Prospective and REtrospective Study of nivolumab thERapy in adjuVant MELanoma) is a real-world observational study evaluating the effectiveness and safety of adjuvant nivolumab in patients with completely resected stage III or stage IV melanoma in clinical practice in Belgium and Luxembourg. Patients received nivolumab for up to 12 months per label. The study enrolled 152 patients. At a minimum potential follow-up of 11.4 months, the 12-month and 18-month relapse-free survival rates were 74.7% and 68.4%, respectively, and median relapse-free survival was not reached. Grade 3 or 4 treatment-related adverse events were reported in 14% of patients. Cancer-specific, disease-specific, and generic health-related quality of life were maintained during and after treatment. These results confirm the real-world effectiveness and safety of nivolumab as an adjuvant treatment for patients with completely resected stage III or stage IV melanoma.PRESERV MEL (Prospective and REtrospective Study of nivolumab thERapy in adjuVant MELanoma) is a real-world observational study evaluating the effectiveness and safety of adjuvant nivolumab in patients with completely resected stage III or stage IV melanoma in clinical practice in Belgium and Luxembourg. Methods: Patients were enrolled prospectively and retrospectively during a 2-year period (January 2019–January 2021), and will be followed for 5 years. The results reported here are for the second interim analysis (cutoff date 31 December 2021). The index date was the date of first administration of adjuvant nivolumab. Patients received nivolumab for up to 12 months per label. Outcomes included relapse-free survival (RFS), adverse events (AEs)/treatment-related AEs (TRAEs), and health-related quality of life , Functional Assessment of Cancer Therapy—Melanoma (FACT-M), and EQ-5D-3L instruments). HRQoL was evaluated at group level  and individual patient level . Outcomes were analyzed descriptively. Results: The study enrolled 152 patients  at 15 hospitals in Belgium and Luxembourg. Minimum potential follow-up at time of analysis was 11.4 months. Median age was 60 years (range 29–85), and 53% of patients were male. At 12 and 18 months, the RFS rates were 74.7% : 66.9–80.9) and 68.4% (95% CI: 60.0–75.5), respectively. Median RFS was not reached. Grade 3 or 4 TRAEs were reported in 14% of patients. AEs led to treatment discontinuation in 23% of patients. Deaths occurred in 3% of patients and were not related to treatment. Questionnaire completion rates for HRQoL were high at baseline (90–94%) and at 24 months (78–81%). In the group-level analysis for HRQoL, mean changes in scores from baseline remained stable and did not exceed prespecified thresholds for minimally important differences during and after treatment, except for a clinically meaningful improvement in FACT-M surgery subscale scores. In the individual patient-level analysis for EORTC QLQ-C30 subscales, the percentages of patients who reported clinically relevant scores for fatigue and cognitive impairment increased during treatment (at 9 months) compared with baseline. After treatment cessation (at 18 months), the percentage of patients who reported clinically relevant scores for fatigue decreased. However, the percentages of patients who reported clinically relevant scores for emotional, cognitive, and social impairment increased at 18 months compared with during treatment. Most patients with emotional impairment at 9 and 18 months did not experience disease recurrence . Conclusions: These results confirm the real-world effectiveness and safety of nivolumab as an adjuvant treatment for patients with completely resected stage III or stage IV melanoma. Cancer-specific, disease-specific, and generic HRQoL were maintained during and after treatment. The percentage of patients reporting emotional and cognitive impairment increased after treatment cessation, emphasizing the need for further investigation and tailored supportive care in these patients.Background: Nivolumab, an anti–programmed cell death 1 immuno-oncology therapy, is approved as an adjuvant treatment for patients with completely resected stage III or stage IV melanoma. PRESERV MEL ( AJCC Cancer Staging Manual, seventh edition (AJCC-7)) for a maximum of 1 year .,19.18,19In the individual patient-level analysis for EORTC QLQ-C30 subscales, the percentage of patients who reported scores that exceeded the TCI  for fatiMost patients with emotional impairment at 9 and 18 months did not experience disease recurrence . In a non-protocol, exploratory analysis, stabilization or improvement  in individual global health/QLQ scores with the EORTC QLQ-C30 was documented after recovery from grade 3 or 4 TRAEs in 12 patients who completed their patient-reported outcome questionnaires during and after those TRAEs.At any time point, least squares mean changes from baseline in FACT-M total scores were stable , not exceeding thresholds for MIDs  Figure . AlthougAt any time point, least squares mean changes from baseline in EQ-5D-3L VAS and utility index scores were stable  and did not exceed thresholds for MIDs .Results of the PRESERV MEL confirm the real-world effectiveness and safety of adjuvant nivolumab in patients with completely resected stage III or stage IV melanoma. PRESERV MEL included a cohort of 125 prospectively enrolled patients in Belgium and Luxembourg, representing one of the largest real-world HRQoL datasets reported for an I-O therapy as adjuvant treatment for patients with completely resected stage III or stage IV melanoma. Within this cohort, cancer-specific, disease-specific, and generic HRQoL were maintained during and after treatment. Results from PRESERV MEL were generally consistent with those from the two phase III RCTs evaluating adjuvant nivolumab in patients with completely resected melanoma: CheckMate 238 and CheckMate 915 . Of noteBRAF mutant disease [Baseline characteristics of patients in PRESERV MEL were largely consistent with those of patients in CheckMate 238, which enrolled patients with completely resected stage IIIB, stage IIIC, or stage IV melanoma (per AJCC-7) . Among p disease . However disease . This fi disease ,30,31,32Effectiveness with adjuvant nivolumab in PRESERV MEL was comparable to efficacy in CheckMate 238 and CheckMate 915 ,33, furtSafety was also similar in PRESERV MEL and the CheckMate 238 and CheckMate 915 trials , suggestn = 90) with advanced melanoma who survived > 1 year after initiating I-O therapy frequently reported fatigue, but otherwise demonstrated moderate symptom burden and good HRQoL [PRESERV MEL is the first real-world study to evaluate HRQoL in patients with resected melanoma who were treated with adjuvant nivolumab. Baseline patient-reported outcome scores for patients enrolled in PRESERV MEL were similar to scores for the general population, suggesting that baseline scores in PRESERV MEL were an adequate point of reference for evaluating the impact of treatment on HRQoL. The patient-reported outcomes analysis was also supported by very high questionnaire completion rates at baseline 90–94%) and at 24 months 78–81%). Cancer-specific, disease-specific, and generic HRQoL outcomes remained close to baseline levels after initiation of adjuvant nivolumab, with no clinically meaningful change over time for symptoms, functioning, or global health/QLQ, except for a clinically meaningful improvement in FACT-M surgery subscale scores. Given that the FACT-M surgery subscale addresses the impact of immediate postoperative swelling from surgery, those scores were expected to improve over time. Results for HRQoL in PRESERV MEL are consistent with those in CheckMate 238 and CheckMate 915, in which there were no clinically meaningful differences (based on prespecified thresholds for MIDs) from baseline in HRQoL in patients treated with up to 1 year of adjuvant nivolumab treatment [%. Cancer% and at Although remaining within the thresholds for MIDs at all time points, EORTC QLQ-C30 global health/QoL scores in PRESERV MEL deteriorated slightly during the first 9 months of treatment