Mobile instant messaging could deliver real-time, personalized, interactive smoking cessation support. Nicotine replacement therapy (NRT) is effective in increasing quit attempts and abstinence but is underused. We assessed the feasibility of mobile chat-based intervention combined NRT sampling (NRT-S) on abstinence.In this two-arm, single-blinded, randomized controlled trial, adult (≥18 years) daily cigarette smokers were proactively recruited from Hong Kong community settings using ‘foot-in-the-door’ approach during December 2017 to March 2018. All participants received brief advice on quitting, 1-week of NRT-S, active referral to smoking cessation services, and were individually randomized (1:1) at baseline. The intervention group received two months of chat-based support via instant messaging. The control group received general smoking cessation text messages. The primary outcome was smoking abstinence validated by exhaled carbon monoxide (<4 ppm) and salivary cotinine (<10 ng/mL) at 3 and 6 months using intention-to-treat analysis.A total of 119 participants  were randomized and analyzed. Among the 14 and 13 self-reported quitters at 3 and 6 months respectively, only 3 and 1 had biochemical validation. The 3 months validated abstinence rate was 2/62 (intervention) vs 1/57 (control) . At 6 months follow-up (68.9% of participants retained), more participants in the intervention group reported quitting , smoking reduction , and quit attempts . Significantly more NRT-S use  was observed in the intervention group. Participants engaged in mobile chat support (21/62) reported more NRT-S use (76.2% vs 56.1%), although not statistically significant.Mobile chat-based support plus NRT-S was feasible and showed preliminary evidence of increased quitting, smoking reduction, quit attempts, and NRT-S use in proactively recruited community smokers.ClinicalTrials.gov: NCT03574077. Further reduction is challenging in many countries as most remaining smokers have low intention to quit and low use of smoking cessation (SC) services2. Although SC services are effective, most (96.8%) smokers try to stop smoking without any assistance2. More proactive recruitment and treatments are needed. Our previous trials showed that SC treatments were effective in promoting abstinence and preventing relapse4. Most trials, including ours5, proactively recruited smokers and delivery of interventions such as counselling, brief advice and active referral to SC services relied on limited contacts and follow-up9.Smoking is declining worldwide but remains a threat to public health10. A 2019 Cochrane review of 13 studies (14133 participants) concluded that mobile phone-based automated text messaging via short messaging services (SMS) increased long-term (6 months from baseline) quit rate by about 54% compared with minimal SC support (9% vs 6%)11. Mobile instant messaging apps (e.g. WhatsApp and WeChat) have become popular because they are cheaper and more attractive than SMS12. Our formative qualitative study, which interviewed 21 smokers in 5 focus groups, supports the acceptability and feasibility of mobile instant messaging for chatbased SC support13. Our recent randomized controlled trial (RCT) on 1185 community-based smokers has shown that combined 3 months of chat-based support with brief advice intervention  increased validated smoking abstinence  and SC service use  at 6 months follow-up14. In a cluster RCT of 136 recent quitters, we found fewer relapse among participants joining counsellor-moderated WhatsApp groups discussion than usual care 15. The reduction of relapse is probably due to the enhanced social support15.Recent trials have shown the beneficial effects of mHealth on SC16 but the use of and adherence to the 12-week full course is suboptimal2 partly due to misinformation on risks and benefits, and lack of behavioral support17. NRT has been misperceived to be as dangerous as cigarettes and not efficacious18. NRT prescription with counselling and side-effect monitoring is a routine treatment for SC service users who have planned to quit19 but rarely provided to community smokers who are mostly not ready to quit. However, a substantial proportion of successful cessation was from unplanned quit attempts, and most of these unplanned attempts are unsupported21. Effective methods to provide behavioral and pharmacological support for these attempts are needed21.Nicotine replacement therapy (NRT) is an effective pharmacological support for quitting and clinical guidelines recommend that NRT should routinely be used to support quitting attempts23. Our recent feasibility trial, which offered 1 week of NRT-S and brief advice  to community smokers, did not achieve effective quitting outcomes  at 6 months follow-up but the results suggest that sustainable post-recruitment support should be added24. Since our previous non-pharmacological chatbased intervention increased quitting by providing social support14, the use of NRT-S for promoting quit attempts might also be enhanced by personalized mobile chat-based support via instant messaging (IM) to quickly respond to smokers’ questions and concerns. Whether chat support combined with NRT-S would increase quitting among community smokers is unknown and warrants investigation.Previous RCTs found NRT sampling (NRT-S) was effective in increasing quit intention, motivation, confidence, quit attempts and duration of abstinenceNo similar trials evaluating the effect of the mobile chat-based support plus NRT-S on quitting are found in PubMed and trial registries . We examined the feasibility of offering the mobile chat-based support plus NRT-S integrated with brief cessation advice, and active referral to SC services on quitting in proactively recruited community smokers in Hong Kong. The preliminary effects of chat-based support (vs SMS control) on quit attempts and NRT-S use were also tested.25, SC ambassadors proactively approached smokers at the smoking hotspots and asked about their smoking behaviors , assessed their exhaled carbon monoxide level, and invited them to join the study to quit or reduce smoking. Interested smokers were then screened for eligibility for participation. Inclusion criteria were: 1) being a Hong Kong resident aged ≥18 years; 2) having smoked ≥1 cigarette daily in the past 3 months, validated by an exhaled carbon monoxide level of ≥4 ppm26 (using a Smokerlyzer); 3) having a smartphone; and 4) having an instant messaging app (e.g. WhatsApp). Individuals who were using SC medication or other SC services, had any contraindication for NRT  or were physically or mentally unable to communicate in Cantonese or Putonghua, were excluded. This two-arm pilot randomized controlled trial proactively recruited community smokers from smoking hotspots in Hong Kong, where ashtrays were available and smokers gathered to smoke, including vicinities of public transport stations, housing estates, and outside shopping malls. University students were trained as SC ambassadors to approach and recruit smokers. Using a ‘foot-in-the-door’ approachhttps://www.sealedenvelope.com/simplerandomiser/v1/lists) with a block size randomized among 2, 4 and 8 in the 1:1 allocation ratio and concealed using a sequentially numbered, opaque and sealed envelope (SNOSE). The co-investigator (WY) prepared the SNOSE for group assignment. All envelops were labelled with serial numbers and SC ambassadors were concealed from the allocation sequence. Group allocation was determined by opening the SNOSE on site once the consent form was signed. Masking of the interventionist is not possible due to the nature of the interventions. But the participants were not informed about the intervention in the other group. Outcome assessors were blinded to the group allocation.The allocation sequence was generated using an online tool  model in which participants were asked about smoking behaviors (Ask); warned about smoking hazard using a health-warning leaflet (Warn); advised to quit or reduce smoking as soon as possible and to set an initiation date (Advice); actively referred to SC services for standard treatment (Refer); and repeat the intervention during the chat-based interaction and follow-up calls . The AWARD process usually took one to two minutes to complete and has been validated in community smokers7. Among smokers who were willing to try NRT-S27, an NRT use card containing the instructions and potential side effects were given with a brief oral explanation17. Ambassadors introduced the SC services (free-of-charge) using a 2-sided color printed information card and actively referred the participants to their preferred SC services. With a separate consent form signed, names and telephone contacts of the agreed participants were sent to the service provider for appointment booking of clinical treatment within a week of enrollment .Participants in the intervention group received brief SC advice, 1 week of NRT  sampling, and were actively referred to SC services at baseline upon completion of the baseline questionnaire. NRT-S  was provided according to their preferences and daily cigarette consumption. Participants who consume ≤20 cigarettes daily received the 14 mg patch, 2 mg gum, or 1 mg lozenges while participants who consume >20 cigarettes daily received 21 mg patch, 2 mg gum, or 2 mg lozengesParticipants received two months of fix scheduled personalized regular messages combined with one-to-one interactive mobile chat support via instant messaging after enrolment. The mobile chatbased intervention was conducted by a trained SC counsellor with two years of experience in smoking cessation research. Messages were sent twice per week in the first four weeks and once per week for the following four weeks. Two additional follow-up reminders were sent before the follow-up call at 3 and 6 months, a total of 14 regular messages. Each message was personalized according to sex, age, and smoking pattern of the participants. The contents of the messages included benefits of quitting, tips for quitting, coping with craving, and the effectiveness of SC services. To initiate and facilitate the interactive conversations, some simple questions related to their smoking behavior were asked after sending the informative message, e.g. ‘How many cigarettes have you smoked today?’. Participants engaged in the interaction received synchronous feedback and personalized behavior support. Counsellors also monitored participants’ quitting progress, advised about the safety and effectiveness of NRT, and checked for NRT side effects via the interaction. All incoming messages were replied as soon as possible during working hours (9:30–18:30) on weekdays. For non-engaged participants, 3 additional prompt messages were sent in the first month to encourage engagement. Messages included asking their reasons for continuing smoking, history of quit attempts and attitudes towards successful quitters. The instant messaging dialogues were recorded and checked, by a research nurse trained in smoking cessation, biweekly to ensure intervention fidelity.13. The motivational interviewing (MI) behavior change model underpinned the design of the regular messages and the conduct of chat-based support. MI model focused on enabling change through the enhancement of intrinsic motivation and the exploration and resolution of ambivalence28. Consultation strategies adopted in the intervention included identifying discrepancies between participants’ thought and action, supporting their autonomy, and positive encouragement. Two participants who refused to receive instant messaging were contacted via SMS messages or telephone calls to remind them of follow-up calls.The frequency and content of the messages were informed by our formative qualitative studyAs an extension of the AWARD model, participants were encouraged to use SC services, helped with rebooking appointments and actively referred to SC services (for those who refused at baseline) during the chat-based interaction (Refer and Do-it-again). At the 1 and 2 months follow-up, a booster intervention of brief (one to two minutes) SC advice was delivered through telephone. Upon completion of the followup questionnaire (Ask about smoking behaviors), non-quitters were warned about the health risks of continue smoking (Warn), instructed about the use of NRT, monitored for potential side effects of NRT, and advised to quit again or reduce smoking (Do-itagain).29. Counsellors did not respond to any messages from participants and no telephone booster was given at follow-up.Participants in the control group also received brief SC advice, NRT-S and active referral at baseline. During the following 2 months, participants only received general SC messages through SMS with frequency similar to the regular instant messaging received by the intervention group. The SMS contents included brief SC advice, tips for coping with craving and reminders for