was used as the dependent variable. The significant background characteristics were controlled for the model. Goodness of fit was tested using chi-square tests, the comparative fit index (CFI), the Tucker-Lewis index (TLI), and the root mean square error of approximation (RMSEA). Standardized path coefficients (β) and unstandardized path coefficients (B) were reported. The asymmetric CIs based on the bootstrap method  that were used for significance testing of mediation hypotheses with the 95% bootstrap CI did not include zero, indicating a statistically significant mediation effect. The level of statistical significance was set at The CBOs approached 10,845 PLWHA in their WeChat groups, 8692 accessed the online survey, 2740 completed the survey, and 170 received at least one dose of COVID-19 vaccination at the time of the survey. This study was based on 2570 eligible participants who had never received COVID-19 vaccination; the flowchart of this study is presented in The majority of the 2570 participants were 18 to 39 years of age , identified themselves as male , were currently single , had a full-time job , and only had basic health insurance . Regarding characteristics related to HIV infection, 17.2% (n=443) of the 2570 participants received their diagnosis within 1 year, 97.3% (n=2501) were receiving ART, 67.9% (n=1746) reported undetectable viral load, and 46.0% (n=1181) reported their CD4+ T cell count level to be above 500/µL. Less than 5% of the participants self-reported having severe AIDS-related symptoms  and 2.Over half of the participants were willing to receive free COVID-19 vaccinations in the future . A shortage of COVID-19 vaccines was encountered in Shenyang, Guangzhou, and Shenzhen. Among the participants, 19.0% (488/2570) identified themselves as belonging to a priority group that would receive COVID-19 vaccination. The Cronbach α of the scales for perceptions related to COVID-19 vaccination ranged from .83 to .92; single factors were identified by exploratory factor analysis, explaining 61.1% to 76.4% of the total variance .In the univariate logistic regression analysis, transgender persons, those with chronic conditions, and those with more severe AIDS-related symptoms showed lower willingness to receive COVID-19 vaccination. Having commercial health insurance only and a history of other vaccinations in the past 3 years were associated with higher willingness to receive COVID-19 vaccination  and 2.P<.001), stronger perceived support from significant others , and higher perceived behavioral control  to take up the vaccination were associated with higher willingness to receive COVID-19 vaccination. A negative association was found between negative attitudes toward COVID-19 vaccination and the dependent variable . At the interpersonal level, receiving advice supportive of COVID-19 vaccination from doctors , CBO staff , friends and/or family members , and other PLWHA  were associated with higher willingness to receive COVID-19 vaccination. The overall opinion supporting COVID-19 vaccination for PLWHA on the internet and social media was also positively associated with the dependent variable . All the factor loadings were significant at P<.001, with standardized coefficients ranging from .27 to .50. The hypothesized mediation model showed good fit to the data .Confirmative factor analysis showed that perceptions fit the data well . All the factor loadings were significant at P<.001), while their association with willingness to receive COVID-19 vaccination was nonsignificant . Perceptions were positively associated with willingness to receive COVID-19 vaccination  were indirectly associated with willingness to receive COVID-19 vaccination via perceptions. Perceptions fully mediated the association between interpersonal-level variables and willingness to receive COVID-19 vaccination.Bootstrap analysis showed that interpersonal-level variables . This level was lower than that of PLWHA in France and the United States as well as the general population in most parts of the world  and ChinThis study examined associated factors at all three levels suggested by the socioecological model; the findings could inform tailored interventions promoting COVID-19 vaccination among PLWHA. This was the first study to demonstrate that the individual-level variables  and interpersonal-level variables  were determinants of willingness among PLWHA. The findings extended the application of the socioecological model. More importantly, this study examined the potential mechanism of the associations between interpersonal interactions and willingness to receive COVID-19 vaccination. The results suggest that exposure to advice and information supporting PLWHA in receiving COVID-19 vaccination might enhance perceptions favoring COVID-19 vaccination, which, in turn, may increase their willingness to receive such vaccination. The significant mediation effect supported the mechanism proposed by the social learning theory .This study also had numerous practical implications for developing tailored vaccination strategies for PLWHA. First, more attention should be given to PLWHA who are transgender persons, with chronic conditions, and who have severe AIDS-related symptoms, as these subgroups reported lower willingness. Transgender people are often marginalized, encountering difficulties to access health care services . Future Second, modifying perceptions related to COVID-19 vaccination is potentially useful in health promotion, as they were significantly associated with willingness in the expected directions. It is useful to increase positive attitudes toward COVID-19 vaccination, as this was a facilitator. Health communication messages should emphasize the physical and psychological benefits of COVID-19 vaccination. About half of the participants had concerns related to side effects, exposing PLWHA’s identities, and potential interactions between COVID-19 vaccines and HIV and ART. Having more concerns was associated with lower willingness. Testimonials on positive experiences shared by vaccinated PLWHA might be useful in reducing their concerns related to side effects and privacy. Health communication messages should also emphasize that there is no evidence showing that ART and COVID-19 vaccination would have negative impacts on each other . Less thThird, the significant mediation effect of perceptions on the association between interpersonal-level variables and willingness to receive COVID-19 vaccination suggested that future programs should involve clinical doctors, CBO staff, friends and/or family members, and peers of PLWHA to give supportive advice. Health authorities should also disseminate clear recommendations for PLWHA to receive COVID-19 vaccination through official online channels, which are considered influential and credible sources by Chinese people . These sThis study also had some limitations. First, policies and guidelines related to COVID-19 vaccination are changing rapidly. Our findings are most applicable to the early phase of COVID-19 vaccination implementation in China. Second, participants were recruited in large Chinese cities. Generalizations to PLWHA living in smaller cities or counties in China should be made cautiously. Third, we were not able to collect information from PLWHA who refused to participate in the study. PLWHA who refused to complete the survey may have different characteristics from the participants. Selection bias existed. Fourth, most items and scales used in this study were self-constructed based on those used in the general population. The internal validity of these scales was acceptable. However, external validation data were seldom available. Fifth, it was a limitation that we did not ask whether participants anticipated or had experienced challenges in making an appointment to receive the COVID-19 vaccine. The procedures to make an appointment are easy. Most Chinese people did not encounter difficulties when they were using the appointment system. We believe that the impact of anticipated and experienced challenges in making an appointment on PLWHA’s willingness to receive COVID-19 vaccination was limited. Sixth, we did not study PLWHA’s preferences for different types of COVID-19 vaccines. Although inactivated vaccines were the only available COVID-19 vaccines in China during the study period, it is worthwhile to look at their preferences for other types of vaccines  . MoreoveIn sum, PLWHA in China reported a relatively low willingness to receive COVID-19 vaccination compared to PLWHA in other countries and the general population in most parts of the world. Perceptions related to COVID-19 vaccination and interpersonal-level variables, such as receiving advice from others or information exposure through the internet and social media, were determinants of willingness. Information exposure on the internet and social media and interpersonal communications with doctors, CBO staff, friends, family members, and other PLWHA may be major sources of influence on PLWHA’s perceptions and willingness to receive COVID-19 vaccination. The study findings could be used to design tailored interventions with the aims of improving vaccination coverage and reduce risks of COVID-19 among PLWHA.