and later improved, approaching baseline level. The early decline and subsequent improvement/stabilization in EORTC QLQ global health/QLQ scores may have been due to the onset and resolution of immune-related TRAEs. In PRESERV MEL, stabilization or improvement  in individual EORTC QLQ-C30 global health/QLQ scores was documented after recovery from serious and/or grade 3 or 4 TRAEs in 12 patients who completed their patient-reported outcome questionnaires during and after those TRAEs.Mean FACT-M total scores deteriorated during the first 3 months of treatment and later stabilized while remaining within the thresholds for MIDs at all time points. As the FACT-M total score is the sum of the FACT-G and the FACT-M, this decline may be explained by an improvement in symptoms related to surgery.Even though fatigue was the most common any-grade TRAE in PRESERV MEL, EORTC QLQ-C30 fatigue mean scores remained stable over time and did not exceed prespecified thresholds for MIDs. Moreover, when considering the TCI  in the iImpaired objective and subjective cognitive functioning are also common in patients with cancer and may be related to the disease itself, cancer treatments, fatigue or emotional distress, and interference with the ability to return to work . In PRESThis study has certain limitations. Firstly, this was a non-comparative, observational, descriptive study that did not control for multiple comparisons. In addition, results of this study may have been influenced by differences in characteristics between the baseline population and patients remaining on treatment at later time points, who were more likely to have responded to or tolerated treatment. Although patient-reported outcome completion rates  were high throughout PRESERV MEL, the number of patients completing the patient-reported outcome questionnaires decreased at later time points. This decrease may have been due in part to patients having been enrolled consecutively; thus, some patients had not yet reached the later time points at the time of the analysis. In patient-reported outcome studies in general, the effect of treatment on HRQoL may be underestimated because of low patient numbers at later time points resulting from discontinuation related to disease progression, AEs, and death . AdditioIn summary, interim results of the PRESERV MEL study, which was conducted in 152 patients in Belgium and Luxembourg, confirm the real-world effectiveness and safety of adjuvant nivolumab in patients with completely resected stage III or stage IV melanoma and suggest that HRQoL with adjuvant nivolumab treatment is maintained. Patients’ symptoms, functioning, and overall HRQoL remained stable over the course of treatment, as measured by cancer-specific, disease-specific, and generic patient-reported outcomes. Six months after the maximum treatment duration (at 18 months), some patients reported clinical important emotional, cognitive, and social impairment, which needs to be further investigated and justifies offering tailored supportive care/treatment for these patients. Findings from this study were generally consistent with those from the phase III CheckMate 238 and CheckMate 915 trials , further


Effective cross-cultural care is foundational for mitigating health inequities and providing high-quality care to diverse populations. However, medical school teaching practices vary widely, and learners have limited opportunities to develop these critical skills. To understand the current state of cross-cultural education and to identify potential opportunities for improvement, we disseminated a validated survey instrument among medical students at a single institution.Learners across 4 years of medical school participated in the cross-cultural care assessment, using a tool previously validated with resident physicians and modified for medical students. The survey assessed medical student perspectives on (1) preparedness, (2) skillfulness, and (3) educational curriculum and learning environment. Cross-sectional data were analyzed by class year, comparing trends between school years.p<0.05) for 7 of 12 preparedness items and 4 of 9 skillfulness items. Less than 50% of students indicated readiness to deliver cross-cultural care by their fourth year in 9 of 12 preparedness items and 6 of 9 skillfulness items. Respondents identified inadequate cross-cultural education as the primary barrier.Of 700 possible survey responses, we received 260 (37% response rate). Fourth-year students had significantly higher scores than first-year students (Medical students reported a lack of readiness to provide cross-cultural care, with self-assessed deficiencies persisting through the fourth year of medical school. Medical educators can use data from the cross-cultural care survey to longitudinally assess students and enhance curricular exposures where deficiencies exist. Optimizing cross-cultural education has the potential to improve the learning environment and overall patient care. Although cross-cultural education is an integral aspect of undergraduate medical curricula, implementation and assessment remains inconsistent.4 As a result, unexplored and under-considered sociocultural factors in patient–physician communication can lead to unequitable care, poor adherence, mistrust, and worse health outcomes.6 Cross-cultural care training can empower health care professionals to recognize a patient's health beliefs, values, and preferences, thereby improving quality of care and mitigating disparities.7–9 However, such benefits will only be realized if medical education can support development of these skills.10The COVID-19 pandemic has laid bare racial, ethnic, and other sociodemographic health inequities. Improving cross-cultural care is a necessary step in understanding and addressing the sociocultural factors11 A significant barrier to progress is the gap in knowledge around current practice and areas of deficiency. Prior studies have documented variable content, teaching, timing, and assessment of learners.12–15 Thus, although significant efforts are directed at improving cross-cultural competency, relatively few learners are likely to graduate medical school equipped to provide patient-centered care across diverse cross-cultural contexts.17 For example, fewer than half of students at Harvard Medical School felt comprehensively prepared to deliver cross-cultural care.18 Thus, although cross-cultural care has the promise of improving outcomes, more purposeful efforts to teach and assess cross-cultural education are necessary.Cross-cultural care curricula are required by the Accreditation Council for Graduate Medical Education, in recognition of the need to reduce bias and provide safe, high-quality care to diverse patients.19 We hypothesized that fourth-year students would have greater preparedness and skills than first-year students, years 2 and 3 were clinical. These data serve as a roadmap for curricular innovation, including opportunities for enhancing didactic and experiential learning.To gain a deeper understanding of medical students' perspectives on cross-cultural care and education, we adapted and distributed the validated cross-cultural care survey (CCCS) for medical students at the University of Michigan Medical School. We assessed self-perceived skills and preparedness as well as the cultural climate and learning environment in medical students all 4 years. We did not implement a specific intervention as part of this study; rather, we used the CCCS to assess the existing curriculum, found on the University of Michigan Medical School website.18 to investigate the preparedness and skillfulness of medical students at providing cross-cultural care. We also asked about the learning environment at the medical school and captured personal and professional characteristics. We obtained permission to adapt and use the modified CCCS for medical students18 and made minor modifications to ensure relevance of all questions to the institution.We used the validated Internet-based survey, previously utilized at Harvard Medical School® and