follow-up calls30. Demographic information was collected.Smoking behavior including daily cigarette consumption, time to first cigarette upon waking up in the morning, past quit attempts (yes/no), and readiness to quit , and self-efficacy on quitting  was assessed at baseline . The levAll participants were followed up at 1, 2, 3 and 6 months from baseline. Smoking behavior was collected at each follow-up. We additionally measured for self-efficacy of quitting, intervention satisfaction at 3 and 6 months follow-up. Participants reporting smoking abstinence (not even a puff) in the past 7 days, at 3 and 6 months, were invited for biochemical validation.31) and saliva cotinine concentration of <10 μg/L (by NicAlert test strip32) at 3 and 6 months. Secondary outcomes included self-reported 7-day point prevalence abstinence (PPA), smoking reduction by at least 50% of baseline cigarette consumption (quitters included), quit attempts (abstinence for ≥24 h), NRT-S use and SC service use since the baseline. Participants in both groups received a small cash incentive of HK$200 (about US$25.6) for passing each validation at 3 and 6 months, which was found to have no effect on abstinence in our previous trial33.The primary outcome was biochemically validated abstinence, defined by an exhaled carbon monoxide level of <4 ppm (by Smokerlyzerpost hoc) were conducted using the effect size of the primary outcome to estimate the power of sample we achieved.In this feasibility study with integrated interventions of brief advice, NRT-S, active referral and sustained chat-based interaction, we aimed to recruit as many participants as resources allowed during December 2017 and March 2018 to generate preliminary estimates for the intervention efficacy. Analyses  for primary and secondary outcomes. We tested the differences in NRT-S use, quit attempt, smoking reduction, and self-reported quitting by intervention engagement, defined by having interaction in the IM chat-based intervention (verified by conversation log), controlling for sex and perceived importance of quitting at baseline35. The absolute number in some outcomes was relatively small (i.e. n<5), suggesting the analysis may be subjected to the sparse data issue. We thus conducted post hoc sensitivity analyses by incorporating weakly informative prior to penalize regression estimates and calculating profile likelihood-based CIs. Change-in-estimate procedure [ΔOR = /ORoriginal×100%] was adopted to compare the ORs obtained from the penalization analysis (model 3) with the ORs in adjusted regression model (model 2) (Supplementary file Tables S2 and S3). A value of p<0.05 indicated statistical significance in the 2-tailled analysis. All analyses were conducted in Stata (version 15.1).Intention-to-treat (ITT) analysis was conducted by treating participants with missing outcome measures as having no changes in smoking behaviors from baselineFrom December 2017 to March 2018, 164 potential participants were screened for eligibility, 121 were found eligible and provided written consent . Two parAmong the 14 and 13 self-reported quitters at 3 and 6 months, respectively, only 3 (21%) and 1 (7.7%) participated in the face-to-face biochemical validation. The intervention group showed non-significant higher validated abstinence than the control group of 3.2% vs 1.8%  at 3 months, and 1.6% vs 0% at 6 months. In model 2, we adjusted for unbalanced variables between groups (sex and perceived importance of quitting). The intervention group (vs the control group) reported more quitting at 3  and 6  months. The smoking reduction and cessation rate was also greater in the intervention group at 6 months prior change-in-estimates (<10%), suggesting that .Consistently, more participants in the intervention group reported quit attempts  and SC service use . Significantly more NRT-S use  was observed in the intervention group. Results from sensitivity analyses (Supplementary file Tables S2 and S3) show that the observed estimates remained statistically stable and largely unaffected by the weakly informative prior change-in-estimates (<10%), suggesting that estimates are with moderate to high precision and not overly sparse across the comparisons .This first pilot trial on NRT-S combined with chat-based support for SC to proactively recruited community smokers was feasible and showed increased self-reported abstinence compared with the control group which received NRT-S plus text messaging. More participants in the intervention group reduced smoking by at least 50% at 6 months. The results might be attributable to the increased quit attempts and NRT-S use. Although the findings were not conclusive due to the pilot nature of the trial and the small sample size, this preliminary evidence supports the feasibility and the need for full-scale trials on more community smokers.12. Our findings indicate that with cost-effectiveness in providing the interactive, real-time behavior support, mobile chatbased support is feasible to replace SMS in delivering SC intervention, especially in many regions with high or increasing smartphone penetration rate (89% in Hong Kong)36. We have used a proactive approach combined with brief advice and active referral, which has been well-tested in clinical and community settings37. NRT-S were provided to encourage quit attempts22. Most participants received the NRT-S at baseline, and significantly more participants in the intervention group had ever used it. Among the intervention group, participants engaged in the mobile chat support reported a consistent increase in the NRT-S use, quit attempt, smoking reduction and quitting. Higher knowledge level was a strong predictor of higher NRT adherence38, and exposure to health information through instant messaging was associated with less smoking39. Our participants were often exposed to the health information in the chatbased interaction, hence more likely to use NRT and stop smoking, probably via increased confidence, motivation, and self-efficacy in quitting40.Recent review of 26 studies (33849 participants) supported that mobile phone-based text messaging was efficacious in promoting SC42. Misinformation and lack of social support may act as barriers against NRT use. In contrast, confidence in NRT use increases when concerns are correctively addressed42. Many SC clinics provide physician consultation for full treatment of NRT with weekly follow-up, yet most smokers are not willing to attend3. Before the present trial, we found that smokers perceived instant messaging as an information center for quitting advice including NRT use13. In this study, we recorded the perceived effectiveness of the message content on quitting on a scale 0–10 at the 6 months follow-up. Most participants in the intervention group perceived that messages relating to quitting methods  and NRT information  as useful (score >5), followed by the contents of benefit of quitting  and encouragement . Real-time interactive discussion may also contribute to the increased use of NRT by providing safety advice, using guidance, reducing concerns and monitoring side effects during the treatment17. According to the conversation dialogue, three participants had discussed their NRT usage and one had shared successful quit experience from using NRT.Smokers perceived safety and effectiveness of NRT was found to influence the adoption of NRT for quit attempts and to affect the compliance to treatment43. Participants less motivated to quit are less likely to use mHealth, and more than half of the smokers  in our trial were not ready to quit in 30 days. Consistent with our previous large trial using chat-based support on community smokers that engaged 17% of the participants14, ‘too busy’ and ‘too boring of the content’ were the reasons for non-response to the messages. According to the trans-theoretical model, cessation support should be provided following the behavior-change stages of the participants44. Personalized mHealth support based on the stages of quitting process to improve the motivation to quit could be adopted in future studies for higher intervention engagement. Other behaviorchange theories (e.g. PRIME theory of motivation) could also be incorporated in the chat-based realtime interactions to increase the motivation to quit and support immediate desire of a quit attempt45. Our counsellors only sent out, replied to messages and engaged participants to communicate during working hours, and were not able to provide the chat support outside office hours. Whether the development of a more technologically advanced interactive SC program, such as an automatic dialogue system (Chatbot) that can provide interactive, personalized, synchronous psychosocial SC support, 24 hours per day 7 days per week, would improve the engagement warrants further investigation.The percentage of participants engaged in the chatbased intervention was low with only 33.9% (21 of 62) having ever replied to messages (single words or emoji were counted) and 16.1% (10 of 62) engaging in interaction on SC. This result is consistent with the law of attrition, which states that in any mHealth trial a substantial proportion of participants drop out or stop using the treatment before completion46. A recent Cochrane review of 5 studies (3079 participants) compared a smoking cessation smartphone app with minimal cessation support and found that self-help mobile applications were not effective in increasing abstinence12. Chat-based support through IM are less complex compared to self-help apps. With rapidly increasing use of smartphones, chat-based support can be used in larger pragmatic trials as an additional tool for other behavioral change interventions . Pharmacological treatment combined with behavioral support may also be a feasible and effective intervention model for other clinical services. Other modifiable harmful behaviors such as alcohol drinking and substance abuse might consider adding chat-based support when providing pharmaceutical treatment.The complexity of the intervention may also influence the attrition rate and the effect. A large sampled SC trial using smartphone-based application in young adults in Canada reported a relatively low retention rate of 60.5% at the 6 months follow-up and found no intervention effect in increasing quitting14. We offered small financial incentives and scheduled the validation at their preferred location and time, but some quitters reported too busy to attend, and some perceived the validation unnecessary. The adoption of self-reported abstinence in interpretation of mHealth intervention and the generalizability of the findings when providing different or no incentive for validation participation warrants confirmation47. The retention rate at 6 months (68.9%) was comparable to that of similar community trials on SC24. For those unreachable participants at the scheduled follow-up time, we made further calls, but limited to a maximum of 7 calls and 1 voice message as a reminder. Future mHealth studies might consider taking advantage of the connection with participants of instant messaging to obtain a higher retention rate. In this pilot trial with small sample and multiple outcomes, the estimated odds ratios may be subject to sparse data and multiple testing biases. Although the sensitivity analysis supported the moderate to high precision of adjusted estimates, further studies with larger sample size are needed to confirm the effect of the integrated intervention. Given the prevalence of current quitting outcomes, we re-estimated the sample size required for a definitive trial (Supplementary file Table S1). Results show that an estimated sample size of 382 (191 each group) would be enough to detect significant long-term (6-month) effects of increased self-reported quitting, smoking reduction, quit attempts, and NRT-S use at 80% power, using similar interventions. However, the effect remained unstable in the short-term (3-month) and a larger sample size would be needed in trials reporting validated quitting. The calculation was based on unplanned post hoc sensitivity analyses, so the data should be interpreted with caution. A factorial trial is warranted to examine the contributions of different intervention components. We used a proactive approach in the community setting where most smokers were not ready to quit, thus the generalizability to smokers in clinical settings is uncertain.This trial had several limitations. Firstly, the participation rate of biochemical validation was low in self-reported quitters. However, the self-reported quit rate at 6 months (16.1% vs 5.3%) was comparable to the previous trial (19% vs 11%) using chat-based SC interventionNRT-S plus mobile chat-based support was feasible and showed preliminary evidence of increased NRT-S use, quit attempts and quitting, in proactively recruited community smokers. A full-scale trial is warranted.