The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from  This therapeutic strategy has significantly expanded in the setting of advanced and early-stage breast cancer (BC), but much more work is needed to optimize patient selection based on tumor-based biomarkers. The presence of tumor-infiltrating lymphocytes (TILs) is believed to be predictive of response to immunotherapy, chemotherapy, and other targeted therapies3 in addition to their role as a prognostic biomarker5. Moreover, TILs in the tumor and the surrounding microenvironment are thought to reflect ongoing anti-tumor host immune response. Three main categories of the tumor microenvironment (TME) have been defined across different tumor types: immune-desert (“cold” tumors largely devoid of lymphocytes), immune-excluded , and immune-infiltrated/inflamed (“hot” tumors)7. Conceptually, each of these TME categories reflects a specific interaction between the tumor genotype/phenotype and the host immune system, which can impact the response to both conventional anticancer therapies and ICI8. However, there is considerable heterogeneity within each TME category, adding uncertainty to the reproducibility of the current classification of “cold” vs. “hot” vs. “intermediate” immune-subtypes. Also, there are no validated criteria to define these subtypes, either using morphology, immunostaining, transcriptomics, or their combination, limiting the impact of these descriptors in clinical trials and daily practice.The use of immune-checkpoint blockade (ICI) in clinical oncology has revolutionized patient care and improved survival outcomes in many patients with malignancies10. However, all BC subtypes include cases with TIL-infiltration. The degree of TIL infiltration has been hypothesized to reflect the tumor mutational burden (TMB), which is lower in HR-positive BC12. Higher TMB is linked to the expression of more neoantigens and has been shown to predict survival after ICI therapy in several cancer types, and recent evidence indicates this may also be the case for TNBC15. However, the correlation between mutational burden and immune composition is complex, with the degree and nature of clonality of the mutations playing a key role in determining whether they favor or hinder immune-mediated tumor control16. In TNBC, higher TMB and greater genomic heterogeneity have been associated with lower TILs17. Conceptually, this can be explained by immunoediting, which is a result of a selection of cancer cell clones with decreased immunogenicity despite the presence of many mutations18. This escape from immune surveillance is associated with a reduced TIL component and increased tumor clonal heterogeneity, explaining the negative association between TMB and TILs21.In BC, the molecular subtype of the tumor has a major influence on its interaction with the immune system. Triple-negative BC (TNBC) and HER2-positive BC are more frequently infiltrated by higher numbers of TILs than hormone receptor (HR)-positive tumors24. Generally, intra-epithelial TIL density tends to be lower than stromal TIL density23, raising the question of whether a tumor nest-stromal barrier precludes more robust T cell infiltration25, and/or whether the intra-epithelial TILs have always been present (in an inactive state), as tissue-resident T-cells. CD8+ tissue-resident memory T (TRM) cells were shown to mediate BC immunosurveillance26. Notably, high BC infiltration by TILs contained CD8+ T lymphocytes with TRM patterns which highly express immune-checkpoint molecules26.In TNBC, scoring TILs in the stromal compartment (sTILs) is demonstrably reproducible and generally well-correlated with intra-epithelial TILs, with higher stromal TILs (sTILs) predicting longer survival27. TLS has been detected in the stroma of up to 60% of BC, with the highest frequencies in HER2+ and TNBC29. These findings support a critical role for the stroma in shaping the TME of BC. For example, fibroblastic reticular cells, concentrated in the T cell zone of TLS, promote, maintain or suppress T cell activities via their cytokine and chemokine secretion30. TLS architecture is distinguished by a T cell zone adjacent to a B cell follicle, similar to secondary lymphoid organs27. Immune responses generated in tumor-associated TLS would thus produce immunological memory to multiple BC neoantigens and could potentially control the growth of disseminated tumor cells34. However, there are significant concerns that TLS cannot be assessed in a reproducible manner by analysis of HE-stained slides, and that B- and T-cell immunostains are needed34. In addition, TLS is frequently found at the tumor perimeter, often contain high endothelial venules, and when present in the tumor area are generally considered as an aggregate when quantifying TILs in BC. Their role in BC still remains to be addressed.Tertiary lymphoid structures (TLS) are lymph node-like structures that arise in tissues at sites of chronic inflammation35 coupled with computational deconvolution of immune cell populations37, global and single-cell transcriptomics39, and multiplex imaging40, to quantify TIL distribution, functional orientation and relative frequencies in individual tumor types. However, these cutting-edge investigational tools are often limited by reproducibility across studies, particularly when attempting to resolve specific immune cell subtypes. In addition, tumors with low TILs seem to pose a significant challenge to any of these techniques and platforms41. Machine learning techniques are in development to evaluate TIL distribution patterns and integrate the spatial information with sTILs and with molecular profiling data43. A study examining clonal heterogeneity, TMB, copy number variations, somatic mutations, and germline polymorphisms, as well as the neoantigen load for their association with immune metagene expression in the BC subtypes, did not find any distinct recurrent single gene or pathway level mutations associated with immune infiltration21. However, lower clonal heterogeneity was observed in TNBC and HER2+ BC associated with higher immune gene expression, which is consistent with immunoediting21. There is also evidence for immune escape during the in situ to invasive BC transition, with a decrease in immune activation measurable using a combination of global profiling and single-cell transcriptomics44. It may be hypothesized that similar immune evasion mechanisms also occur during the transition from stage I TNBC to more advanced stages of the disease. This paradigm was also recently reported in lung cancer in which immune-evasion seems to be triggered by neoantigen editing during tumor evolution45. In the remainder of this review, the analytical and clinical impact of sTILs in BC management will be discussed based on recent updates from human studies, particularly clinical trials.The importance of expanding our current understanding of the complex TME in breast and other cancers has driven the development of diverse techniques, including molecular multi-omics profiling24. There is overwhelming evidence for the clinical validity of this evaluation47. However, analytical validity cannot be formally demonstrated due to the lack of a “gold standard” against which to assess the proposed method. This may not be that important for the clinical implementation of sTILs. However, for machine learning approaches this question is crucial. Comparing two methods, for example, a pathologist vs. an automated image-analysis system provides evidence on concordance, but not accuracy43. All the previous TIL-RING studies have been performed with the assumption of histological accuracy47.Analytical validity is defined as how accurately a test predicts the presence or absence of a biological variable. In other words, can the “test” correctly distinguish between TILs and other immune cells?. Previous TIL inter-pathologists-reproducibility studies (RING-studies) have shown that pathologists (using an H&E slide) can reliably assess TILs with very high concordance between many pathologists on a powered number of cases and at different cut-offshow often is what a pathologist calls a TIL actually a TIL?”. This is crucial when applying machine learning methods to identify TILs42, since the unequivocal differentiation of a myofibroblast, an invasive lobular carcinoma (ILC) cell, and lymphocyte is not always possible on an H&E slide. Moreover, the concordance between TIL assessment by machine learning-based methods and pathologist-TIL scores is unlikely to be 100%48. Currently, a TIL is defined as being a lymphocyte or a plasma cell, and both are scored and defined according to classic morphological definitions. Then, we need to define a “stromal TIL” (sTIL), i.e., what is the maximal distance between a tumor cell and a TIL to define it as an sTIL. Furthermore, uncertainty amongst pathologists exists about the inclusion of stroma abutting the tumor, or all of the stroma within the total tumoral area to include intervening stroma with low/very low sTILs. Indeed, accumulating evidence suggests that sTILs touching the tumor cells may have a different molecular phenotype than sTILs distant to the main tumor bulk25. Kos et al.24 have recently reported further factors that may impact the assessment of TILs including pre- and post-analytical histology factors, particularly in the setting of retrospective analysis of trial material, and the recognition of common artifacts. In an effort to improve concordance, the International Immuno-Oncology Biomarker Working Group, also called the TILs Working Group  recently provided reference images and digital slides as well as accessible guidance regarding the analysis of heterogeneous immune cell infiltrates.In order to define accuracy in the context of assessing TILs using an H&E-stained slide, it is necessary to define a “gold standard” against which to assess the proposed method, i.e., an orthogonal-type method which is used as the “gold standard”. For example, a pan-lymphocyte marker can establish the accuracy of lymphocyte identification, answering “will when implemented, impact patient management. Simply put, a test only has clinical utility when it impacts physician