distributed the survey, by e-mail listserve for years 1–4, in early February to all medical students. We chose to exclude dental and MD/PhD students from the current study due to differences in training schedule and curriculum. Participation in the online survey was voluntary for all students. To enhance our response rate, we sent out three reminder e-mails to students separated by 5 days.The survey was conducted in accordance with the University of Michigan institutional procedures (IRB No. HUM00174766). We inserted the survey into QualtricsXMTwo constructs related to readiness to provide cross-cultural care were measured: (1) preparedness to care for specific types of patients and (2) self-assessment of specific cross-cultural skills. We also included questions assessing the educational curriculum and climate around cross-cultural care.To assess preparedness, medical students indicated how prepared they felt to care for a series of types of patients . The list includes patients from cultures different from their own; patients with health beliefs at odds with Western medicine; patients with religious beliefs that might affect treatment; patients who were new immigrants to the United States; patients with limited English proficiency; lesbian, gay, bisexual, and transgender patients; patients with disabilities; and patients who use alternative or complementary medicines.Students were asked to assess their skill level in performing tasks relevant to treating culturally diverse patients (a response of 1=Not at All Skillful to 5=Very Skillful). These include determining how to address patients from different cultures, assessing patients' understanding of their illness, identifying mistrust, negotiating treatment plans, assessing English proficiency, identifying relevant cultural and religious beliefs, understanding decision-making roles, working with interpreters, and counseling patients about their use of complementary or alternative medicine.Respondents also responded to questions relating to how the educational curriculum incorporated cross-cultural learning and the overall climate for cross-cultural learning. To assess the curriculum, students were asked to specify the courses completed and how each class prepared them to interact with culturally diverse patients using the following rating scale .To assess the educational climate, we asked medical students to rate “how much of a problem”  the following has been during medical school: (1) lack of practical experience caring for diverse patients; (2) inadequate cross-cultural care training; (3) absence of good role models or mentors in cross-cultural care; (4) dismissive attitudes about cross-cultural care among physicians; and (5) dismissive attitudes about cross-cultural care among fellow students. We also asked students to rate  their level of agreement on the inclusion of cross-cultural care curricula into courses and clinical practice. Other questions assessed medical student demographic characteristics, including gender, race/ethnicity, language, and school year.p<0.05 was considered statistically significant for all analyses.Chi-square analyses were used to analyze differences in preparedness and skillfulness by medical student year in the curriculum. All preparedness and skillfulness item responses were dichotomized into “Unprepared/Unskilled”  and “Prepared/Skilled” (response of “4” or “5”) and examined for change between year 1 and 4. Furthermore, responses about the extent of problems during medical training were dichotomized into the categories of “No Problem” (response of “1”) and “Problem”  and analyzed with Crosstab analyses. Demographics were examined using frequency analyses. Of 700 possible survey responses across the medical school, we received 311 responses. Of those, we excluded 51 due to incomplete responses. After accounting for these exclusions, the final sample was 260 (response rate 37.1%).p<0.05) between year 1 and 4 students were found for 7 of 12 preparedness items. Exceptions involved lack of difference in caring for patients who either have distrust of the U.S. health system, use alternative medicines, harbor religious beliefs that affect treatment, have health beliefs at odds with Western medicine, or are new immigrants. The top 3 items that showed the greatest improvement across years were caring for patients either in general (+55.3%) or caring for patients with cultures different from one's own (+25.9%), or who are transgender (+22.2%).The mean differences between first-year students and fourth-year students for the 12 reported preparedness items was 17.8% (range 2.7–55.3%). For the nine skill items, the mean difference was 19.6% (range 7.6–33.4%). Statistically significant differences (p<0.05) between year 1 and 4. The three lowest item differences between first-year and fourth-year students included identifying religious beliefs that affect clinical care (7.6%), counseling on alternative medicine use (10.2%), and identifying cultural customs that affect clinical care (10.4%).For the nine skillfulness items, four items  showed statistically significant differences  when first-year students were compared with third-year students.Within the learning environment, only 3 medical courses of 12 queried were reported as useful for cross-cultural education; those deemed useful included doctoring small groups, clinical rotations, and clinical electives. Respondents ascribed high importance to seeing a diverse mix of patients and highlighted the need for greater integration of cross-cultural education into existing curricular offerings. On a scale from 1 to 10, first year medical students responded with a mean of 9.1 on the importance of seeing a diverse racial/ethnic patient mix, versus 8.7, 8.5, and 7.8, in years 2, 3, and 4, respectively. Notably, 51.2% of all students disagreed that didactic course directors incorporate cross-cultural considerations into teaching and 39.3% of students disagreed with a statement that clinical faculty incorporate cross-cultural education into teaching . Overall18 Many patient health outcomes are known to be heavily influenced by social determinants of health, and improving cross-cultural care is part of the larger moral imperative to widen our lane in medicine.20 Cross-cultural education equips doctors to care for individuals across diverse backgrounds, including race, ethnicity, gender orientation, socioeconomic status, cultural affiliation, and religious beliefs.21 The COVID-19 pandemic has underscored the importance of teaching students cross-cultural care to counter social and medical inequities.Despite growing awareness of the importance of cross-cultural care to medical education, there remains a gap between the aspiration of medical educators to instill such skills and the perceived readiness of learners to apply them. We found that our medical students do not feel adequately prepared to provide patient-centered care in a cross-cultural context, and prior work suggests that such lack of readiness persists among physicians-in-training and at later career stages.11 However, the results of our survey reinforce prior observations noting a lack of robust and multifaceted experiences to promote cross-cultural care.18 An unanswered question is whether these shortfalls reflect inadequacy of the resources made available or inadequate integration of such resources into curricula.Some of the available resources to improve cross-cultural care include checklists or assessment tools, which can guide medical educators in the design of courses that enable students to engage with patient diversity.Our survey data also reveal the ambivalence of students regarding efforts in this area. For example, 43% of students felt that learning about culturally diverse patients was not emphasized as a priority, and half felt that course directors did not consistently incorporate cross-cultural issues into teaching. This insight is crucial, as most medical students cited inadequate cross-cultural education and lack of experience as the foremost barriers to their maturation in