In the USA and Europe, questionnaires that allow patients to directly report chemotherapy side effects can improve their quality of life and clinical outcomes. No similar tools exist for Africa, so we aimed to design and validate a paper-based tool for use in oncology clinics in South Africa: The Patient Reported Symptoms-South Africa (PRS-SA) survey. The PRS-SA included questions on overall feelings of distress and severity of 11 common chemotherapy side effects. By comparing responses to the PRS-SA to responses to other quality of life questionnaires and to patients’ performance status, we found the PRS-SA to be valid and responsive for measuring all included symptoms. Compared to a standard instrument for measuring depression and anxiety, the PRS-SA’s distress thermometer had 88% sensitivity and 55% specificity for identifying distress. The PRS-SA may be a useful tool for efficiently assessing distress and chemotherapy symptoms in South Africa’s overburdened public oncology clinics.p = 0.03). All but cough showed responsiveness to change in QLQ-C30 score (highest p = 0.045). In South African cancer patients, the PRS-SA’s stress scale behaves similarly to the distress thermometer in other populations, and the symptom items demonstrated construct validity and responsiveness. Of note, 46% and 74% of participants who completed the PRS-SA in English or isiZulu, respectively, required assistance reading half or more of the instrument.Patient-reported outcome measures (PROM) for monitoring treatment toxicity improve quality of life (QoL) and clinical outcomes. However, no such PROMs exist for sub-Saharan African cancer patients. We aimed to validate the Patient Reported Symptoms-South Africa (PRS-SA) survey, a novel PROM for measuring distress and chemotherapy-related symptoms in South African cancer patients. We enrolled patients at the oncology clinic at Charlotte Maxeke Hospital, Johannesburg. At three separate visits, participants simultaneously completed the PRS-SA survey and several previously validated questionnaires. We constructed a receiver operator characteristics curve for distress levels predicting a Hospital Anxiety and Depression Scale (HADS) score ≥15. We evaluated construct validity for symptom items by comparing severity to the EORTC Core Quality of Life Questionnaire (QLQ-C30) summary score (Pearson correlation tests) and ECOG performance status (Mann–Whitney U tests). We assessed symptom item responsiveness by comparing change in severity to change in QLQ-C30 summary score and comparing standardized mean scores with negative, no, or positive change on the Global Impression of Change (GIC) questionnaire (Jockheere–Terpstra trend test). Overall, 196 participants with solid tumors completed instruments. A distress score of 4 had 82% sensitivity and 55% specificity for clinical depression/anxiety. All symptom items showed construct validity by association with either QLQ-C30 score or performance status (highest  Use of patient-reported outcome measures (PROMs) has been expanding into routine clinical oncology practice, particularly for monitoring cancer- and chemotherapy-related symptoms. Various academic cancer centers have developed electronic, web-based programs that allow patients to directly record the types and severity of symptoms they are experiencing while undergoing therapy ,2,3. WhePROMs can also have clinical impact . At MemoIn addition to physical symptoms, mental and emotional distress is prevalent among cancer patients, with broad meta-analyses suggesting up to 40% of cancer patients may simultaneously experience depression, anxiety or adjustment disorder . HeighteAs far as we know of, there are no reports from sub-Saharan Africa (SSA) of systematic PROM use in cancer care. Frequently, in the resource-constrained healthcare settings of SSA, patient volumes are extremely high, and time spent on each patient-provider interaction is limited. However, given the potential of PROMs to improve patient–provider communication and identify patients appropriate for additional supportive interventions, they may be an ideal, low-tech tool for strengthening those interactions and improving patient QoL and outcomes.We designed a paper-based PROM for monitoring physical symptoms of chemotherapy toxicity and patient distress levels called the Patient-Reported Symptoms-South Africa (PRS-SA) survey. We aimed to validate that instrument for use in South Africans undergoing treatment for solid tumors.We designed our novel instrument by adapting PROM tools that have been successful in other, high-income settings. The authors prepared an initial draft instrument containing the elements described below. An informal group discussion was held with a convenience sample of South African cancer and palliative care physicians, nurses, and patient navigators to review the draft and elicit feedback on completeness and relevance of the content and appropriateness of the language. That feedback was then incorporated to create a finalized version .To capture overall distress, we used a version of the Distress Thermometer (DT) instrument. The classic DT is a visual analogue scale labelled with scores from 0 (‘no distress’) to 10 (’extreme distress’) and was developed by Roth A.J. et al. in 1996 as a simple screening tool for identifying cancer patients needing further review for depressive or anxiety symptoms . The verOur group of providers believed that the term “suffering” would be best understood by the local population to represent the combination of physical, emotional, and spiritual discomfort characterized in the United States as distress, so we labelled our DT as ranging from “No Suffering” to “Severe Suffering.” The list of practical problems was reduced and consolidated as well.To capture the severity of common chemotherapy-related symptoms (CRS), we modeled the approach of Basch et al. . First, The final English draft underwent forward–backward translation into isiZulu, the most commonly spoken local language in the Gauteng province. Both English and isiZulu versions were then piloted with five patients each to gauge understanding of the content; no changes were made based on this last step.Similar to the approach taken by the National Institute of Health PRO-CTCAE Study Group, we evaluated the construct validity and responsiveness of each CRS item by comparing participant responses to previously validated “anchor” instruments and to pre-defined clinical sub-groups expected to have differing symptom burden . Our ancThe QLQ-C30 is a cancer-specific quality of life instrument that measures patient experience within five functional categories, eight symptom categories and global health status; the instrument specifically asks about experience of the past week. A single health-related QoL summary score can be calculated by linearly transforming and then averaging the individual scores from each functional and symptom category, excluding financial difficulties ,23. The The GIC simply asks patients to describe their “overall status” since the last time they were surveyed according to 7 response choices ranging from “Very Much Worse” to “Very Much Improved.” We prepared an isiZulu version of the GIC, also using the forward–backward translation method.Validation of the modified DT focused on establishing a threshold value with balanced sensitivity and specificity for detecting clinically meaningful depression and anxiety. As with the original and most of the subsequent validation studies of the DT, we used the Hospital Anxiety and Depression Scale (HADS) as a comparator instrument ,26. The Taken in total, South Africa is an upper middle-income country, but the stark degree of income inequality means that 56% of the population live below the national poverty line . South ALow-cost cancer care is available to uninsured and underinsured patients at public tertiary hospitals. For this study, we enrolled participants from the medical oncology clinic of one such hospital: Charlotte Maxeke Johannesburg Academic Hospital (CMJAH). The hospital is an affiliated teaching hospital of the Faculty of Health Sciences at the University of Witwatersrand. The clinic provides chemotherapy and other systemic cancer therapies to most public patients in Johannesburg, including those who receive their surgical care at Chris Hani Baragwanath Academic Hospital, an enormous public hospital serving Johannesburg’s majority Black neighborhood of Soweto. Patient–provider ratios are high in South Africa’s public settings . Long waParticipants were eligible if they were ≥18 years of age, had a histologically confirmed diagnosis of a non-hematologic cancer, were either currently receiving chemotherapy at CMJAH’s oncology clinic or scheduled to initiate chemotherapy within 14 days of enrollment, were not receiving their last planned dose of chemotherapy on the day of enrollment and were able to communicate in either English or isiZulu. Patients with both metastatic and non-metastatic disease were allowed to enroll.Potentially eligible clinic patients were identified by study staff and recruited on the day of their normally scheduled clinic visit. No randomization was performed for this single arm study and consenting patients were enrolled consecutively. Patients were approached in the waiting area and, if they expressed interest in participation, were taken to a private room for the consenting process and the administration of study questionnaires.On the day of enrollment, consenting and eligible