and patient choice on treatment or management options. The level 1 requirement for clinical validity clearly differs according to the setting49. However, it is clearly not optimal to assess the clinical validity of TILs without first framing the question of clinical utility correctly. Once the correct clinical question is framed, appropriate studies must be designed to generate evidence to support the clinical validity of TILs in the setting of clinical utility. In this respect, we agree with Simon et al. that prospective trials not originally designed to address tissue biomarker studies can be used to “accommodate biomarker utility” using archived samples49. Since only simple tools, like a microscope, are needed for the assessment of TILs, this provides tremendous opportunities to test the “clinical utility” of TILs in various settings.Clinical validity refers to the presence of sufficient evidence, usually level 1 evidence of the effect of a test (biomarker) to demonstrate its validity in a clinical setting. That is, there is robust statistically validated evidence that the test (biomarker) relates to a clinical outcome (prognostic or predictive) or a specific phenotype (TILs), etc. However clinical validity alone, whilst required for changes in clinical practice, does not drive a change in practice. For this to occur, the test must have clinical utility. Clinical utility essentially requires that the test in question addresses a direct clinical need  and 49 can either be reached by incorporating a biomarker into a properly powered prospective clinical trial (level 1A) or by achieving reproducible results in archived tissues from independent randomized trials, designed, conducted, and analyzed as per REMARK criteria (level 1B)50. Using these widely accepted criteria, level 1B evidence for clinical validity of TILs as a prognostic biomarker in early-stage TNBC is well established22. Two pooled analyses of TILs, in the adjuvant setting for TNBC22, and in the neoadjuvant setting across BC-subtypes9, included studies that have evaluated TILs on archived tissue samples based on our published guidelines23.Level 1 evidence for a biomarkern = 2148), the clinical value of TILs in predicting prognosis of early-stage TNBC, including adjuvant trials of anthracycline-based chemotherapy alone or in combination with taxanes was investigated22. The average age of enrolled patients was 50 years, and 33% of them were lymph node-negative. The quantification of TILs showed that their average was 23%, and 77% of patients had at least 1% sTILs. Notably, sTILs were found to be significantly reduced with advanced age, larger tumor size, more positive lymph nodes, and lower histological grade. In the multivariable Cox regression model, sTILs were an independent prognostic predictor for all endpoints; each 10% increment in sTILs corresponding to an invasive disease-free survival (DFS) hazard ratio (HR) of 0.87 (95% CI: 0.83–0.91), a distant DFS HR of 0.83 (95% CI: 0.79–0.88) and an overall survival (OS) HR of 0.84 (95% CI: 0.79–0.89) (p < 10−6)22. Histological grade was not a prognostic factor in this study. The second pooled analysis included women with primary BC treated with neoadjuvant chemotherapy (NACT) in six randomized trials conducted by the German Breast Cancer Group9. It assessed the predictive value of sTILs for chemotherapy response and prognostic estimation in patients with TNBC, HER2-positive, and luminal A/B–HER2-negative BC. sTILs quantified in diagnostic core biopsies from 3771 patients were associated with pathologic complete response (pCR) after NACT across all BC subgroups. For instance, based on three predefined groups of low , intermediate (11–59%), and high TILs (≥60%), pCR was achieved in 31% (80/260) of TNBC patients with low TILs, 31% (117/373) of TNBC patients with intermediate TILs, and 50% (136/273) of TNBC patients with high TILs (p < 0.0001). OS was analyzed in 2560 patients across all BC subtypes from five of the six neoadjuvant clinical trial cohorts. However, increased sTILs were associated with longer OS only in TNBC .In a pooled analysis (51 investigated the prognostic impact of sTILs in early-stage TNBC based on four multicenter cohorts (476 patients) who were not treated with (neo)adjuvant chemotherapy. The presence of sTILs at baseline was correlated with several clinical endpoints including OS. Multivariate analyses demonstrated that sTILs are an independent prognostic biomarker of OS (p = 0.015), invasive DFS and distant DFS for TNBC (p < 0.001 for both)51. A 10% increase in sTILs also positively correlated with OS , invasive DFS  and distant DFS . In a subgroup of TNBC patients with stage I tumors and sTILs ≥ 30%, excellent 5-year survival outcomes were reached including 98% 5-year OS51. Indeed, the expert opinion at the 16th St. Gallen International Breast Cancer Conference has endorsed the routine reporting of sTILs in TNBC as a prognostic factor52 although guidelines have not yet recommended de-escalation of standard systemic therapy according to TILs. The WHO Classification of Tumors: Breast Tumors, 5th Edition has also endorsed histopathological TIL quantification in TNBC and HER2-positive BCs, expressed as a mean percentage of lymphoplasmacytic infiltration of the tumor stroma53.Recently, Park et al.invasive breast carcinoma of no special type with medullary pattern53. These carcinomas have been characterized as showing high immune-related gene54 expression and it is likely that the high TILs component of these tumors contributes significantly to their favorable clinical outcomes56. The role of TILs in the histological spectrum of TNBC is yet to be fully elucidated although early data on metaplastic carcinoma suggest that TILs may have prognostic relevance58. The role of TILs in TNBC subtypes recognized as low grade and with good prognosis histological features, such as adenoid cystic carcinoma of the breast59, is at present unknown. The importance of histological grade in TNBC is likely to be of utility in identifying the so-called low-grade TNBC, including the rarer subtypes (for example adenoid cystic carcinoma), that have low TILs but an excellent outcome compared to the lack of clinical utility and prognostic significance of histological grading alone in TNBC NOS.The 5th Edition of the WHO classification also re-classifies medullary carcinoma, in which prominent TILs have long been recognized, into 60. This proof-of-concept study showed that patients with higher sTILs in both arms of the trial had significantly improved pCR rates (p < 0.01), although the sTILs were not specifically related to durvalumab-response. A recent translational analysis of this trial demonstrated that both continuous TMB and TILs were independent predictors of pCR61. Patients with high TMB had excellent pCR rates of 82% (95% CI: 60–95%) highlighting the potential of this emerging biomarker in tailoring therapy in this setting61. The German phase II trial (NCT03289819) compares neoadjuvant pembrolizumab combined with nab-paclitaxel vs. pembrolizumab with epirubicin and cyclophosphamide in patients with early TNBC will also investigate sTILs at baseline, in addition to other potential biomarkers such as mutational load and microbiota. In this setting, the recently released findings of IMpassion03162, KEYNOTE-52263, and I-SPY264 studies suggest that response to ICIs is independent of PD-L1 status. Thus, other biomarkers of response are needed to predict outcomes in TNBC patients treated with NACT and immune-checkpoint blockade.The phase 2 double-blind placebo-controlled GeparNuevo study (NCT02685059), randomized 174 early stage TNBC patients to receive durvalumab (a PD-L1 inhibitor) combined with standard NACT and used sTILs as a biomarker for patient stratification during randomization65, phase II KEYNOTE-086 (NCT02447003)67, and phase III IMpassion130 (NCT02425891)68 clinical trials. TILs were used to assess the activity of atezolizumab as monotherapy in a phase Ia expansion cohort in metastatic TNBC69. Patients’ stratification based on TILs at baseline indicated that median OS was improved in those with >10% of sTILs cut-off 69. In the phase III study, Schmid et al. showed that the addition of atezolizumab to nab-paclitaxel improved progression-free survival (PFS) in TNBC as compared with the addition of placebo to nab-paclitaxel, particularly in those with PD-L1 positive tumors. Median OS in this subgroup was consistently improved in both interim analyses70, but no formal statistical testing of OS in the PD-L1 positive subgroup could be performed due to the absence of OS benefit for the entire study population70. Hence, the FDA approval for this particular drug and assay was based on PFS, not on OS. The available tumor samples from this study were also re-assessed for the analytical concordance of various assays for PD-L1 immunohistochemistry (IHC) and the clinical utility of these assays71. This post-hoc analysis demonstrated that all subgroup results based on different PD-L1-based assays were suggestive of some clinical benefit , with different HR and low concordance between the immunohistochemical assays used (SP142 vs. 22C3 vs. SP263)72. This analysis also raises confusion as to whether HR or underpowered subgroup-analyses combining different antibodies, should be used to make claims on the performance of assays or not; taking into consideration that only a biomarker-treatment interaction analysis, in the biomarker positive vs biomarker negative population and only in powered subgroup-analysis, can inform the performance of assays. It was also shown that when >20% of sTILs were present, nearly all patients had a positive PD-L1 essay, irrespective of the assay