this area. In addition, two-thirds of respondents noted a lack of role models. Half reported dismissive attitudes about cross-cultural care among attending physicians and fellow students. Such concerns were most marked in first- and second-year students, who were judging the learning environment based on lecture and small group dynamics, perhaps signaling a need for update of course materials and need to bring cross-cultural clinical experiences to earlier years.The early effects of curricular improvements might be present within our data, especially during the clinical years, which include the second half of second year and all of third year. The third-year class demonstrated greater self-reported preparedness and skills in multiple items compared with the fourth-year class, but no measures were found significant . When st22 This ethos and limited exposure to patients with other beliefs might partially account for low levels of preparedness to care for those who use alternative medicine or distrust the health system. For example, in our study, students reported seeing less than 20% of racial diverse patients and less than 10% of patients with limited English-speaking proficiency and were more likely to report lack of preparedness or skillfulness.Medical students' exposure to diverse cultural groups influenced their perception of readiness to engage in cross-cultural care. During medical training, students are socialized into a professional ethos that privileges Western medicine and normalizes institutional health care.18 the LGBT and racial categories of patients received the highest levels of students' self-reported preparedness. This finding might reflect increasing social tolerance, or exposure, especially among the increasingly diverse medical student body. Nonetheless, unconscious bias can also adversely affect outcomes,23 and future study is necessary to verify whether self-reported perceptions will translate into equitable care and outcomes.Similar to the Harvard medical student study,25; experiences in diverse communities afford breadth of understanding on the culture, needs, and challenges of marginalized groups. Such experiences can also instill a passion for change, encourage advocacy, and teach physicians the skills to counter the negative health outcomes imposed by poverty, inequality, and discrimination.26Our data corroborate the link between clinical experiences and readiness for cross-cultural care. Experiential learning is a critical aspect of cross-cultural education27Our results also highlight the importance of integrating cross-cultural education into early didactic years, which can be achieved through community experiences, service learning, and simulations. Course providers can explore ways to increase medical student exposure to immigrants, indigenous people, and other cross-cultural groups. Standardized patient simulations involving patients from different backgrounds and beliefs can offer a controlled environment for students to gain skills and confidence. Simulations can also teach shared decision making, which improves patient adherence, satisfaction, and outcomes.28 Case-based learning can also help students understand how building trust between a patient and the medical team can improve care.27Community experiences can promote confidence in building rapport with patients who mistrust the health system or have different religious beliefs.18 In our survey, learners revealed limited growth from first to fourth year in three areas: identifying religious beliefs that affect clinical care, counseling on alternative medicine use, and identifying cultural customs that affect clinical care. Strategies that can address this limitation include practicing open-ended questions, shared decision making, and teach back methods. Cultural humility affords sensitivity to patients from different backgrounds and supports collaborative physician–patient decision making by diminishing power differentials and promoting trust; a stance of humility avoids the pitfall of assuming one has achieved cultural expertise or completed learning.12 The ideal atmosphere actively invites patient contributions and values patient experience and perspective alongside physician expertise.29Communication lies at the heart of cross-cultural care, which requires open dialog and cultural humility.12 Community outreach clinics and service experiences can also promote cultural humility and advocacy to reduce health disparities.26A few pedagogical approaches to developing patient–physician dialog and cultural humility have met with success: small-group discussions, personal journals, constructive professional role models from cultural groups, and videotaping with feedback.22 and doing so requires a willingness to go beyond the immediate biomedical considerations of patient care. In the busy clinical setting, cross-cultural education is often fragmented or omitted, perhaps partly because some clinical teachers themselves lack sufficient cross-cultural awareness.30 The limited literature on faculty development in this area suggest a paucity of faculty with expertise in designing and implementing cross-cultural curricular content31; comprehensive training of faculty and promoting mentorship across differences can provide a pathway for educators to offer structured cross-cultural experiences for learners.32Clinical teachers have a vital role in modeling cross-cultural care,16 In the future will consider further tracking of students longitudinally over the course of their schooling and ideally into residency. Although the results presented in this study are consistent with prior work in resident physicians and at Harvard Medical School, a future multi-institutional rollout of the survey would improve generalizability.Our study has several limitations, primarily relating to potential sources of bias and limited generalizability. These include potential for selection bias, recall bias, or subjective responses. In addition, the cross-sectional design precluded assessment of individual classes over time. Also, we assessed students near the middle of the year, foregoing an assessment of students before entering medical school and omitting any additional improvement in the final portion of the fourth year. Although the survey instrument was validated, students may under- or overestimate their readiness.Last, the study assessed current state without investigating specific interventions, but the work from this study was part of the impetus for ongoing innovations at our institutions in diverse areas, including the rollout of a health disparities curriculum, introduction of a history of racism course, and innovations around interprofessional education, all of which await formal study. The present study supports the feasibility of using the cross-cultural care validated survey as a diagnostic tool that can pinpoint areas for improvement and thereby assist in cultivating empathetic, and culturally aware clinicians who can provide safe, high-quality care for diverse patients.The CCCS, originally developed for assessment of physicians-in-training during residency, is a validated tool that can identify areas where medical school curricula can be improved. At University of Michigan, medical students perceived limited preparedness and skillfulness in care for culturally diverse patients, most marked in early years, but persisting through the fourth year of medical school. Respondents also highlighted limitations in cross-cultural curriculum and climate, suggesting a potential role for moving some cross-cultural education upstream to ensure meaningful exposures both early in medical school and sustained across the continuum of clinical experiences. The CCCS can guide curricular innovation in cross-cultural care through experiential learning, simulation, discussion, and practice. Finding from this survey provided an impetus to expand our own offerings and will hopefully also spur medical educators to use this tool to identify opportunities to improve cross-cultural education at their own institutions.