participants completed, in order, the PRS-SA, QLQ-C30 and HADS questionnaires. The study interviewer collected self-reported data on demographics, literacy, and basic clinical information. Participants were then followed for up to two subsequent clinic visits. At those visits, they completed the PRS-SA, QLQ-C30 and GIC questionnaires . InterviStudy data were collected and managed using REDCap electronic data capture tools hosted at the University of Witwatersrand . To be cPatient characteristics, literacy skills and reported levels of distress and physical symptoms were described using simple counts and percentages.Construct validity for each symptom item was assessed separately by comparing individuals’ reported severity for each item on the PRS-SA to the QLQ-C30 summary score and ECOG PS (PS 0–1 vs. 2–4) measured at the same visit. Pearson correlations with 95% confidence intervals were calculated for the comparison to QLQ-C30, and the Mann–Whitney U tests were used to evaluate association with performance status. In addition to testing for significant association, we determined the strength of association between each PRS-SA item and the anchor item. Applying the same standard as the PRO-CTCAE Study Group, for the QLQ-C30 and ECOG PS comparisons we, respectively defined r values of 0.1, 0.3 and 0.5 and Cohen d effect sizes of 0.2, 0.5 and 0.8 as small, medium, and large effects. If a PRS-SA symptom item showed a significant association with at least a small effect size for either anchor item, we considered it to have shown construct validity. As sensitivity analyses, we also calculated Pearson correlations between all PRS-SA symptom item scores and the individual QLQ-C30 functional sub-scale  and any relevant symptom sub-scale scores.Responsiveness for each symptom item was assessed in two ways. First, for each item, we calculated the change in reported severity on the PRS-SA and change in QLQ-C30 between pairs of consecutive visits. Pearson correlations were calculated to evaluate for association between the changes in those two values. In addition, individual visits were sorted into three subgroups based on response to the GIC questionnaire . Within each group, for each individual symptom item, we calculated the standardize mean change in severity from the prior visit. Those standardized means were compared across GIC subgroups using a 1-sided Jockheere–Terpstra test for trend. We considered items responsive if we found significant correlation for change in severity and QLQ-C30 score and found the Jockheere–Terpstra test to be significant . All staTest characteristics for various DT threshold values were determined using the approach of Jacobsen PB et al. . We defiStatistical calculations were performed using SAS v9.4  and our ROC curve was generated using the web-based ROC Plot app .From September 2020 through May 2021, 211 patients consented to and were screened for participation. Nine lacked a non-hematologic cancer diagnosis; two were not currently receiving or starting chemotherapy within 14 days of enrollment; two were receiving their last planned cycle of chemotherapy on the day of enrollment; one was felt by study staff to be unable to consent to participation; and one participant was too ill to participate after consenting. Of the remaining 196 participants, all completed study procedures at enrollment, 173 completed procedures at second visit, and 150 completed procedures at a third visit. The median time between visits was 22 days (interquartile range: 21–28 days).n = 157, 80.1%). IsiZulu was the most common primary language , while English was the primary language of just 38 (19.4%). Participants with breast cancer made up over half of the study cohort , and those with colorectal cancer were the next most common . By self-report, 48 participants (24.5%) had comorbid hypertension, and 33 participants (16.8%) had comorbid HIV.Median age for all participants was 52.3 years, and 152 (77.6%) were female . Most ofp-values < 0.0001)  score  . Fatigue 0.0001) . The paip = 0.03)  (highest significant  = 0.03) . All symp-value = 0.045) (p = 0.03)  . The sta = 0.03) .Of the 175 participants who completed the PRS-SA after receiving at least one cycle of chemotherapy, 110 (62.9%) reported at least one symptom of grade 3–4 severity . Grade 3n = 90, 59.6%), stress  and transportation .The receiver operating characteristics (ROC) curve for the modified distress thermometer’s (DT) identification of clinically meaningful anxiety and depression, as measured by the Hospital Anxiety and Depression Scale (HADS), showed an area under the curve (AUC) of 0.76 . DT scorOf the 167 participants who preferred to complete the PRS-SA and other study instruments in English, 153 (91.6%), 152 (91.0%) and 147 (88.0%) reported understanding, reading and writing English well or very well, respectively. However, during the 458 times the PRS-SA was administered in English, the study interviewer reported needing to read half or more of the instrument to the participant in 210 (45.9%) cases. Similarly, among the 29 participants choosing to complete study instruments in isiZulu, 29 (100%), 26 (89.7%) and 26 (89.7%) reported understanding, reading and writing isiZulu well or very well, respectively. For this group, study interviewers reported reading half or more of the PRS-SA in 41 (73.8%) of the 61 instances it was completed. In a post hoc sensitivity analysis, neither the distribution of DT scores or of reported severity of any symptom item significantly differed between participants who did and did not require any assistance with reading the PRS-SA.This study is the first in South Africa to validate a locally relevant PROM for patients with solid tumors. We demonstrate construct validity for all symptom items, and all items showed at least a medium-sized association with either QLQ-C30 summary score or ECOG PS, except for diarrhea which showed a small-sized association with both. All individual symptom items except cough also showed responsiveness to change in health-related quality of life (HRQOL). The full set of items was responsive to changes in patients’ GIC score. Our approach to establishing construct validity and responsiveness of our symptom items was modeled after that of the PRO-CTCAE Study Group . That grResponses to the modified DT were also remarkably consistent with other populations worldwide. Using an international standard to detect clinical depression or anxiety, our ROC curve for the DT has an AUC of 0.76. Reported AUCs from DT validation studies in 33 other countries range from 0.63 to 0.88 . Using aWhile not the primary interest of this study, it is notable that >60% of participants reported at least one grade 3 or higher chemotherapy-related symptom, and >75% of participants reported DT scores ≥4. Chemotherapy adverse event (AE) rates in sub-Saharan African populations are rarely published, but our findings are consistent with the >70% rate of grade 3–5 events at one hospital in Ethiopia and much higher than the ~30% grade 3–4 event rate among breast cancer patients treated in Senegal ,36. SelfFollowing validation, it will be necessary to pilot and study implementation of general use of the PRA-SA at our clinic and similar settings. In high-income environments, provider-level barriers to PROM implementation have included skepticism regarding their value or necessity, underdeveloped strategies for interpreting or reacting to symptoms and distress, insufficient time or staffing to review reports, underuse of information technology solutions for presenting and tracking response over time and fear that PROMs depersonalize doctor–patient interactions ,46,47,48Our results point to another major potential barrier to implementation of PROM use in general clinical care in South African oncology clinics: literacy. Despite ~90% of participants reporting they could read and write well or very well in either English or isiZulu, 46% and 74% required assistance reading at least half of the English and isiZulu PRS-SA, respectively. Formal literacy evaluation in South African patients has shown wide discrepancies between the ability to read or pronounce English medical terms and the ability to understand their meaning . To reduGiven the myriad of other challenges resource-constrained healthcare systems face in providing effective, high-quality cancer care, it will also be necessary to demonstrate that PROM use for monitoring chemotherapy toxicity maintains the ability to improve patient quality of life, treatment adherence and clinical outcomes. As that work proceeds in South Africa, we offer our validated tool here in hope that researchers and clinicians working in similar settings will take up its use and simultaneous study.In conclusion, our novel PROM, the PRS-SA, demonstrated construct validity and responsiveness in the measurement of chemotherapy-related symptom severity and typical sensitivity to clinically meaningful distress among solid tumor patients receiving chemotherapy at a public oncology clinic in Johannesburg, South Africa. Patients reported both a high burden of grade 3–4 symptoms and distress. Incorporating the PRS-SA into clinical use in South Africa may provide a low-cost, low-tech way to improve cancer patient QoL. Based on these initial findings, the instrument’s implementation, and impact merit further study. We anticipate that low patient literacy will present a major barrier to the PRS-SA’s routine use; creative solutions will be needed to increase accessibility.