used. This indicates that sTILs are important drivers of response and that sTILs could help mitigate the well-known assay- and reproducibility issues associated with PD-L1 assessment72. In fact, numerically higher counts of sTILs were noted in TNBC patients with a positive FDA-approved SP142 assay71. Furthermore, in the recent biomarker-analysis of Impassion13073, it was shown that TILs predict benefit to atezolizumab for any PD-L1-expression.In metastatic or locoregionally advanced TNBC, several immunotherapy trials have demonstrated the value of immune cell infiltrate in estimating survival outcomes including phase I (NCT01375842)66. Similarly, the cohort of the trial treated with first-line pembrolizumab displayed a durable response in patients with positive-PD-L1 67. These findings confirm the previously noted improved outcomes in this setting with higher TIL levels74. In fact, higher sTILs were associated with significantly increased ORR  and disease control rates . PD-L1 expression was also significantly correlated with the levels of sTILs (p < 0.001) in metastatic TNBC treated with pembrolizumab74.KEYNOTE-086 enrolled 170 patients with advanced TNBC who were treated with at least one prior line of therapy and showed a 5% response rate (RR) and 20% stable disease of the subgroup with PD-L1 positive tumors (based on the 22C3 pharmDx assay) when treated with pembrolizumab monotherapy13. This study did not demonstrate significantly prolonged OS in the overall cohort nor in the PD-L1-positive subgroup (combined positive score -CPS- ≥1 or ≥10)13. Exploratory analysis revealed that a marked increase in the efficacy of pembrolizumab was seen when the cut-off for a combined positive score was increased to ≥20. In this study, TILs were then evaluated according to our established guidelines23. In the pembrolizumab arm, high distribution of TILs was observed in responders with better survival outcomes, an effect not observed in the chemotherapy arm75. Since sTILs were not prespecified in the trial protocol, sTILs were not considered “regulatory-proof”, despite the OS benefit demonstrated in this phase 3 setting. TILs and combined positive scores were moderately correlated and were independent predictors of outcome in patients treated with pembrolizumab. More recently, KEYNOTE-355 (NCT02819518) randomized 847 women with metastatic TNBC to receive pembrolizumab in combination with chemotherapy vs. chemotherapy plus placebo76. This demonstrated that patients with CPS ≥10 treated in the immunotherapy arm had improved median PFS as compared to the placebo group  but not in those with CPS of 1 or more 76.A recent advance resulted from the KEYNOTE-119 phase III trial (NCT02555657) in which 622 previously treated metastatic TNBC patients were randomized to receive pembrolizumab as monotherapy or cytotoxic chemotherapyNevertheless, biomarker analyses of TILs in such studies are all exploratory. These findings should, therefore, ideally be validated in independent prospective studies that use suitably powered phase III randomized and controlled trial design to accelerate the clinical validation of TIL-guided therapy and to provide Level 1A evidence. We strongly propose pre-specifying the use of TILs as an integral biomarker in future trial protocols. However, the FDA has recently approved pembrolizumab for adults and children with TMB-H solid tumors. Treatment efficacy was studied in a prospectively planned retrospective analysis of 10 cohorts of patients with solid malignancies with unresectable or metastatic TMB-H tumors. These patients were enrolled in a multicenter, non-randomized, open-label clinical trial . This approval raises interesting questions, for example: is the old paradigm for obtaining level 1A evidence for biomarkers becoming obsolete or is it simply underused?. Are prospective-retrospective analyses for biomarkers that are predictive for immune therapy and indicate OS, with the level of evidence IB, and with proven analytical and clinical validity (like TILs) sufficient for regulatory approval as a predictive marker for selection of patients for immune checkpoint inhibition? Will level 1B evidence drive clinical change? Regulatory agencies approve assays and drugs, but the scientific community still must apply that knowledge in a clinical context based on a synthesis of all evidence. Is it the role of regulatory agencies to approve clinical practice changes or should this be the role of the scientific community, in a partnership with industry and the regulatory agencies?78. For example, the mean TILs count is significantly lower than in TNBC and HER2-positive BC80. The average TMB, which frequently correlates with neoantigen load, is also lower in these tumors81. Small studies of single-agent immune checkpoint blockade have yielded low responses in patients with pretreated metastatic ER-positive cancer83. Nevertheless, there is marked heterogeneity for TILs infiltration and mutational burden seen in ER-positive BCs, with a considerable proportion above the mean observed in triple-negative tumors77. The importance of TILs in ER+/HER2-ve BC can be most easily demonstrated by combining two aspects of BC; firstly, recent population-based surveys of over 350,000 BCs in the UK and USA suggest that 73% of newly diagnosed BCs are ER+/HER2−85. Given that TILs have been detected in the stroma of up to 60% of BC29 the majority  of all BCs with TILs must therefore be in the ER+/HER2- subgroup. Put simply, the population of ER+/HER2− BCs with TILs exceeds the combined population of HER2+/TNBCs with this feature. This demonstrates the large unmet potential for exploiting immune targeted agents in ER+/HER2− patients.Although a high percentage of sTILs in primary TNBC and HER2-positive BCs suggests a favorable prognosis, the significance in estrogen receptor (ER)-positive, HER2-negative tumors remains uncertain. This is partly because there has been less focus on ER-positive carcinomas, which are traditionally considered to demonstrate lower immunogenicity88. These studies retrospectively examined histological slides either from randomized controlled trials or annotated clinical cohorts. However, the material varied considerably (tissue microarray vs. core biopsy vs full face section) as did the TILs analysis methodology (H&E staining vs. IHC) and the TILs quantification statistical methods . With these caveats in mind, the most notable finding from these analyses is that no study demonstrated a favorable impact of TILs on disease-free, BC-specific, or OS in ER-positive BC. In some analyses, TILs were associated with an unfavorable prognosis. For example, in a recent meta-analysis of six NACT trials, higher TILs levels were associated with a significant reduction in OS9. Furthermore, two studies analyzing mixed trial and/or clinical cohorts  reported that higher levels of intratumoral CD8+ TILs were associated with a significant reduction in BC-specific survival86.Several studies have examined the impact of TIL levels on patient prognosis in primary ER-positive BC, and five of the largest studies are summarized in Table 189. Luminal A tumors have both lower TIL infiltration and TMB than luminal B tumors79 and luminal B cancers are associated with worse clinical outcomes90. In keeping with this, the significant associations between CD8+ TIL infiltration and worse prognosis in the studies described above were lost after adjusting for tumor grade. Similarly, the significant reduction in OS observed in the ER+/HER2− cancers in the BIG 02-98 analysis was not upheld in a multivariate analysis adjusting for other prognostic features78.These observations must be interpreted with caution given that the molecular subtype of ER+ BC is a major confounder in all these analyses. With the introduction of PAM-50 assays in the last decade, ER-positive cancers have been divided into luminal A and luminal B tumors—the latter characterized by higher proliferation and reduced dependence on endocrine signaling91. Nevertheless, ILC has been shown to harbor lower TIL levels when compared to IDC93, although gene expression profiling has indicated that the transcriptomic immune response signatures may in fact be upregulated in ILC94. High TIL ILC has been associated with poor prognostic factors and, to date, emergent data suggest that high TILs are suggestive of less favorable clinical outcomes in ILC96. Indeed, it has been suggested that high TILs may drive aromatase inhibitor resistance which may in part account for some of the ILC-TIL signal identified98. However, the role of TILs in ILC is still unsolved, and more and larger, preferably pooled studies are needed to define the importance of TILs in this specific subtype.ILC is the second most common histological subtype of BC and is frequently ER+/HER2−. Compared to IDC, fewer data are currently available on the immune microenvironment in ILC88. Patients with high CD8 infiltration in the tumor had a significant unfavorable prognosis; however, this effect was seen regardless of tamoxifen treatment. In addition, a study of 79 patients demonstrated a smaller reduction in tumor cell proliferation after two weeks of neoadjuvant aromatase inhibitor treatment in tumors with a significant lymphocytic infiltrate91. Another analysis from a prospective study of neoadjuvant letrozole ± lapatinib (n = 73) showed that significant Ki-67 reduction was observed in both patients with high and low baseline TILs, with high TILs tumors achieving more frequently a relative Ki-67 suppression >50% (55% vs. 35% of patients with low TILs)99. The long-term clinical significance of these observations remains unclear. There is also very limited data regarding the impact of targeted systemic