Knowledge on physical activity recovery after COVID-19 survival is limited. The AFTER (App-Facilitated Tele-Rehabilitation) program for COVID-19 survivors randomized participants, following hospital discharge, to either education and unstructured physical activity or a telerehabilitation program. Step count data were collected as a secondary outcome, and we found no significant differences in total step count trajectories between groups at 6 weeks. Further step count data were not analyzed.The purpose of this analysis was to examine step count trajectories and correlates among all participants (combined into a single group) across the 12-week study period.Linear mixed models with random effects were used to model daily steps over the number of study days. Models with 0, 1, and 2 inflection points were considered, and the final model was selected based on the highest log-likelihood value.P<.001) steps per week, culminating in an average daily step count of 7658  at the end of week 3. During the remaining 9 weeks of the study, weekly step counts increased by an average of 67  steps per week, resulting in a final estimate of 8258  steps.Participants included 44 adults (41 with available Fitbit [Fitbit LLC] data). Initially, step counts increased by an average of 930 (95% CI 547-1312; Participants showed a marked improvement in daily step counts during the first 3 weeks of the study, followed by more gradual improvement in the remaining 9 weeks. Physical activity data and step count recovery trajectories may be considered surrogates for physiological recovery, although further research is needed to examine this relationship.ClinicalTrials.gov NCT04663945; https://tinyurl.com/2p969ced Low physical functioning and physical inactivity are observed among people who were hospitalized with severe COVID-19 . UnderstFew studies have detailed data on physical activity levels after SARS-CoV-2 infection and hospitalization. A large-scale population-based study found that those who previously reported performing aerobic and muscle strengthening activities that met or surpassed physical activity recommendations in the 2 years prior to the COVID-19 pandemic had a lower risk of infection, severe illness when infected, and COVID-19–related death . AnotherWearable fitness trackers, including smartwatches, enable objective, continuous, long-term monitoring and have various applications in health care settings, such as health monitoring and medication adherence monitoring . HoweverThe AFTER (App-Facilitated Tele-Rehabilitation) program for COVID-19 survivors was a pilot study that randomized participants to either education and unstructured physical activity or a telerehabilitation program following discharge for COVID-19–related hospital admissions . Step coA detailed description of the AFTER study methods can be found elsewhere . BrieflyThe education and exercise components of the app were only prescribed to the intervention group, although participants in the control group could access educational modules and physical function testing on their own.The AFTER trial was approved by the University of Colorado Institutional Review Board (reference number: COMIRB 20-2415) and registered on ClinicalTrials.gov .P<.05, and there was no adjustment for multiple comparisons. R software  was used for all data cleaning, summary statistics, and graphics.We analyzed all participants, who were combined into a single group. Linear mixed models with random effects were used to model daily steps over the number of study days. Models with 0, 1, and 2 inflection points were considered, and the final model was selected based on the highest log-likelihood value. The final model included a random intercept and random effects on both the pre– and post–inflection point trajectories. All participants with any synced Fitbit activity data were included. Model robustness was evaluated by reviewing results after excluding records with a daily step count beyond 2 SDs from the participants’ overall average. The adjustment variables considered in the analysis included treatment group, sex, age, and BMI. Baseline demographics and COVID-19 hospitalization information were presented by treatment group and were compared by using a chi-square test or the Mood median test, as appropriate. All statistical analyses were performed in SAS 9.4 (SAS Institute Inc); we assumed a significance level of Participants included 44 adults who enrolled between December 2, 2020, and July 2, 2021. Fitbit data were available for 41 participants, with a median of 78 (IQR 64-83) contributing days. The median age of participants was 53 (IQR 44-61) years, and 46% (19/41) were female. The median number of days that participants spent in the hospital was 4 (IQR 2-8), and 22% (9/41) of participants were admitted to the intensive care unit. The average length of time between hospital discharge and enrollment was 3.5 weeks. The median number of steps for the cohort at baseline was 4928 (IQR 3083-7574).P<.001) steps per week, culminating in an average daily step count of 7658  at the end of week 3. During the remaining 9 weeks of the study, weekly step counts increased by an average of 67  steps per week, resulting in a final estimate of 8258  daily steps  compared to that of the intervention arm. Female participants had 1153  fewer daily steps compared to those of male participants. On average, older participants and those with a higher BMI had a lower daily step count. For every 1-year increase in age, the daily step count was lower by 67  steps on average. Similarly, for every 1-point increase in BMI, the average daily step count was lower by 182  steps.Covariates that were considered potentially predictive of an increase in daily steps over the 12-week study period were put into a fully adjusted model, including sex, BMI, age, and treatment group. Given substantial variation in steps, differences between treatment groups and sex failed to reach statistical significance. Participants in the control group had a higher average daily step count  that were provided to the control group and the regular check-ins with the study team may help to explain the similar between-group results . In the P=.003) [Our study is among the first to report objective physical activity data (step counts) from COVID-19 survivors. In the only other study that we are aware of, Hunter et al [P=.003) . Our parA limitation of our data set is that we do not have metrics regarding wear time for those who had Fitbit devices; thus, our daily average step counts may not reflect true activity and may have been underestimated. Moreover, this trial occurred at a single center in Colorado; therefore, our results are most generalizable to similar populations. Finally, our sample is small, and a 12-week follow-up is considered short-term. However, given that this is the first study to report post–hospital discharge step count data from a heterogeneous group of COVID-19 survivors, we believe that our results are noteworthy.Our findings, in combination with prior studies, suggest that physical activity data and step count recovery trajectories may be considered surrogates for physiological recovery, although further research is needed. Further, while activity trajectories may differ based on the presence or absence of post–COVID-19 conditions, monitoring changes over time can provide immediate, objective feedback to patients and support patient-specific goals in the return to prior levels of function following a hospitalization.


Drug-induced liver injury (DILI) presents significant diagnostic challenges, and recently artificial intelligence-based deep learning technology has been used to predict various hepatic findings. In this study, we trained a set of Mask R-CNN-based deep algorithms to learn and quantify typical toxicant induced-histopathological lesions, portal area, and connective tissue in Sprague Dawley rats. We compared a set of single-finding models (SFMs) and a combined multiple-finding model (MFM) for their ability to simultaneously detect, classify, and quantify multiple hepatic findings on rat liver slide images. All of the SFMs yielded mean average precision (mAP) values above 85%, suggesting that the models had been successfully established. The MFM showed better performance than the SFMs, with a total mAP value of 92.46%. We compared the model predictions for slide images with ground-truth annotations generated by an accredited pathologist. For the MFM, the overall and individual finding predictions were highly correlated with the annotated areas, with R-squared values of 0.852, 0.952, 0.999, 0.990, and 0.958 being obtained for portal area, infiltration, necrosis, vacuolation, and connective tissue (including fibrosis), respectively. Our results indicate that the proposed MFM could be a useful tool for detecting and predicting multiple hepatic findings in basic non-clinical study settings. The diverse histological findings of DILI can include inflammation, necrosis, cholestasis, fibrosis, nodular regeneration, vascular injury, and bile duct destruction. In a histological sample, these lesions may exist in combinations that can be difficult to discriminate3. Therefore, pathologists must provide an expert interpretation of the tissue changes in light of the patient’s medical and pharmaceutical history when they evaluate a case of DILI. Because of the potential complexity of these cases, a systematic approach is recommended4. Examination of animal models is a critical step in the preclinical investigation of DILI5. Several animal models have been studied for predicting DILI, with acetaminophen (APAP) hepatotoxicity having been extensively investigated6. Although animal studies are useful, they can only predict about 70% of human hepatotoxicity outcomes5.Drug-induced liver injury (DILI) is the most frequent safety-related cause of drug withdrawn from the market7. Researchers have used deep-learning techniques for both clinical and non-clinical applications, particularly those for which classical imaging analysis methods cannot be automated9. In recent years, artificial intelligence (AI)-based digital pathology has shown great promise as a means to increase healthcare availability and accuracy in many aspects of medicine12. In addition to the importance of its growing sub-segments, such as digital molecular pathology and pathology informatics, digital pathology could potentially alter the traditional core functions of pathology and improve the workflow of pathologists14.Digital pathology is a sub-field of pathology that uses tools and systems for digitizing, evaluating, and analyzing pathology slides and associated metadata15. A technique that has shown great promise is the mask region-based convolutional neural network (Mask R-CNN), a detection-based segmentation model17 comprised of two main stages: (i) object detection and localization; and (ii) using the features of the detected regions to classify them, assign their final localization, and segment them16. Recently, Mask R-CNN-based