Data supporting hypofractionated preoperative radiation therapy (RT) for patients with extremity and trunk soft tissue sarcoma (STS) are currently limited to phase II single-institution studies. We sought to understand the type and thresholds of clinical evidence required for experts to adopt hypofractionated RT as a standard-of-care option for patients with STS.  An electronic survey was distributed to multidisciplinary sarcoma experts. The survey queried whether data from a theoretical, multi-institutional, phase II study of 5-fraction preoperative RT could change practice. Using endpoints from RTOG 0630 as a reference, the survey also queried thresholds for acceptable local control, wound complication, and late toxicity for the study protocol to be accepted as a standard-of-care option. Responses were logged from 8/27/2020 to 9/8/2020 and summarized graphically.  The survey response rate was 55.3% (47/85). Local control is the most important clinical outcome for sarcoma specialists when evaluating whether an RT regimen should be considered standard of care. 17% (8/47) of providers require randomized phase III evidence to consider hypofractionated preoperative RT as a standard-of-care option, whereas 10.6% (5/47) of providers already view this as a standard-of-care option. Of providers willing to change practice based on phase II data, most  would accept local control rates equivalent to or less than those in RTOG 0630, as long as the rate was higher than 85%. However, 51.3% (19/37) would require wound complication rates superior to those reported in RTOG 0630, and 46% (17/37) of respondents would accept late toxicity rates inferior to RTOG 0630.  Consensus building is needed among clinicians regarding the type and threshold of evidence needed to evaluate hypofractionated RT as a standard-of-care option. A collaborative consortium-based approach may be the most pragmatic means for developing consensus protocols and pooling data to gradually introduce hypofractionated preoperative RT into routine practice. Radiation therapy (RT) is an integral component of limb-salvage therapy for patients with high-risk soft tissue sarcoma (STS) . RT can Single-institution phase II data have emerged  suggestiDespite the appeal of a hypofractionated approach that is also potentially more cost effective , additioExperts in the management of soft tissue sarcoma were identified either by word-of-mouth, through authorship of high-impact publications in sarcoma, through lead design of ongoing clinical trials in sarcoma, or by reviewing the websites of sarcoma centers of excellence.N > 250), multi-institutional, single-arm phase II study of 5-fraction preoperative RT, based on a recent single-institution phase II study ). A majority of providers  indicated they would either accept local control rates equivalent to those in RTOG 0630  or local control rates less than those in RTOG 0630 but higher than 85%  . The remWhen primed with the wound complication rate from 71 evaluable patients on RTOG 0630 as a reference (36.6%), survey respondents had more conservative attitudes regarding acceptable wound complication rates. To incorporate 5-fraction preoperative RT as a standard-of-care approach, 51.3%  of respondents would require a wound complication rate superior to that reported in RTOG 0630; only 27% (10/37) would accept a slightly higher wound complication rate than reported in RTOG 0630 . The remRespondents were then primed with the late toxicity rate among 57 evaluable patients from RTOG 0630 (10.6%). To incorporate 5-fraction preoperative RT as a standard-of-care approach, 32.4%  of respondents would require lower late toxicity rates compared to RTOG 0630 and 46% (17/37) of respondents would accept a late toxicity rate inferior to RTOG 0630 . The remAmong the three clinical endpoints assessed, local control was most frequently chosen by respondents as the endpoint most important in determining whether 5-fraction preoperative RT would be considered a standard-of-care option . AdditioWe report surveyed perspectives from sarcoma experts regarding hypothetical clinical trial endpoints that, if met, would be sufficient to enable the inclusion of hypofractionated preoperative RT as a standard-of-care option. We found that just 17% of providers require randomized phase III evidence to consider hypofractionated preoperative RT as a standard-of-care option. Moreover, respondents indicated that a large, single-arm multisite phase II study would be sufficient to incorporate this approach as a standard-of-care option. In fact, 10.5% of providers already use this approach despite the availability of only single-institution phase II nonrandomized studies. Finally, local control is the most important clinical outcome for sarcoma specialists when evaluating whether a radiotherapy regimen should be considered standard of care, though there is variability around the precise local control threshold.Based on our data, there is no strong consensus on how to best evaluate hypofractionated preoperative RT as a standard-of-care option for the treatment of sarcoma. While our survey predominantly reflects opinions from experts in North America and is likely impacted by regional biases, it nonetheless highlights an important need for early consensus building through networks of sarcoma experts as a first step toward engineering new therapeutic approaches. Here, we discuss several potential pathways for evaluating this patient-centered and value-driven radiation treatment as a standard-of-care option.The first pathway would be a multisite randomized phase III trial assessing the noninferiority of hypofractionated preoperative radiotherapy compared to conventionally fractionated preoperative radiotherapy. This approach has been the gold standard for changing practice in oncology. However, several hurdles, both logistical and financial , temper Another concern is that despite the power of a properly accrued randomized trial approach, such a design still may not offer a conclusive clinical answer, potentially due to the heterogeneous biologic profiles  of soft Moreover, the high risk of a randomized study is unlikely to accompany high reward in this setting, given our survey results suggesting that over 10% of surveyed sarcoma experts already consider 5-fraction preoperative radiation to be a standard approach. Thus, as with other areas of research , the priA multi-institutional, nonrandomized, phase II trial is an alternative approach with a potential for faster accrual. Such a study would enhance the current evidence base surrounding hypofractionation, which mostly relies on single-institution studies. However, even if local control and wound toxicity were found to be on par with historical standards, the benefits of such a trial are again uncertain in the setting of widespread preexisting enthusiasm for preoperative hypofractionation off-study and the heterogeneous thresholds among clinicians.A more grassroots approach might involve establishing a consortium of institutions that are comfortable performing hypofractionated RT in service of pooling outcomes from these centers so that we can continue to learn about the benefits and tradeoffs of this approach outside of the confines of a rigorous clinical trial protocol. This approach would be cost effective, promote knowledge and experience sharing among sarcoma experts, and would obviate the need for centralized IRB requirements. The obvious shortcoming here is that variable practices across centers may make data pooling and subsequent interpretation a challenge. This might be proactively managed by developing consensus protocols that aim to optimize the endpoints highlighted by this study as important for sarcoma experts, prior to inception of the consortium.Finally, the most passive strategy would entail pausing research efforts and relying on natural forces and incentives  to driveWe eagerly anticipate the long-term results of several completed and ongoing single-institution studies of hypofractionated RT. As discussed above, paradigm-shifting studies for radiotherapy can be challenging to orchestrate in sarcoma, and our survey results indicate the need for consensus building among clinicians on the type and threshold of evidence needed to evaluate hypofractionated RT as a standard-of-care option. One avenue will be cooperative-group sponsored multisite studies . However, given the existing obstacles around large, multisite, and randomized studies, the most pragmatic way forward may be a collaborative consortium-based approach for developing consensus clinical protocols with an eye toward pooling data from these individual institutional efforts.


Furthermore, this soluble factor appears to be an immune complex, which may be composed of COVID-19 Spike protein and related antibodies. We suggest that these Spike-specific immune complexes contribute to COVID-19 platelet activation and thrombosis in a manner similar to heparin-induced thrombocytopenia. Understanding this underlying pathobiology will be critical for advancement of future research and therapeutic options.Coronavirus disease 2019 (COVID-19) is a highly prothrombotic viral infection that primarily manifests as an acute respiratory syndrome. However, critically ill COVID-19 patients will often develop venous thromboembolism with associated increases in morbidity and mortality. The cause for this prothrombotic state is unclear but is likely related to platelet hyperactivation. In this review, we summarize the current evidence surrounding COVID-19 thrombosis and platelet hyperactivation. We highlight the fact that several studies have identified a soluble factor in COVID-19 patient plasma that is capable of altering platelet phenotype  Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) –5. The pPlatelets have long been known to function as mediators of thrombosis and hemostasis but have only recently gained recognition in their role as immune mediators , 11. TheIn this review, we summarize the general thrombotic nature of COVID-19 and the importance of understanding this process in regards to therapeutic options. Subsequently, we review platelet physiology and their current recognized functions as immune cells. We also review platelet activation in heparin-induced thrombocytopenia (HIT) and how this parallels platelet activity in COVID-19. Finally, we discuss the novel entity of vaccine-induced thrombotic thrombocytopenia (VITT) and the role of platelets in this unique COVID-19 associated disorder.9/L was the lower limit of normal)  . The deg normal) .Thrombosis is particularly prevalent amongst critically ill COVID-19 patients, with some studies identifying deep vein thrombosis in up to 79% of patients through ultrasound screening . Up to 2This observation has resulted in several randomized clinical trials demonstrating a benefit of therapeutic-dose low molecular weight heparin in hospitalized COVID-19 patients. In the ATTACC, ACTIV-4a, and REMAP-CAP multi-platform trial, therapeutic doses of heparin increased the probability of organ support-free days in non-critically ill patients  . HoweverPrior to delving into platelet activation in COVID-19, it is important to gain a basic understanding of how platelet synthesis and function are intimately related to immunity. Platelets are produced in the bone marrow from progenitor cells, termed megakaryocytes, through a complex process of hematopoietic stem cell differentiation . Their pOnce released into circulation, platelets are equipped with various intracellular materials over 300) to mediate their effector functions . These i0 to mediImmune complexes are important initial defenses against pathogen infection and are formed from antibody binding to soluble antigen. They often consist of immunoglobulins (Ig) of the IgG or IgM type but can also be IgA , 