therapy on TILs infiltration in ER-positive BC. Recently, CDK4/6 inhibitors have been reported to enhance antitumor immune responses associated with the upregulation of interferon-driven gene expression signatures100. However, this has not been associated with an increased TILs level in tumors101.The data on TIL levels and benefits from anti-estrogen endocrine therapy is even sparser. Sobral-Leite and colleagues evaluated CD8 expression in over 500 ER-positive BC patients who were randomized between no adjuvant endocrine treatments or one or three years of tamoxifen102 and should report data for luminal A and B tumors separately, In addition, a more refined understanding of the ER-positive BC immune environment, including the significance of immunosuppressive regulatory T cells, the spatial distribution of lymphocytes, and the role of estrogenic signaling will be required to define meaningful immune biomarkers in ER-positive disease105. Notably, a recent retrospective analysis (n = 563) of the multicenter IKA trial that randomized stage 1–3 ER-positive BC patients to receive tamoxifen or no adjuvant therapy showed that CD8-positive sTILs were significantly higher in patients with PIK3CA-mutated tumors 88. In addition, this population of BC patients had an increased risk of recurrence  on multivariate analysis as compared to those with CD4 and FOXP3 sTILs that were not statistically associated with prognostic outcomes88. This enrichment of TILs in tumors with mutated PIK3CAand their association with outcomes provided additional evidence on the crosstalk between mutational status and TME.At present, there is no role for routine assessment of TILs in primary ER-positive BC, and their presence cannot be used to guide prognosis nor as a predictive biomarker. We propose that new studies should take a uniform approach to the assessment of TILs, such as those outlined in recent guidelines107. Moreover, improved event-free survival in primary disease treated with trastuzumab and lapatinib was also noticed107. The predictive and prognostic value of TILs as a continuous variable in HER2-positive BC in the neoadjuvant setting was demonstrated in another analysis (n = 498) that evaluated TILs from two trials (GeparQuattro and GeparQuinto)108. In the group of patients with lymphocyte-predominant (≥60% sTILs) phenotype, a marked increase of pCR rates was noted108. Moreover, an increment of TILs by 10% was found to be an independent predictor of pCR in multivariate analysis (p = 0.014)108. Notably, this effect was more relevant in patients with ER+, PR+, and HER2+ tumors108. In the N9831 phase III trial (NCT00005970), tumor samples from patients with early HER2-positive BC were collected at baseline for TILs quantification in deciles with a prespecified categorical cutoff of ≥60%109. TILs were associated with recurrence-free survival in the cohort treated in the control arm with chemotherapy alone. Unexpectedly, higher TILs predicted a lack of response to trastuzumab in HER2 positive BC patients109. However, an immune gene-expression analysis on the same dataset did show a strong association with benefit110. A recent meta-analysis of 5 randomized and controlled trials that pooled data from 1256 patients provided additional evidence of the prognostic impact of TILs in the neoadjuvant setting111. Higher TILs at baseline were predictive of pCR irrespective of the neoadjuvant treatments used in HER2 positive BC111.Patients with HER2-positive early BC have a higher infiltration of TILs and improved pCR with NACT and trastuzumabp = 0.006)112. At five years of follow-up, distant DFS rates were significantly higher in patients with TILs ≥ 20% (p = 0.025)112. Importantly, the 10% TIL increments were significantly associated with distant DFS only in patients randomized to receive nine weeks trastuzumab-based regimen 112.Recently, supporting evidence of the association of sTILs with distant DFS in this setting has emerged from the Short-HER phase III randomized study that compared different adjuvant regimens combined with trastuzumab (9 weeks vs. 12 months) (NCT00629278). On multivariate analysis, TILs predicted improved distant DFS for each 10% increment 47. The association between TILs and prognosis in the advanced HER2-positive setting is less well established. A recent analysis of the pre-treatment samples from the CLEOPATRA study population revealed that higher quantities of TILs predicted an improved survival benefit in first-line treatment with taxane-based chemotherapy, trastuzumab, and pertuzumab113. Conversely, analysis of the samples from MA.31, a randomized clinical trial of lapatinib or trastuzumab in combination with taxane-based chemotherapy, reported that 35% of the population were categorized as high TIL but that this did not show significant prognostic or predictive effects114. It is worthwhile noting that the cutoff used for high TILs was 5%, much lower than the 20% cutoff used in the binary component of the CLEOPATRA analysis. MA.31 assessed TILs also on the primary tumor samples and found a mean of 9.2%; median, 5%; IQR, 1–10%. Provision of tumor tissue from a metastatic site pre and post-study treatment was an optional subsidy with low uptake—hence metastatic samples were not included in the TIL analysis. Conversely, the CLEOPATRA TILs analysis used samples of either the primary (93%) or metastatic (7%) sites and found the mean stromal TILs value was 21.07% and the median stromal TILs value was 10% (IQR 5–30). Although only a small number of metastatic samples were analyzed, the TILs values of samples from lung and lymph nodes were significantly higher than those from primary tumor, liver, bone, or skin. Furthermore, in the PANACEA-trial115, combined TILs and PD-L1 predicted benefit for pembrolizumab combined with trastuzumab in trastuzumab-resistant advanced HER2 positive BC. Whilst the early TNBC studies used a similar cutoff for high TIL groups, more recent analyses using TIL as a continuous variable has shown a linear association with survival outcomes. The ideal threshold for high TILs remains unclear in advanced HER2-positive disease, but variation in cut-offs may account for the conflicting results seen thus far114.In the National Surgical Adjuvant Breast and Bowel Project B-31 stromal TILs were also assessedTILs Working Group does not currently recommend the use of sTILs in isolation to guide treatment decisions in clinical practice. However, sTILs could be used to help identify patients with an excellent outcome to study de-escalation approaches and thereby reduce toxicity. In addition, based on the data of Luen et al., escalation of treatment regimens may be clinically beneficial for TNBC patients with a high post-neoadjuvant residual disease burden and low sTILs due to the poor prognosis116. Along those lines, the retrospective evaluation of sTILs in trials that have evaluated adjuvant treatments for those patients who did not reach a pCR, such as the CREATE-X trial—is planned117. In practice, some clinicians are using TILs in conjunction with other clinical variables to decide on the type and duration of cytotoxic chemotherapy. It may be emphasized that for most of the prognostic factors currently used for decades in BC daily practices, there is no level of 1A-evidence. To achieve clinical utility in TNBC, TILs must be included as pre-specified biomarkers in clinical trials and/or prospectively/retrospectively evaluated in randomized trials.It should be emphasized that formally speaking, the clinical utility of sTILs is not proven since no prospectively designed phase 3 TILs-driven trials have yet been performed, and evidence is lacking that treatment decisions based on sTILs favorably affect patient outcomes. Therefore, the 118. A total of 67 patients with metastatic TNBC were randomized to nivolumab (anti-PD-1) without induction or to one of four induction treatments, consisting of irradiation of one metastatic lesion (3 × 8 Gy) or a 2-week low-dose regimen of cyclophosphamide, cisplatin, or doxorubicin, all followed by nivolumab (NCT02499367)118. Concordance values between four pathologists were >90% in this trial, showing the applicability of this concept in real-world trial-settings119. The added value of web based-tools is that the biomarker scores of local pathologists can be integrated into the flow of a clinical trial, thereby enhancing local pathologists’ compliance and experience within a trial, bridging trial- with daily pathology practices. This is in contrast to the current situation, where the tissue may be sent to a central laboratory with limited to no involvement of local pathologists. Is the current trial-paradigm of “single-reference laboratory-approach” therefore in need of revision?To successfully integrate TILs as a stratification factor in clinical trials, it is necessary to develop digital pathology tools to ensure accurate and rapid sTILs scoring by independent pathologists. Web-based platforms that allow easy scanning, viewing, and scoring of H&E slides should be optimized. A risk-based framework, aiming to reduce variability in TIL assessment within a clinical trial was used in the first stage of an adaptive non-comparative phase II trial 71 is still debated, illustrating the complexity and confusion that exists on these assays in the scientific community. In addition, the TILs Working Group, in a partnership with the College of American Pathologists, the European Society of Pathology, the Latin American Society of Pathology, and the European Working Group of Breast Screening Pathology argued how the current assay-approval policies, exemplified by the current PD-L1-assay situation, are leading to (unintended) imprecision medicine, which is