approaches have been used in medical research19.Medical imaging research has explored various machine-learning techniques, including many classifier and clustering algorithmsMost DILI-related liver lesions are complicated, and recognizing such complex lesions is critical to diagnosing DILI and assessing its progression and/or severity. In the present study, we applied Mask R-CNN-based deep learning to develop a more efficient tool for screening and examining complex liver lesions in a rat model of liver injury. To test the real-world usefulness of our prediction method, we established a set of single-finding models (SFMs) and a multiple-finding model (MFM). We trained, validated, and tested the AI algorithms by comparing the SFM- and MFM-predicted findings with ground-truth annotations generated by an accredited toxicological pathologist.The Mask R-CNN was trained to identify various hepatic injury lesions  and normal features  to enable the algorithm to discriminate among these findings. To induce liver injury, N-nitrosodimethylamine (NDMA), APAP, or corn oil was applied for the periods listed in Table The procedures used to establish the SFMs and MFM for liver injury are shown schematically in Fig. To perform real-world testing of the trained SFMs, we assessed their ability to predict hepatic lesions and normal features in a total of 100 slide images, each of which corresponded to one third of a whole-slide image. Each SFM was used to predict the corresponding findings Fig. B.Figure Several deficiencies were found in the SFM prediction results, as shown in supplementary material 2 = 0.914) with the annotated results; by contrast, the combined group  showed a low correlation (R2 = 0.022)  with the annotated results than did the combined group. For the SFM prediction of infiltration, no strong correlation was observed , infiltration (green), necrosis (white), connective tissue plus fibrosis (blue), and vacuolation (red). The results obtained using the MFM showed patterns comparable to those of the ground-truth annotation  SFMs generally overestimated or misestimated the areas of the features for which they were trained compared to the annotated areas Fig. A. In conIn the present study, we applied Mask R-CNN-based deep learning to assess several types of liver lesions and normal features in a non-clinical study setting. The models were established through training and validation for single or multiple hepatic findings and the performance was assessed by comparison with pathologist-annotated slide images. Additionally, we compared the set of SFMs with the MFM. Each SFM yielded a mAP value above 85%, while the total mAP for the MFM was 92.46%, suggesting that the models had been successfully established. Compared to the SFMs, the MFM showed a higher correlation with the ground-truth annotation results assigned by an accredited toxicologic pathologist. Our results suggest that this new Mask R-CNN-based algorithm could be a useful tool that applies deep-learning to digital pathology, and that the MFM is more useful than individual SFMs in predicting different and complicated liver lesions and normal features.22, our results indicate that the MFM could be useful for predicting real-world histopathological liver lesions.All of the slide image results of the SFMs and MFM are shown in Supplementary material 23 and image analysis of various hepatic lesions related to liver injury and liver toxicity is considered to be critical for the diagnosis and severity assessment of this disease24. Recently, deep learning based on deep neural networks has been applied to the assessment and diagnosis of various lesions in non-clinical and clinical studies26. The use of deep-learning algorithms in non-clinical studies can allow rapid, quantitative, and consistent histopathological assessment in novel drug development processes and toxicological studies with implications for clinical settings. Therefore, a classification network capable of detecting and discriminating particular lesions may be favored over object detection and/or segmentation, which require high calculation loads and labor-intensive annotation procedures. Deep learning-based diagnosis or quantification of liver lesions has been attempted in several studies29. Puri tested the ability of automated machine-learning models to classify DILI injury patterns from whole-slide images, and obtained an average precision of 98.6%30. Xu et al. employed deep learning for DILI prediction in response to 198 drugs and observed good accuracy, sensitivity, and specificity2. In addition to these studies, there are several reports on the performance of the model in the dataset environment; however, AI-based pathological analysis has not yet been sufficiently studied using real-world slide images31. In the present study, we confirmed the possibility of applying AI-assisted image analysis in a drug-induced liver injury rat model. The results suggested that the proposed trial has potential for use in clinical applications. However, the proposed approach was not validated in human samples, untrained complex lesions, severity scoring, or various real-world settings. Moreover, in the present study we compared the number of pixels, not the actual concordance of pixel location between ground truth annotation and model prediction. Therefore, further studies will be needed before the method can be applied to human diagnosis.The histological spectrum of DILI is broadHere, we used a Mask R-CNN algorithm to assess hepatic lesions induced by NDMA, APAP or corn oil in rats. The study results suggest that the Mask R-CNN-based MFM could be a useful tool for detecting and predicting multiple and complicated toxic liver lesions in basic non-clinical study settings.Sprague–Dawley (SD) rats  were obtained from Orient Bio, Inc. (Republic of Korea). The rats were maintained at a temperature of 23 ± 3 °C and a relative humidity of 30–70% under a 12 h light/dark cycle, and allowed to acclimate for 7 days prior to the first administration of drug or vehicle. All experimental procedures involving animals were approved by the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and Institutional Animal Care and Use Committee (IACUC). All animal experiments were conducted in accordance with the ARRIVE guidelines and all methods were carried out in accordance with the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The rats were randomly assigned to four groups: control (distilled water), N-nitrosodimethylamine (NDMA)-treated, acetaminophen (APAP)-treated, and corn oil-treated. The animal study design and liver tissue slide preparation procedure are detailed in Table 19. Briefly, the slide images (20×-magnified) were cropped into 448 × 448 pixel tiles, and VGG Image annotator 2.0.1.0  was used to label all lesions and normal features. An accredited toxicologic pathologist assessed the annotated lesions and normal features, and verified findings were labeled and used to first train and then test the Mask R-CNN algorithm. To distribute the annotated image tiles into training, validation, and test datasets (approximately 7:2:1), we used the train_test split function of the scikit-learn package. To improve the training dataset, we used data augmentation: eight-fold augmentation was enabled using image-augmentation techniques such as reversal, rotation, and brightening. For SFM and MFM, total datasets of 46,284 and 48,772 image tiles, respectively, were used to train, validate, and test the algorithm on liver injury lesions and normal features. The numbers of image tiles used for each training, validation, and testing set are presented in Supplementary material A total of 100 liver samples representing 25 sections per group were H&E-stained and digitized into slide images using an Aperio XT slide scanner . Slide images of liver sections were scanned using an Aperio ScanScope XT  with a 20 × objective and bright-field illumination. The scan resolution was 0.4993 μm per pixel and the images were saved as JPEG image-compression files. The data were prepared for detection of portal area, connective tissue, vacuolation, infiltration, necrosis, and fibrosis, as previously described19. Briefly, an open-source framework for machine learning (Tensorflow 2.1.0 with a Keras 2.4.14 backend) powered using an NVIDIA RTX 3090 24G GPU was used for training, along with Matterport Mask R-CNN 2.1 . The Mask R-CNN algorithm (https://github.com/matterport/Mask_RCNN) consisted of two stages: first, a region proposal network (RPN) proposed candidate object-bounding boxes; and second, RoIAlign extracted features that would be used to predict pixel-accurate masks. In RoIAlign, bi-linear interpolation is applied to determine the exact values of input features at four regularly sampled locations for each RoI bin, and the results are aggregated using max pooling.All algorithm-training procedures, including the distribution of data, were undertaken as previously described19, reflects the ratio of the area overlaid by the union of the predictions to that overlaid by the ground truth. The mAP value, which reflects model accuracy, was generated. We used the transformed mAP, which goes to 0 when an image is identified as containing a misprediction. This transformation was used to analyze the error cases in more detail, investigate the basis for correct and incorrect predictions, and evaluate the model performance more strictly.In the SFMs, four lesions and two normal features were trained on separate Mask R-CNNs to create a model for each lesion and normal feature. The MFM was created as a single model by learning all four lesions and two normal features in one Mask R-CNN. The hyperparameters applied for training are presented in Table One-hundred slide images were used as the confirmation set. Each slide image, which was 10,752 × 10,752 pixels and had not been used during the training, was cropped by Aperio Image Scope version 12.4.0  from whole-slide images that were scanned by an Aperio ScanScope XT  using a 20 × objective and bright-field illumination. Before being submitted for confirmation, each hepatic lesion, normal feature, and tissue type was annotated by an accredited toxicologic pathologist; this was used as the ground truth that would later be compared with the algorithm-based prediction. The ground-truth annotations for all lesions and normal features were applied using VGG Image annotator 2.0.1.0 , as described for the annotation of findings used in model training. The annotated areas were calculated based on pixel counting. Each cropped slide image was divided into tile images of 448 × 448 pixels. The trained algorithm was used to predict each hepatic lesion and normal feature, and the prediction mask-bearing cropped images were merged into an overall slide image. Linear regression was used to compare the calculated prediction mask areas with the ground truth annotated areas. The histopathological annotations used to label the images are shown in Supplementary material Supplementary Information.