36. ImmHIT is a prothrombotic autoimmune disorder characterized by the presence of thrombocytopenia (low platelets) and thrombosis that shares many features with COVID-19. It most commonly presents in hospitalized patients who are receiving unfractionated heparin anticoagulation and is characterized by antibodies targeting platelet factor 4 (PF4)-heparin complexes . PF4 is 14C-serotonin allowing uptake into platelets. These modified platelets are subsequently exposed to patient plasma, in the presence and absence of heparin, which allows formation of anti-PF4/heparin immune complexes . Briefly, platelets from healthy donors are incubated with radioactive omplexes . These itivation . This istivation Figure 1To this point, many studies have confirmed that platelets in COVID-19 patients display a hyperactivated phenotype with altered gene expression. In a cohort of 115 COVID-19 patients, featuring both non-severe and severe disease, platelets secreted increased IL-1beta and soluble CD40 ligand compared to healthy controls . FurtherOne mechanism by which platelets may contribute to COVID-19 presentation is through neutrophil recruitment and aggregation. As previously noted, platelet P-selectin is able to bind neutrophil ligands to induce rolling and aggregation at sites of activation . This inIt is still unclear as to what triggers such drastic platelet changes in critically ill patients. Some have hypothesized that SARS-CoV-2 directly interacts with platelets to mediate these observed effects. Evidence for this is supported by the presence of viral RNA in platelets of infected individuals, although this is only seen in up to 24% of patients , 49, 55.Immune complexes are one potential circulating factor that could contribute to platelet activation in COVID-19. As previously mentioned, immune complexes activate platelets through the FcγRIIa and may be formed from antibodies against self or exogenous antigens. Viral illnesses are well documented to produce antibodies against self-antigens, such as antiphospholipid antibodies, through a process called molecular mimicry. Early reports in COVID-19 patients highlighted the presence of these antibodies in association with thrombosis, including anti-beta-2 glycoprotein and non-specific inhibitor –59. Injein vitro study confirmed that recombinant anti-Spike IgG is able to activate platelets through the FcγRIIa . Interestingly, anti-Spike IgG and Spike protein alone did not lead to significant thrombus formation. How exactly von Willebrand factor interacts to promote platelet activation is unclear but may be through facilitating platelet aggregation. Glycosylation status of anti-Spike IgG was also found to be a significant factor in the ability of these complexes to activate platelets. Therefore, certain anti-Spike IgG activate platelets in the context of COVID-19 infection, but this remains to be validated in the clinical context.Another potential hypothesis is that HIT antibodies are contributing to the IgG-mediated platelet activation seen in COVID-19 patients. This is supported by the observations that COVID-19 and HIT share many clinical similarities; COVID-19 patients are often exposed to heparin in the context of hospitalization; and a high proportion of COVID-19 patients test positive for anti-PF4/heparin antibodies on further testing , 61. How FcγRIIa . This wain vitro  in the context of platelet activation and COVID-19. Although this platelet activation is not directly related to SARS-CoV-2 viral infection, it has important clinical and public health implications. VITT is a novel, “drug” related disorder attributed to vaccination by adenoviral vector-based SARS-CoV-2 vaccines. This primarily includes the ChAd-Ox1 (produced by AstraZeneca) and Ad26.COV2.S (Johnson and Johnson) vaccines. VITT was first described in eleven patients, predominantly female, who presented with unusual thromboses  and thrombocytopenia . PatientAlthough the field of COVID-19 thrombosis is in its infancy, there is sufficient evidence to support a major role for platelets in disease pathogenesis. Platelets have been shown to be hyperactivated in critically ill patients and secrete excessive procoagulant molecules. Furthermore, they are able to interact with other immune cells to mediate the immune response. However, this excess inflammation may also contribute to tissue damage and ultimately, mortality. Targeting these pathways in order to dampen the excess immune response may thus present attractive therapeutic targets.Immune complexes also appear to contribute significantly to these platelet changes in a manner similar to HIT and other immune-complex mediated disorders. The antigen specificity and additional characteristics of these immune complexes remain to be determined but will be crucial to the development of therapeutic targets. Identifying the specific antibodies involved may also allow clinicians to risk stratify patients who are at high risk of severe disease or thrombosis, thus offering the potential for prophylactic anticoagulation.Both SJ and IN contributed equally to this work, including initial manuscript preparation, editing, and final drafting. All authors contributed to the article and approved the submitted version. Funding was received from the Canadian Institutes of Health Research (CIHR 452655) awarded to IN.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Triple-negative breast cancer (TNBC) is considered one of the highest-risk subtypes of breast cancer and has dismal prognosis. Local recurrence rate after standard therapy in the early breast cancer setting can be upwards to 72% in 5 years, and in the metastatic setting, the 5-year overall survival is 12%. Due to the lack of receptor expression, there has been a paucity of targeted therapeutics available, with chemotherapy being the primary option for systemic treatment in both the neoadjuvant and metastatic setting. More recently, immunotherapy has revolutionized the landscape of cancer treatment, particularly immune checkpoint inhibitor (ICI) therapy, with FDA approval in over 20 types of cancer since 2011. Compared to other cancer types, breast cancer has been traditionally thought of as being immunologically cold; however, TNBC has demonstrated the most promise with immunotherapy use, a timely discovery due to its lack of targeted therapy options. In this review, we summarize the trials using checkpoint therapy in early and metastatic TNBC, as well as the development of biomarkers and the importance of immune related adverse events (IRAEs), in this disease process. With the number of predicted breast-cancer cases in the United States reaching 250,000 in the year 2020, it is estimated that 10–15% of these newly diagnosed cases will be triple-negative breast cancer (TNBC) ,4. TNBC Immunotherapy has prolonged survival in other solid tumors, including melanoma, lung, and kidney cancers with the most successful immunotherapeutic agents consisting of immune checkpoint inhibitors (ICIs). ICIs block immune checkpoints or immunosuppressive receptors, such as cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4) and programmed death receptor-1 (PD-1) to improve the cytotoxicity and proliferative capacity of tumor-infiltrating lymphocytes (TILs) ,33,34,35Unlike other solid organ cancers, breast cancer has long been considered more immunologically “cold” with low T-cell infiltration. However, several lines of evidence have demonstrated the immune system’s role in the prognosis and outcomes of TNBC. First, TNBC has more TILs, with several studies demonstrating that these are associated with a higher response to ICIs in other tumors and improved prognosis in early stage TNBC ,43,44,45In this review, we focus on the role of immune checkpoint inhibitors in early stage and metastatic triple-negative breast cancer either in the neoadjuvant or adjuvant settings. In addition, we summarize the effects of combination treatments with chemotherapy and immune checkpoint inhibitors, as well as recent and ongoing trials attempting to elucidate the role of promising therapeutic strategies that target specific subsets of TNBC. We briefly outline other potentially immunotherapy strategies and future directions.Triple-negative breast cancer is uniquely heterogeneous at the clinical, histologic, and molecular level. Breast tumors that do not overexpress the estrogen, progesterone, or Her2neu receptor are histologically known as TNBC. The American College of Pathology, the American Society of Clinical Oncology, and the St. Gallen have established that ER and PR negativity are defined by 1% positivity of either receptor ,51. MoleImmune checkpoints are molecules that protect against normal tissue damage caused by over-activity of T-cells . PD-1 anOne of the most studied ICIs, pembrolizumab is a humanized IgG4 antibody targeting PD-1. Pembrolizumab first gained initial FDA approval for unresectable or metastatic melanoma in 2014  and has A humanized IgG1 antibody targeting PD-L1, called Atezolizumab, has also been explored as ICI monotherapy in metastatic TNBC. It selectively targets PD-L1 to prevent interaction with the receptors PD-1 and B7-1, reversing T-cell suppression . In a phIt is generally accepted that most TNBC is chemotherapy-sensitive, but the optimal treatment regimen continues to be investigated. Most chemotherapy regimens include anthracyclines, taxanes, and/or platinum compounds, dose-dense AC (doxorubicin/cyclophosphamide), or TC (docetaxel/cyclophosphamide). The addition of platinum to standard chemotherapy has shown to increase the pathologic complete response rate ,62. MultWith the introduction of ICIs, the therapeutic landscape has changed. Generally, ICI monotherapy was effective against metastatic TNBC when there was limited disease present; however, in women with advanced disease and a high metastatic tumor burden, there was minimal or no response. Therefore, most studies began to focus on combination therapy of ICI with chemotherapy. The hypothesis was that chemotherapy could augment tumor-antigen release and antitumor responses to ICI. Specifically, taxanes were thought to have the potential to activate toll-like receptor activity and promote dendritic-cell activity . The KEYThe Impassion130 trial demonstrated significantly prolonged median PFS with a regimen of atezolizumab plus nab-paclitaxel compared to placebo plus nab-paclitaxel. (7.2 months and 5.5 months respectively); the PD-L1 positive immune cells subgroup demonstrated an even more dramatic difference in PFS. The difference between PFS of atezolizumab and control groups was 7.5 months vs. 5.0 months) . There wDue to the promising results in the metastatic setting of combination chemotherapy and ICIs, studies have now been conducted in early stages of TNBC. They have demonstrated preliminary success thus far. In the I-SPY 2 trial, patients with stage II/III disease treated with combination chemotherapy and pembrolizumab had estimated the pCR rate to be nearly three times that of those individuals with chemotherapy alone . The KEYThere are several ongoing early stage disease trials that will further clarify the efficacy of ICIs in the neoadjuvant setting for TNBC and whether these agents should be utilized as adjuvant treatments. Two key trials—namely SWOG S1418 and the A-brave trial—are investigating if adjuvant anti-PD-1/L1 therapy prolongs event-free survival (EFS) or disease-free survival (DFS). Concurrently, two large trials are investigating if the addition of atezolizumab to both neoadjuvant and adjuvant therapy prolongs EFS or invasive DFS-the NSABP B-59 trial and the Impassion030 trial.KEYNOTE-119 is a phase 3 trial that compared pembrolizumab with chemotherapy for second-line or third-line treatment of patients with metastatic TNBC. The trial included 1098 patients who were randomly assigned to receive