not in the best interest of our patients. Hence, this pathology partnership proposed concrete solutions to improve the current assay approval pathway120. A framework for better evaluation of risks associated with suboptimal patient selection and stratification for ICI-based treatments in future clinical trials and real-world settings based on TILs/PD-L1 is proposed by the TILs Working Group72. The use of TILs as a predictive biomarker of response to ICI’s may considerably support cancer care in countries that have difficulty with PD-L1 implementation, mainly related to costs. Many ongoing clinical trials will report data that are expected to support TILs as a valid biomarker for response  does not have plausible potential to be cost-effective in advanced TNBC. Nevertheless, PD-L1 testing and atezolizumab can be offered to patients with advanced TNBC. In Sweden, TILs have been incorporated in the Swedish Breast Cancer Guidelines as of June 2020121. Based on the results of Impassion 131, the New Therapies Council (NT-Rådet) does not recommend the use of atezolizumab in combination with paclitaxel or nab-paclitaxel for the treatment of advanced TNBC. The implementation of PD-L1 testing (SP142 assay) in Denmark has been hampered by the lack of analytical validity and reproducibility of immunostaining. Consequently, PD-L1 IHC is not in widespread use. In addition, the process concerning approval of atezolizumab for metastatic TNBC is ongoing in the Danish Medicines Council. TILs are included in the national Danish Breast Cancer Cooperative Group (DBCG) pathology guidelines. In Romania, PD-L1 testing is not routinely used. The testing of patients is made primarily by private pathology laboratories on request. National Health Insurance does not reimburse the bill for testing, so patients can enroll in a clinical trial or get free testing from the Pharmaceutical Industry accessing special testing programs. Atezolizumab is currently not approved for BC in Romania. Atezolizumab is approved and reimbursed in Italy for the treatment of patients with locally advanced or metastatic TNBC, and the SP142 assay has been identified as a companion diagnostic.In the United Kingdom (UK), the National Institute for Health and Care Excellence (NICE) reported that atezolizumab combined with nab-paclitaxel . In Brazil, some central pathology laboratories perform PD-L1 testing based on principal investigator-sponsored agreements. PD-L1 IHC is tested mostly in private institutions with some programs offering pro bono testing to assess eligibility for approved ICI. The Brazilian Pathology Society follows WHO recommendations; hence the inclusion of TILs in daily practice can be envisaged in the near future. In Chile, PD-L1 testing is available on several platforms but their cost is high and this is not covered by public health insurance which covers 75% of the population. Anti-PD-L1 drugs remain at a high cost and are usually only funded by additional insurance or in clinical trial settings. In Argentina, PD-L1 testing is not covered by public funds. A few central private pathology laboratories perform PD-L1 testing on-demand funded by industry. Some pathologists have started to incorporate TILs into pathology reports for TNBC; however, there are no current national guidelines. In Perú, TILs evaluation has been included in TNBC protocols by pathologists in public and private centers. SP142 IHC is available in a few private labs while Dako 22C3 is being processed for non-breast malignancies in private and public centers. There are a few BC patients who have received atezolizumab funded via private insurance or clinical trials. It is not expected that the public system will support anti-PD-1 drugs in the near future.In the United States, the PD-L1 SP142 assay has been approved as a companion assay for the selection of patients with TNBC to offer atezolizumab and taxane chemotherapy . OptiView PD-L1 (SP142) on the Ventana platform was approved in Japan as the companion diagnostic for metastatic TNBC treatment with atezolizumab. Other antibodies including 22C3, 28-8, and SP236 or OptiView PD-L1 (SP-142) on other platforms are un-funded. Pathology laboratories can use any PD-L1 assay for clinical research but only PD-L1-testing using SP142 on the Ventana platform companion diagnostic is permitted for funded treatment with atezolizumab in clinical practice. Many Japanese pathologists have limited clinical experience interpreting PD-L1 staining with no current national guidelines. The Japanese Society of Pathology does not provide oversight for quality control, which leaves this mostly to the pharmaceutical industry and individual laboratories. In Taiwan, PD-L1 assays are obligatory when considering immune checkpoint inhibition for BC. The fee for a PD-L1 assay is very high and the assay is not covered by the national health insurance and has to be paid at the patients’ own expense. The PD-L1 essay is only available in medical centers and some large hospitals.In India, PD-L1 and TIL assessments are not being performed routinely with significant discrepancies of results due to the different clones available to date. PD-L1 reporting is assessed on the platform as recommended by the standard reporting guidelines. TILs reporting is performed simultaneously with PD-L1 testing. Insurance companies do not offer reimbursement for PD-L1 testing. In April 2020, atezolizumab in combination with nab-paclitaxel received Drug Controller General of India (DCGI) approval as a first-line treatment for metastatic TNBC patients. This may pave the way for its use in selected patients if affordable  pertaining to PD-L1 testing. There is a considerable difference between the costing of the standard PD-L1-antibodies which are for most patients unaffordable. The high cost of PD-L1 assays in LMIC can be mitigated by selecting patients using the sTILs on H&E-slides to include cases for further analysis. Furthermore, as argued in detail beforewww.tilsinbreastcancer.org, “NEW PD-L1 Help Desk) to help oncologists and pathologists select and interpret appropriate anti-PD-1/PD-L1 therapies and associated companion diagnostics. This will be updated regularly to reflect the most current standards for eligibility for ICI for all solid tumors.The International Immuno-Oncology Biomarker Working Group supports efforts to advance knowledge and best practices for immune-based therapies in all solid tumors. In this regard, the TIL Working Group has created an online tool  much of our data exist in unstructured reports that future clinicians do not access, either because they do not know it exists or they can’t find it amidst the hundreds of files that we generate. If patients had a checklist of markers that were important to their disease, we could help ensure evidence-based treatment plans. Patients suffer unnecessarily; it is stressful enough to face your own mortality but to trawl social media comparing your treatment to that of strangers creates more stress. Patient-ownership of pathology reports would help remove fears of age-bias and variation in care, and furthermore could help generate complete datasets for audits and real-world evidence studies.We know that cancer is an ecosystem of cancer cells and the stromal cells between them. We know the well-defined markers for the cancer cells that direct chemotherapy and hormone therapy. What markers do we have for the intra-tumor stroma? PD-L1 SP142 is an expensive assay and notoriously difficult to achieve consensus on, but nevertheless if you ever once had PD-L1 recorded for any one of your TNBC primary tumors or metastases, you are eligible for one of the latest drugs, atezolizumab. Would it be wise therefore for all TNBC patients to request PD-L1 testing of their primaries for the event that their metastases can’t be biopsied? Shouldn’t we be seeking an easier and cheaper marker  to understand the stroma and direct treatment? I still feel very lucky that my TILs were scored in my pathology report. They have given me a more positive outlook and this alone is a reason for scoring them. I’m also very aware (through atezolizumab’s licensing) that details on my pathology report today might be my gateway to a life-prolonging drug tomorrow.”TILs are an inexpensive, robust prognostic biomarker that represents a surrogate for anti-tumor T cell-mediated immunity. Incorporating TILs into standard clinical practice should be strongly considered in both early and advanced TNBC and HER2-positive BC. TILs assessment at the time of diagnosis may enable a clinician to assess prognosis and in future inform therapeutic decision-making more accurately, is informative for predictive purposes, can help to interpret PD-L1 assays, and, certainly in LMIC, may be considered as a screening tool before embarking on expensive immune-assays, being PD-L1 or others. Further information on the clinical utility of TILs in HR positive BC is needed to identify their role in this subtype of the disease.Education and standardization of testing across the pathology community by providing centralized training and educational tools are required to up-skill clinicians to utilize this new biomarker. The TIL-WG host’s pathologists from academic hospitals, community hospitals, and industry, supported by expert clinicians and statisticians, incorporating the view of patients also. We encourage transparent and efficient communication with the regulatory authorities. This collaboration of experts and patients is imperative to guide the development of this new biomarker and optimize its role across academia, industry, and the clinical setting. Looking to the future, a collaboration between the oncology and pathology communities across countries and continents is integral to define how best TILs can be integrated into a multivariate prognostic model with standard variables such as age, tumor size, and nodal status to optimize outcomes of patients with BC. In addition, trials being developed using baseline, on-treatment, and post-treatment TILs may improve our understanding of the complex interaction between host immunity and the TME and will improve our approach to fit as best as possible our patient’s needs.