Patterns of analgesic use before and after total joint replacement (TJR) in patients with knee/hip osteoarthritis (OA) is not well reported.This retrospective longitudinal analysis used JMDC claims data of patients who underwent knee/hip replacement surgery from 2010 to 2019. Primary outcome was proportion of patients using analgesics pre-surgery, immediately post-surgery, and in post-surgery period. Factors affecting post-surgery analgesic withdrawal and opioid prescriptions were assessed using logistic regression.Of all (N = 3168) patients, those with knee OA (91.1 %) and hip OA (82.5 %) used analgesics pre-surgery, and 96.1 % with knee OA and 84.9 % with hip OA required analgesics even 3 months post-surgery. NSAIDs were most commonly used pre- and post-surgery in both OA groups. Before surgery, 15.6 % (knee OA) and 13.7 % of patients (hip OA) used weak opioids, and 23.1 % (knee OA) and 10.5 % (hip OA) of patients continued them post-surgery. Strong opioid use was noted in 2.2 % and 1.2 % of patients pre-surgery, and 5.8 % and 3.4 % of patients post-surgery in the knee and hip OA groups, respectively. Using pre-operative oral NSAIDs  and weak opioids  associated with withdrawal of post-surgery analgesics in patients with hip OA, and using intra-articular hyaluronic acid pre-surgery  was significant in patients with knee OA. Using weak  and strong opioids  pre-surgery associated with post-operative opioid use in patients with hip OA, and weak opioid use was significant in patients with knee OA .This study reported difference in analgesic use before and after TJR, and that many patients required analgesics even 3 months after TJR surgery in Japan. Pre-operative analgesic use associated with continued use after surgery. Optimal pain management before and immediately after TJR is important to reduce post-operative analgesic use, especially opioids. These patients were followed-up for up to 3 months before and after surgery, and had ≥2 prescriptions (with ≥1-month gap) for analgesic-use after the initial diagnosis of OA. The study observation period was from January 1, 2010, to December 31, 2019. Index date was the date when TJR was performed. Analgesics included oral and non-oral non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, intra-articular hyaluronic acid and steroid injection, weak  and strong opioids , duloxetine, and vaccinia virus-inoculated domestic rabbit inflammatory skin extract. Analgesics not indicated in Japan for OA were excluded. Key exclusion criteria included patients with THR/TKR during both pre-surgery (from index date −91 days to index date −1 day) and post-surgery periods (from index date +7 days to index date +89 days), and those with ICD-10 codes of malignancy , and rheumatoid arthritis  during the observation period.The present study involved use of data within a pre-existing database, and primary data collection was not performed. The Japanese Ethical Guidelines “Medical and Health Research Involving Human Subjects” do not apply to studies that use anonymized secondary data (Part 3 “Scope of Application” on page 10); therefore, approval from an institutional review board/research ethics committee was deemed not necessary. No informed consent was sought from patients.However, the study was conducted in accordance with the Guidelines for Good Pharmacoepidemiology Practices (GPP) issued by the International Society for Pharmacoepidemiology (ISPE). The study was reported in compliance with the REporting of studies Conducted using Observational Routinely-collected health Data (RECORD) statement.The primary outcome of the study was the proportion of patients using analgesics before and after surgery, defined as drug use during the pre-surgery period, immediate post-surgery period (from index date to index date +6 days), and post-surgery period. Secondary outcomes included factors affecting withdrawal of analgesics drugs during the post-surgery period and those affecting opioids prescription during the post-surgery period. These outcomes were calculated separately for patients with TKR and those with THR. The percentage of prescription for analgesics in patients discharged from facilities where TJR was performed but who visited other facilities post-surgery were also calculated. This analysis was performed assuming that fewer analgesics would be prescribed when patients would visit other facilities after surgery.2.2All patients who met the eligibility criteria were included in the analysis set. For categorical data, frequency and percentages of patients were summarized. For continuous variables, mean, standard deviation (SD), median, and interquartile range (IQR) were calculated. The percentage of medication used before and after TJR were summarized with their 95 % confidence interval (CI). Two separate multivariate logistic regression analyses were performed: one was to identify the factors affecting the prescription of analgesics post-operatively for hip and knee OA patients separately. The other was to identify the factors affecting the opioids prescription post-operatively for hip and knee OA patients separately. The model variables included patients’ characteristics: age, gender, comorbidities, and pre-surgery treatment. An alpha level of 0.05 was considered statistically significant. Analysis was performed using SAS Release version 9.4 .33.1Of the 6308 patients who underwent TJR of the knee/hip between January 2010 and December 2019, 4082 patients met the inclusion criteria. After excluding 914 patients who had THR or TKR or a record of malignancy or rheumatoid arthritis during the observation period, 3,168 patients were included in the final analysis .Fig. 1Pa3.2Patient demographics and clinical characteristics are described in 3.3As shown in The aforementioned results corroborated with the mean number of analgesics used by patients as the number of analgesics used per patient increased in the immediate post-surgery period compared to that before surgery in the knee and hip OA groups . Moreove3.4Use of pre-operative oral NSAIDs  and weak opioids  were associated with post-surgical use of analgesics in patients with hip OA. Pre-operative use of intra-articular hyaluronic acid was a significant factor in patients with knee OA  . Certain3.5Use of opioids before surgery was associated with post-operative use of opioids, thus affecting opioid prescription post-surgery: weak opioids for knee  and hip OA  and strong opioids for hip OA  . Comorbi4This is the first real-world study to report the difference in the use of analgesics before and after TJR, and to analyze the factors influencing the prescription patterns of these analgesics in patients with knee/hip OA in Japan. Our study showed that a significant number of patients were using analgesics even 3 months after TJR. NSAIDs were the most commonly used analgesics in the pre- and post-surgery periods, whereas strong opioids were the most common analgesics in the immediate post-surgery period in knee/hip OA patients. When compared with pre-surgery, prescription of analgesics, mainly NSAIDs, acetaminophen, intra-articular steroids, and opioids, increased in the immediate post-surgery period and reduced in the post-surgery period. Pre-operative use of oral NSAIDs, opioids, and intra-articular hyaluronic acid