either pembrolizumab or chemotherapy. The primary endpoints were OS in PD-L1 positive patients and all patients. In the PD-L1-positive patients, the median OS was 12.7 months in the pembrolizumab group and 11.6 months for the chemotherapy group 0.057). In the overall population, the median OS was 9.9 months for the pembrolizumab group and 10.8 months for the chemotherapy group (non-significant). The most common adverse events were anemia and neutropenia [. In the p = 0.0012). Among all patients, the median PFS was 7.5 months compared to 5.6 months. The pembrolizumab treatment effect increased with PD-L1 enrichment. Adverse events were seen in 68% of the pembrolizumab group and 67% in the placebo group [Based upon the findings of the KEYNOTE-119 trial, this trial is a phase 3 trial that compared pembrolizumab with chemotherapy versus placebo with chemotherapy untreated locally recurrent inoperable or metastatic TNBC. The trial included 1372 patients who were randomly assigned to the two treatment groups and the primary endpoint was PFS. IN the PD-L1 positive patients, the PFS in the pembrolizumab group was 9.7 months and 5.6 months in the placebo group . The primary outcomes are DFS and OS. The trial is ongoing and continues to accrue patients (NCT02954874).p = 0.01) with placebo with the median OS being 21.3 months compared with 17.6 months (p = 0.01), respectively within the two groups. Among the patients with PD-L1 positive tumors, the median OS was 25.0 months and 15.5 months, respectively. Adverse events that led to the discontinuation of any agent occurred in 15.9% of the patients who received atezolizumab and 8.2% of those who received a placebo [Impassion130 is a phase 3 trial that compared atezolizumab in combination with nab-paclitaxel or a placebo in patients with untreated metastatic TNBC. The trial included 451 patients in each group and was randomized. The intervention was continued until disease progression or an unacceptable level of toxic effects occurred. The primary endpoints were PFS and OS. The median PFS was 7.2 months with atezolizumab, as compared with 5.5 months . In the PD-L1 positive patients, the difference was more pronounced—with 69% achieving a pCR in the atezolizumab group and 49% in the placebo group. The adverse events were balanced across the two groups [Impassion031 is a phase 3 trial, similar to Impassion030, that compared atezolizumab versus placebo combined with nab-paclitaxel followed by doxorubicin plus cyclophosphamide as neoadjuvant treatment for early stage TNBC. The trial included 333 patients who were randomly assigned to either atezolizumab plus chemotherapy or placebo plus chemotherapy, and the primary endpoints were pCR in all patients and in PD-L1 positive patients. In all patients, the pCR with atezolizumab was 58% vs. 41% in the placebo group  . Recent BRCA-1-mutated tumors have been demonstrated to be deficient in DNA-repair, as well as 25% of sporadic breast cancers. PARP (poly (ADP-ribose) polymerase) is a nuclear enzyme that helps repair DNA single-strand breaks and is highly expressed in more than 90% of TNBC . PolymerAnother novel strategy in cancer immunotherapy are cancer vaccines, with the potential to illicit an immune response against tumor-specific and tumor-associated antigens. Ongoing trials in TNBC are investigating using tumor vaccines or oncolytic viruses with csfg312blockade against TNBC  and polyThere are many promising results in past and ongoing trials using immunotherapy for treatment in TNBC. However, there are ongoing challenges with these treatments, including development of biomarkers for optimal patient selection and immune-related adverse events (IRAEs) ,83.Enriching the population with the development of biomarkers to determine who may derive benefit from ICI is an area of active investigation . One of RM) cell differentiation expressing high levels of immune checkpoint molecules has been associated with survival in an early TNBC population and was better prognosticator than the CD8+ TIL population alone [Quantitative and qualitative TIL levels have also shown promise in predicting patient response to ICI. In the Impassion130 trial, metastatic TNBC patients with higher CD8+ TILs demonstrated increased PFS and OS when treated with chemo-immunotherapy. In early stage TNBC patients treated with pembrolizumab-based combination therapy, higher quantities of TILs and PD-L1 expression was associated with a higher pCR rates and improved ORR . Furtheron alone .Other biomarkers that are under investigation include tumor mutation burden (TMB), microsatellite instability (MSI), and BRCA status. TNBC harbors more TMB or non-synonymous somatic mutations than other breast-cancer subtypes. In other cancers, a higher TMB is correlated with improved response to ICI therapy. One study has shown that higher levels of TMB with high TIL infiltrates have been shown to be correlated with better prognosis in TNBC . In addiImmunotherapy hyper-activates the immune system, causing a wide variety of toxicities called immune-related adverse events (IRAEs) ,83. IRAEPredicting who will develop IRAEs is an active area of investigation and may include assessment of personal and/or genetic risk factors. Studies have shown that obesity, kidney disease, and personal or family history of autoimmune disease have been linked with increased risk, while other factors, such as history of steroid use or female sex, may be protective. Currently, the treatment of IRAEs include cessation of the ICI, and use of glucocorticoids and/or monoclonal ab, including TNF (infliximab) or IL-6 (Tocilizumab). Preliminary investigation on how the use of these agents affects the antitumor response of the ICI is reassuring in other malignancies, but requires longer-term follow-up ,99,100.Another challenge of immunotherapy is the wide variety of different response patterns seen across patients with what appears to be the same disease. In other solid organs such as non-small-cell lung cancer, there is a phenomenon known as “pseudo-progression”, where patients appear to be progressing, with tumor enlargement secondary to lymphocyte infiltration, prior to demonstrating regression. With ongoing investigations into immunotherapy in the TNBC setting, hopefully we will gain some answers to these ongoing questions.The understanding of immune-checkpoint-based treatment in TNBC has radically changed the therapeutic options for women who have been diagnosed with this disease. Existing trial data have clearly demonstrated that ICIs should be combined with other agents to improve their benefit. It is also recognized that immunotherapy should be implemented in the first-line setting of metastatic treatment to improve overall survival rates . Additio


Anti-human leukocyte antigen antibodies (anti-HLA) play a crucial role in graft. Detection of anti-HLA, both pre- and post-transplant is a crucial investigation in clinical organ transplantation.Three methodologies for the detection of lymphocytotoxic antibodies were compared to establish which of these is best suited to optimise pre-transplant donor-recipient matching.Serum samples from 15 renal transplant patients were tested for the presence of anti-HLA by i) cytotoxic-dependent cross-match (CDCXM), ii) flow cytometric cross-match (FCXM) and iii) Luminex-based donor specific antibody cross-match (DSAXM) method, Confirmatory tests for the presence of preformed HLA antibodies were tested using Luminex methodology.Two (13%) of the 15 patients had positive HLA Class I antibodies (Ab) using all 3 methods. An additional 2 HLA Class I Ab were identified with FCXM/CDCXM. DSAXM identified 1 HLA Class I positive, not indicated by CDCXM/FCXM.High HLA Class II positivity (40%), identified by CDCXM, while DSAXM and FCXM identified two and one patients, respectively. CDCXM produced 4 false-positive results confirmed by lymphocyte single antigen (LSA) assay.The DSAXM method appears to add value in pre-transplantation screening to identify pre-sensitised patients that may not reject the donor graft due to the absence of donor-specific antibodies. Chronic kidney disease (CKD) is a worldwide public health problem. In this context, renal replacement therapy can be done either by dialysis or organ transplantation. However, dialysis is life-long and is associated with reduced quality of life and increased risk of mortality. Kidney transplantation, on the other hand, offers better survival and quality of life benefit for patients with endstage kidney disease relative to dialysisAccording to the South African Renal Registry Annual Report of 2015, the total number of patients on renal replacement therapies (RRT) was 10 360. Gauteng province had the highest number of patients at 3238 (958 from public sector and 2280 from the private sector). Of the 10 360 patients, 1 440 13.9%) were on peritoneal dialysis and 7 529 (72.2%) were on haemodialysis. Of these patients, only 1 391 (13.4%) underwent renal transplantation.9% were It is imperative therefore, that available organs are optimally utilised by ensuring that best-practice methods are applied when screening for potential rejection risk. In this context, the impact of detection of pre-formed human leukocyte antigen (HLA) antibodies reactive with transplanted organs is a well-established practice in clinical renal transplantationPresently, complement dependent cytotoxicity cross-match (CDCXM) remains the most frequently utilised pre-transplant cross-match technique in the South African settingst for renal transplantation at the Steve Biko Academic State Hospital and Jacaranda Private Hospital, Pretoria, South Africa, were enrolled in this study, which was conducted from September 2014 through April 2015.Fifteen patients and their living – related donors, who were candidates undergoing their 1Unfortunately, there is no known details collected at the start of the study regarding potential multiparous or previous transfusions. Written and signed informed consent was obtained from each patient and healthy donor prior to enrolment into the study. The study was approved by the Research Ethics Committee of the Faculty of Health Sciences of the University of Pretoria, South Africa and conformed to good laboratory practice, as well as with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethics certificate reference number: 242/2013.The patients were cross-matched with their potential donors using three different methods viz the Terasaki microlymphocytotoxicity technique (CDCXM), the flow cytometry cross-match (FCXM) and the Luminex-based donor specific antibodies cross-match (DSAXM).CDCXM: This assay was performed using the standard two-stage National Institutes of Health (NIH) technique . Donor T- and B-lymphocytes were isolated via attachment to CD2- and CD19-monoclonal antibody-bound immunomagnetic beads respectively, followed by elution .Briefly, donor T- and B- lymphocytes were incubated with recipient sera for 30 min at room temperature. Rabbit complement was added to each well and the microplates incubated for 60 min at room temperature. The cells were then stained with a fluorescent dye mix, containing acridine orange (AO) and ethidium bromide (EB)  and observed microscopically for cytotoxicity using a fluorescence microscope. DSA (donor specific antigen) binds to the donor cells, activating the complement cascade via the classical pathway, resulting in lysis of lymphocytes detected by differential fluorescence . CDCXM results for both T- and B-cells were considered positive when cell death exceeded that of the negative control by 20%.FCXM: This assay was performed using patients' serum samples and donor peripheral blood mononuclear cells. The technique was performed by mixing 100µl of a 1x106 cells/ml donor lymphocytes with 20µl of patient or control sera and incubated for 60 minutes at