Kidney transplantation recipients (KTR) with coronavirus disease 2019 (COVID-19) are at higher risk of death than general population. However, mortality risk factors in KTR are still not clearly identified. Our objective was to systematically analyze published evidence for risk factors associated with mortality in COVID-19 KTR. Electronic databases were searched for eligible studies on 1 August 2021. All prospective and retrospective studies of COVID-19 in KTR were considered eligible without language restriction. Since data in case reports and series could potentially be subsets of larger studies, only studies with ≥ 50 patients were included. Random-effects model meta-analysis was used to calculate weighted mean difference (WMD) and pooled odds ratio (OR) of factors associated with mortality. From a total 1,137 articles retrieved, 13 were included in the systematic review and meta-analysis comprising 4,440 KTR. Compared with survivors, non-survivors were significantly older . KTR of deceased donor were at higher risk of death . Comorbidities including diabetes mellitus, cardiovascular disease, and active cancer significantly increased mortality risk. KTR with dyspnea  and pneumonia  at presentation were at higher mortality risk, while diarrhea decreased the risk . Acute kidney injury was associated with mortality . Inflammatory markers were significantly higher in the non-survivors, including C-reactive protein, procalcitonin, and interleukine-6. A number of COVID-19 mortality risk factors were identified from KTR patient characteristics, presenting symptoms, and laboratory investigations. KTR with these risk factors should receive more intensive monitoring and early therapeutic interventions to optimize health outcomes. Immunocompromised patients, particularly solid organ transplantation recipients and those with malignancies, are also at increased risk of severe COVID-19 disease and death3.Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Elderly patients and patients with multiple comorbidities are known to be at higher risk of death7, compared with 10–15% mortality rate amongst admitted patients overall10. The immunosuppressed status in kidney transplantation recipients (KTR) might contribute to the higher mortality rate. Empirical stepwise reductions in immunosuppressive therapy have been recommended in patients at high risk of developing severe disease, and as clinical severity of COVID-19 symptoms increases12. However, temporary reductions in immunosuppression might place these patients at risk for allograft rejection thereafter. More data from clinical studies are urgently needed regarding the management of COVID-19 in KTR, including patient selection criteria for immunosuppressive lowering strategies. Patients with higher mortality risk should be treated more aggressively compared to patients with a lower risk. Moreover, many transplantation programs have been halted during the COVID-19 pandemic. Apart from the strain on hospital facilities by general COVID-19 patients, concerns have been raised regarding donor-derived COVID-19 infection in recipients who might need relatively intensified immunosuppression in the perioperative period, although there are currently no reports of such cases15. Induction therapy might also increase the risk of acquiring COVID-19 in the early post-transplantation period16. A clinical tool that identifies patients who are more likely to have a good prognosis of COVID-19 with minimal clinical symptoms after transplantation, might help transplant programs to continue performing kidney transplantation in these low risk patients.The mortality rate of COVID-19 In kidney transplantation patients was 20–40%To date, many case reports, case series, and cohort studies of COVID-19 in KTR have been published. However, the clinical risk factors for mortality in KTR with COVID-19 infection are still unclear due to many scattered case reports, and inconsistent reporting with varying quality across larger studies. The objective of this systematic review and meta-analysis was to clarify risk factors for mortality in KTR with COVID-19 infection, to improve of quality of care during the ongoing COVID-19 pandemic.17. MEDLINE, Scopus, and Cochrane Central Register of Controlled Trials electronic databases were searched for eligible studies on 1 August 2021. The following search strategy was used for MEDLINE: ("Kidney Transplantation"[Mesh]) AND ("COVID-19"[Mesh] OR "SARS-CoV-2"[Mesh]), and the search terms in Scopus were (TITLE-ABS-KEY (COVID-19) AND TITLE-ABS-KEY (kidney AND transplantation)). The MeSH descriptors which exploded all trees of [Coronavirus] and [Kidney Transplantation] were applied in the Cochrane Central register of Controlled Trials. The reference lists in the qualified articles were also reviewed and studies were manually added if deemed appropriate.This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA)18. Studies with ≥ 50 patients were excluded if they were subsets of other larger studies, based on the study site, start and end date, or if clearly mentioned in the larger studies. If a potentially duplicated population was presented in 2 large studies, the study reporting more information regarding survivors and non-survivors was selected as the main data source. Only studies with adequate information, in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement, were included in the review19. Two authors (S.U. and S.K.) independently screened the titles and abstracts of the electronic citations, and full-text articles were retrieved for comprehensive review. Disagreements were resolved through consensus and arbitration by a third author (N.T.).This systematic review and meta-analysis focused on risk factors contributing to mortality in COVID-19 KTR. Our inclusion criteria were studies of COVID-19 in KTR that included ≥ 50 patients, that reported numbers of survivors and non-survivors, and demographic and/or clinical characteristics by survival group. Baseline demographic characteristics, clinical presentation, laboratory investigations, and treatments related to transplantation and COVID-19 care were extracted separately for survivors and non-survivors. As single case reports and small case series could be subsets of larger clinical registries, we selected only studies with ≥ 50 KTR patients for our review. In addition, meta-analysis of studies that include studies with a small sample size are at risk for bias caused by sampling error and random variation21. The tool evaluates 3 domains which are selection, comparability, and outcome. Each domain is rated total scores of 4 in the selection domain, 2 in the comparability domain, and 3 in the outcome domain.The following information was extracted from each study: author names, publication date, journal title, study site, country of origin, study duration, total KTR included, and the number of COVID-19 survivors and non-survivors. Baseline patient characteristics, clinical presentations, laboratory investigations, and treatments were retrieved separately for each study, and grouped by survival group. The Newcastle–Ottawa scale was used for the quality assessment of each individual included study22, if only the median and range, or interquartile range were provided in the study. Pooled OR were calculated using the logarithm of effect size and standard error from each study. Heterogeneity of pooled effect sizes was assessed using the I2 index and the Q-test p-value. An I2 index higher than 75% indicates medium to high heterogeneity. Even when heterogeneity was low or absent, we reported random effects over fixed effects models because clinical care may have differed by different sites, and care practices likely changed as physicians gained more experience in caring for COVID-19 patients. Regression-based Egger’s test was used to test for small-study effects. The mortality change rate over the study period was calculated by regressing the proportion of deaths against the study end dates, using a generalized linear model with a binomial family and logit link functions, and robust variance estimates, after weighting by study size23. The analyses were performed using Stata Statistical Software Release 16.1 .Using data reported in each individual study, we used random-effects models to calculate pooled weighted mean differences (WMD) of continuous variables, and pooled odds ratio (OR) for binary variables, for non-survivors versus survivors. Mean and standard deviations (SD) were estimated by the method of Wan et al.This meta-analysis and systematic review did not directly obtain data from human or animal subject. All of the included studies’ information was published in the scientific journals without the possibility to identify the individual patients. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the 'Declaration of Istanbul on Organ Trafficking and Transplant Tourism'.36. Details of each study are displayed in Table Figure I2 0%; Q-test p-value = 0.46). KTR with co-existing medical comorbidities were at higher risk for death, including diabetes mellitus , cardiovascular diseases , and active cancer . Patients who received kidney allografts from deceased donors had 1.73-fold higher odds of mortality .Pooled estimates of patient characteristics associated with morality, with the numbers of studies and patients reporting data are displayed in Table I2 84.6%; Q-test p-value < 0.001) and 10.64-fold  higher risk of death, respectively. Acute kidney injury also significantly increased mortality risk . However, diarrhea significantly lowered the risk with OR of 0.61 . Supplementary Figure For presenting COVID-19 symptoms, patients with dyspnea and pneumonia were at 5.68-fold . Other standard laboratory investigations did not show the association with COVID-19 mortality.Table I2 88.9%; Q-test p-value < 0.001), procalcitonin , and IL-6 . Supplementary Figure Details of biomarkers reflecting tissue damage and inflammation at baseline were reported in 3–5 studies representing 473–830 patients in Table I2 93.1%; Q-test p-value < 0.001). Significantly higher proportions of non-survivors than survivors were treated with hydroxychloroquine, steroids, antibiotics, tocilizumab, and convalescent plasma. Antiviral drugs including lopinavir, remdesivir, darunavir, and favipiravir were given more frequently to non-survivors . The forest plot of each significantly associated variable is illustrated in Supplementary Figure Table 36. Statistical methods to analyze multivariable model were different between studies including logistic regression and Cox proportional hazard regression. Thus, the combined or pooled effect sizes of each variable was not executed. The most common variables significantly contributing to mortality after adjustment were age in 8 studies, followed by dyspnea or respiratory rate (5 studies) and renal function (5 studies), and the presence of cardiovascular disease in 3 studies.Ten of 13 studies presented adjusted (multivariable) models for factors associated with mortality Table 24–26, 29The mortality percentages from all 13 studies included in this meta-analysis were plotted against the study end date, with the back-transformed predicted slope from the generalized linear model in Fig. 41, which predisposes patients to an increased risk for severe COVID-19. Non-survivors presented with more dyspnea, pneumonia, and acute kidney injury.This systematic review and meta-analysis are the first to describe and quantitate the degree of risk for factors associated with mortality in COVID-19 KTR from large cohorts and clinical registries. The results show that a number of baseline patient characteristics increased the risk of patient death, including increasing age, medical comorbidities, and being recipients of deceased donor kidneys. The latter likely relates to inferior allograft function relative to the recipients of living donors34. The I2 index of the gastrointestinal symptoms was 0% indicating very low heterogeneity. Diarrhea in COVID-19 patients is proposed to result from direct infection of COVID-19 in the intestinal epithelium cells, via angiotensin-converting enzyme 2 (ACE2) receptors that further increase local proinflammatory cytokines and changes in the intestinal flora43. However, association with lower mortality in patients with diarrhea needs further study to assess whether this association is due to more rapid viral clearance through the gastrointestinal tract, or the stimulation of specific immune cells in the intestinal immune-network.Interestingly, gastrointestinal symptoms significantly more frequent in the survivors. This finding is concordant across studies from France, Spain, the European registry, India, and the international cohort45. Surprisingly, obesity was not associated with mortality in our meta-analysis of KTR, and the studies reporting on this risk factor had low heterogeneity demonstrated by the I2 index of 0% in Table 33. In addition, no multivariable models in Table 46. It is possible that in KTR, immunosuppression or previous exposure to uremic status, might override or interfere with the effect of ectopic fat deposition that also leads to higher mortality of COVID-19 in KTR compared with the general population48. However, further studies are needed to evaluate this hypothesis.Obesity is an established risk factor for COVID-19 mortality in the general population55. Although there is no evidence that interventions decrease mortality rates, some evidence suggests that treatments including remdesivir, convalescent plasma therapy, and tocilizumab can attenuate the clinical course in COVID-19 patients60. Dexamethasone is the only medication proven to lower 28-day mortality in COVID-19 patients who received respiratory support61. The results from this meta-analysis supports that the non-survivors, compared with survivors, exhibit higher inflammatory states, demonstrated from the significantly higher levels of C-reactive protein, pro-calcitonin, and IL-6. This inflammation cannot be explained from the virus itself, and is thought to result from dysregulation of the host immune response leading to a “cytokine storm” and multiple organ dysfunction64. The clinical and laboratory results from this study could help identify the COVID-19 KTR with ongoing inflammation who are at risk for multiple organ dysfunction and death, and these patients might be candidates for anti-inflammatory immunomodulator agents. For example, case reports and case series have demonstrated the benefit of tocilizumab as a treatment of COVID-19 KTR68. More favorable treatment responses might be achieved if IL-6 levels and/or other biomarkers are used as an inclusion criterion for receiving tocilizumab in future RCTs.To date, many large randomized controlled trials (RCT) have failed to demonstrate a benefit of treatment interventions in lowering COVID-19 mortality69.It is important not to misinterpret the associations between treatment effects and mortality found in this meta-analysis. None of the included studies were RCTs specifically designed to evaluate treatment outcomes, the pooled univariable OR presented in the results section do not represent causal relationships, since they are confounded by disease severity, with patients experiencing more severe symptoms more likely to receive aggressive treatment interventions. The bubble plot in Fig. 2 compared with 44.4 mL/min/1.73 m2 in the survivor group). In addition, elevated concentrations of C-reactive protein, procalcitonin, and IL-6 at presentation should flag the patient as having an increased risk of death. In-hospital treatment with more aggressive and earlier immunosuppression reduction strategies would be reasonable in these high-risk patients, who also might be appropriate targets for the future studies of novel therapeutic interventions. Patients without any of these high-risk features could be closely monitored without significant changes in their immunosuppression. This strategy should be tested in future cohorts or clinical trials. Given the slow role out of vaccinations in many countries, patients at high risk of mortality should be prioritized to receive vaccinations. In addition, transplant centers could resume kidney transplantation programs in potential recipients who are at low COVID-19 mortality risk, based on an assessment of risks in baseline patient characteristics, accompanied by proper decision-making systems71.The information from this systematic review and meta-analysis could be used in many ways. Clinician could identify patient characteristics suggesting a poor prognosis, and begin early aggressive monitoring and treatment. According to our results, KTR at higher risk of death included the elderly (mean age of the non-survivors was 67.5 years compared with 54.9 years in the survivor group), patients with diabetes mellitus or cardiovascular diseases, patients with active malignancy, history of deceased donor kidney transplantation, dyspneic patients, presence of pneumonia, and patients with acute kidney injury on initial presentation, or with low eGFR (mean eGFR of the non-survivors was 34.4 mL/min/1.73 m25. However, we carefully selected only large studies with ≥ 50 patients, and thoroughly reviewed all article characteristics to remove studies that were subsets of others, thereby minimizing the chance of data duplication. Using this method, 5 studies were excluded as shown in Fig. 72, therefore the viral load is likely to be another important factor predicting mortality risk in COVID-19 KTR. Third, the included studies were mainly from the European countries and the USA, with only limited data from Asian countries. Fourth, Egger’s test is not reliable for assessing publication bias when small study numbers are involved. However, given the interest of the medical community in reporting studies about COVID-19, we think that the risk of publication bias is mitigated. Lastly, the complications of COVID-19 contributing to patient death, such as superimposed bacterial infection and thromboembolic phenomenon, were not adequately reported in the included studies74. More robust reporting of these complications would raise the awareness amongst clinicians regarding these potentially fatal complications.There are some limitations in our study. First, the possibility of duplicated information in individual patient level data could not be completely eliminated across our included studies, particularly international studyIn conclusion, mortality risk of COVID-19 KTR was increased in older patients, with medical comorbidities and deceased donor kidney recipients. Those with acute kidney injury, dyspnea, pneumonia, and increased of inflammatory biomarkers also had an increased risk of dying. Gastrointestinal tract symptoms were associated with lower risk of death. These risk factors could be used for developing clinical scores to further improve the quality of care in COVID-19 kidney transplant patients.Supplementary Information.