room temperature, followed by three washes with phosphate-buffered saline . To identify alloantibodies and differentiate between T and B cells, the re-suspended pellet was then incubated for 30 min in the dark with 5µl of phycoerithrin-CY5 labelled anti-CD3 (CD3-PC5) , 5µl of phycoerithrin labelled anti-CD19 (CD19-PE) monoclonal antibody , and 10µl of goat F(ab)2 antihuman IgG - fluorescein isothiocynate (FITC) antibody . Two wash steps were then performed as described above and cells analysed following re-suspension in 200µl of PBS.Multicolour flow cytometric analysis was performed using the Cytomics FC 500 instrument . Viable lymphocytes were gated on the basis of their forward and side- scatter characteristics. Flow cytometry crossmatch results were expressed as the median channel florescence shift from the negative serum of either the T- and/or the B-cells.DSAXM: This assay was performed as per the manufacturer's instructions . It is used to detect IgG antibodies to donor-specific class I and class II HLA. Donor lymphocytes were isolated from peripheral blood, solubilised with non-ionic detergents, followed by centrifugation to remove cell debris and the supernatant (lymphocyte lysate) decanted.Donor lysates were then incubated with capture beads at room temperature for 30 min in the dark with intermittent mixing to facilitate binding of the solubilized donor HLA molecules to the immunofluorescent beads. The assay included control beads to monitor the amount of background fluorescence in the assay.After incubation, beads/lysate mixtures were transferred to the filter plate and washed. Diluted recipient serum was then added to the beads and incubated with agitation in the dark for 30 min followed by 3 washes. Diluted anti-human IgG streptavidin-phycoerythrin conjugate (SAPE) was then added and incubated for a further 30 min period. After addition of wash buffer, readings were taken on the Bio-Plex array system, Luminex 100 analyser.Determination if a captured bead was positive or negative for DSAXM was performed according to the manufacturer's instructions. The mean fluorescence intensity (MFI) value of each bead was compared to three cut-off values . The cut-off values were calculated from the measured background of three negative control beads in each test well. The process was repeated for each of the two beads to obtain three results . A sample was considered positive if two or more Adjusted MFI Values were positive and a sample was considered to be negative if two or more Adjusted MFI Values were negativeHLA antibody analysis: In this assay, serum samples from the recipients were analysed for HLA class I and class II IgG HLA antibodies using the commercially available LABScreen mix, single antigen (LSA) class I and class II assay kit  on a Luminex platform . The procedure was performed according to the manufacturer's instructions. Briefly, serum was first incubated with LABScreen® beads coated with HLA antigens , using a 96 V-shaped well plate, followed by washing to remove unbound antibodies. Alloantibodies present in the test serum bind to the antigen-coated beads and are detected by addition of R-Phycoerythrin (PE)-conjugated goat anti-human IgG in the second incubation step. All incubations were performed on a gently rotating platform in the dark at room temperature. After the last wash, buffer was added to each well and samples analysed. Trimmed Mean Fluorescence values were recorded using Luminex 100 IS v 2.3 software  for data analysis. Negative and positive controls were included with each assay. Data was normalised to the background value of the negative control serum and a minimum of 50 beads was required for the assay to pass. Data analysis was performed using HLA Fusion 3.0 software . To determine the presence of HLA antibodies in the patients' sera, Mean Fluorescence Intensity (MFI) of the bead was normalized against the MFI of the negative control serum. A bead was regarded as being positive if the MFI value was 1000 or higher. This procedure enables detection of the entire spectrum of HLA allo-antibodies in recipient serum.The recipient group consisted of 9 (60%) females and 6 (40%) males, while the living related donor group consisted of 6 (40%) females and 9 (60%) males.The mean age of the recipients was 39.3 (range 21–61 years) with Caucasians accounting for 73%. Of these, the mean donor age was 41.7 (range 23–58 years) with Africans accounting for 27% of this group. Recipients and their potential donors were all blood group compatible. Donor and recipient profiles are shown in As shown in CDCXM, FCXM and DSAXM results performed on all recipients included in this study are presented collectively in Renal transplantation is the option of choice in RRT for patients with end-stage kidney diseaseThe presence of these antibodies, especially DSA, plays a crucial role in graft rejection and eventually graft lossLuminex DSAXM is a novel procedure in our local setting. To our knowledge, this procedure is not widely used for routine or diagnostic purposes in tissue immunology centres in South Africa, making our study novel in this regard.Although DSAXM Class II false results have occasionally been reportedThe DSA and FCXM methods showed equivalent performance. However, one patient with a positive FCXM class II reaction was negative with DSAXM. It was established that this patient was pre-sensitised, as the presence of HLA class II antibodies were detected as per LSA as shown in The main limitation of this study was the small numbers of subjects recruited overall, particularly for the post-transplant study. One of the reasons being that most recipients were not residing in Gauteng in close proximity to the hospitals.Hence, they were unavailable for follow-up study. Overall, the time period for this study was also limited being a post-graduate study set within a timeframe of 2–3 years and the fact that the Greater Tshwane transplantation institutions do limited LRD transplantations. Nevertheless, potentially interesting findings with translational potential resulted from the study.There have been reports by others of false-positive DSAXM Class II results


The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had a disproportionate impact on Black, Hispanic, and other individuals of color, although data on the effect of a person’s language on SARS-CoV-2 infection are limited. Considering the barriers suffered by immigrants and non-English-speaking families, we tested whether children with a preferred language other than English was associated with SARS-CoV-2 infection. Children from families with a preferred language other than English had a higher predicted probability of SARS-CoV-2 test positivity  during the first wave of the pandemic. This discrepancy continued into the second wave , although the difference compared with families who prefer to speak English decreased over time. These findings suggest that children from non-English-speaking families are at increased risk of SARS-CoV-2 infection, and efforts to reverse systemic inequities causing this increased risk are needed. All practices share a common electronic health record (EHR) . Data related to caregiver preferred language, age, race/ethnicity, insurance status, and SARS-CoV-2 polymerase chain reaction testing were extracted from the EHR for all care episodes (including telemedicine visits) for patients younger than 19 years old from March 1, 2020 through February 28, 2021. Episodes of care that only included preventive services were excluded. Data from March 1, 2019 through February 29, 2020, were extracted as a comparison. SARS-CoV-2 tests performed as part of preadmission testing, pre-procedure screening, and scheduled screening for other reasons  were excluded. This study was deemed exempt by the Children’s Hospital of Philadelphia Institutional Review Board.Summary statistics were performed to describe SARS-CoV-2 testing results in the patient cohort. Demographic factors associated with SARS-CoV-2 test positivity were evaluated using multivariable logistic regression both with and without splines to adjust for temporal trends during the pandemic .During the study period, 64,382 children received care in the designated practices. The overall patient volume and patient demographics were similar between pandemic and comparison periods . Most paP = 0.03).A total of 10,138 patients were tested for SARS-CoV-2, with 1,284 (12.7%) being positive. Test positivity was higher for Spanish-speaking families than for English-speaking families (22.5% versus 12.3%). In multivariable models adjusted for age and race/ethnicity, the predicted probability of test positivity was significantly greater for children with a preferred language other than English during periods of peak disease activity in our region . At the In the urban primary care practices of a large pediatric health-care network in Philadelphia, children from families with a preferred language other than English were more likely to test positive for SARS-CoV-2 than children from families with English as a preferred language. This finding remained consistent throughout both local “waves” of the pandemic, although the difference between groups decreased over time.,These results are consistent with reported findings in adult patients. In a descriptive analysis of patients tested in the University of Washington medicine system early during the pandemic, the proportion of positive SARS-CoV-2 tests was 4.6-fold higher among patients with a preferred language other than English than English speakers.,,Many factors increase risk of SARS-CoV-2 infection and poor outcomes of infection in non-English-speaking families. Working adults in immigrant families may be overrepresented in essential jobs, face difficulties advocating for occupational health protections, and have increased reliance on public transportation.,,Immigrants also lack adequate access to SARS-CoV-2 testing. Language barriers may prevent identification of appropriate testing sites.To address these issues, every effort should be made to conduct large-scale studies of the impact of language on the risk for SARS-CoV-2 infection.,,,Collaboration with trusted community stakeholders is essential to develop and disseminate pandemic-related public health information in languages that reflect local immigrant demographics.,There are several limitations to this study. We compared positive tests from patients within a single health system within the United States. Our cohort was composed primarily of Blacks, and those with a preferred language other than English spoke Spanish predominantly. These findings may not be generalizable to other populations or clinical settings in the United States or other countries. In addition, we only captured positive test results from a single health system, and testing results in this study may not reflect the true positivity rate in our community. Although there are known limitations in how language is recorded in the EHR,In conclusion, this is the first report examining the impact of family-preferred language on SARS-CoV-2 infection in children. We identified disproportionately elevated SARS-CoV-2 test positivity in children from families with a preferred language other than English, which is consistent with reported findings in adults in the United States and other English-speaking nations. As we continue to track and address the disproportionate impact of the SARS-CoV-2 pandemic on historically underrepresented and marginalized communities across the globe, it will be important to track disparities experienced by communities who speak languages other than English. Including such communities in risk mitigation strategies is not only the equitable approach, but also one that will assist in overall efforts to curb the pandemic and prepare for future pandemics.