was associated with post-operative use of analgesics. Certain comorbidities, such as mental disorders and some gastrointestinal disorders, were associated with increased use of analgesics post-operatively.,,,,In Japan, the prevalence of knee OA of Kellgren-Lawrence grade ≥2 (55.6 %) is higher than that of hip OA (1.4%–3.5 %), and more patients with knee OA undergo arthroplasties than those with hip OA.The results of this study showed that compared to pre-surgery, the use of NSAIDs, acetaminophen, intra-articular steroids, and opioids increases in the immediate post-TJR period and reduces in the post-surgery period. As expected, the number of patients not using analgesics prior to surgery reduced immediately after surgery but the number increased in the post-surgery period. This finding suggests pain relief after 90 days of surgery; however, 96.1 % and 84.9 % of patients with knee OA and hip OA, respectively, still required analgesics to relieve their pain post-surgery. A similar trend of analgesics use was seen in a previous study.,,,,,Almost half of the patients with knee/hip OA who were discharged from the facilities but visited other facilities had received oral and non-oral NSAIDs. Moreover, in this category, the proportion of patients with knee OA was greater than that of hip OA, thus suggesting that patients with knee OA are more likely to have residual pain. In addition, post-operative satisfaction varies greatly, with many TJR patients experiencing chronic pain after surgery. Patient satisfaction was higher with THR than with TKR, and patients rarely refused to undergo THR.Logistic regression analysis in our study showed association between pre-operative use of analgesics and post-operative prescription of these drugs. If a patient's pain becomes too severe before TJR, the patient is less likely to discontinue analgesics. Patients who received opioids before surgery may have experienced severe pain, and this is demonstrated by a significant association between pre-operative use of opioids and continued use of analgesics post-surgery in our study. Pre-operative use of opioids was a significant factor affecting post-operative opioid use in a previous study.,Patients with comorbid mental disorders could use antidepressants and other medications. There have been reports of new-onset depression being associated with pre-operative comorbid anxiety and pre-operative, post-operative, and ongoing opioid use after TJR. Therefore, additional caution is advised while using opioids in these patients.A major strength of this analysis is that data are extracted from a large-scale claims database in Japan and allows patients to be tracked even when they are transferred to other hospitals. Post-operative prescription of analgesic drugs after discharge from facilities to other facilities  included in this JMDC analysis aids understanding of the current situation in Japan. Nonetheless, there are certain limitations of the study that need to be considered. Prescription status of concomitant medications was not examined in this analysis but could be researched further. There is nonavailability of medical chart information, data of the elderly (≥75 years), and disease severity in the database. Information on functional outcome of patients who underwent knee/hip TJR is not available in this database; hence, correlation between analgesic use and functional outcomes could not be determined. Moreover, the reasons for analgesic choices could not be assessed. Generally in Japan, analgesic choice is made by the treating physician based on the overall condition of the patient, or sometimes, analgesics are used in accordance with the pain management protocol of the clinical facility. The database used in this study does not include the reasons for prescribing analgesics; therefore, it is difficult to confirm these observations. There was a higher number of prescriptions for intra-articular hyaluronic acid after surgery observed in this study, which could be attributed to its use to treat OA at non-index joints or for non-OA conditions. As it is highly important to prevent infection after artificial joint surgery, intra-articular injections are generally not performed after surgery. In addition, there was no information regarding the prescription of over-the-counter drugs.This real-world study reports the differences in analgesic use before and after TJR in patients with knee/hip OA in Japan. A significant number of patients were using analgesics even after 3 months of TJR. Use of strong opioids also increased. Pre-operative analgesic use associated with continued use post-surgery, indicating that efficient and optimal pain management before and immediately after TJR may be important to reduce post-operative use of analgesics, especially opioids that pose a risk of drug dependence.10.13039/100010793Pfizer Japan Inc., and hold stock/stock options of Pfizer Inc.; TT is an employee of Clinical Study Support, Inc., and was involved in the study based on their contract with 10.13039/100010793Pfizer Japan Inc.; TS and MD were not financially compensated for their collaboration in this project nor for the development of this manuscript; however, TS and MD have received honorariums from Pfizer Japan Inc. outside of this work. The authors report no other conflicts of interest in this work.NE, HY, and KT are employees of 10.13039/100010793Pfizer Japan Inc. Editorial and medical writing support and publication charges were funded by 10.13039/100010793Pfizer Japan Inc.This work was supported by


Triple-negative breast cancer (TNBC) is an aggressive as well as the most dangerous form of breast cancer. Due to the lack of biomarkers that can be targeted by specific molecular therapeutics, treatment is usually limited to chemotherapy and surgery where applicable. Oncolytic viruses are biologicals that have been engineered in many cases to express different genes that can aid in the recruitment of immune cells to trigger antitumor immune responses in addition to the direct lysis of infected cancerous cells. In this review, the basic biology of TNBC is described, as are the many OVs and their different strategies to attempt to treat TNBC in vitro, in vivo, and in human trials.Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer. TNBC diagnoses account for approximately one-fifth of all breast cancer cases globally. The lack of receptors for estrogen, progesterone, and human epidermal growth factor 2  results in a lack of available molecular-based therapeutics. This increases the difficulty of treatment and leaves more traditional as well as toxic therapies as the only available standards of care in many cases. Recurrence is an additional serious problem, contributing substantially to its higher mortality rate as compared to other breast cancers. Tumor heterogeneity also poses a large obstacle to treatment approaches. No driver of tumor development has been identified for TNBC, and large variations in mutational burden between tumors have been described previously. Here, we describe the biology of six different subtypes of TNBC, based on differential gene expression. Subtype differences can have a large impact on metastatic potential and resistance to treatment. Emerging antibody-based therapeutics, such as immune checkpoint inhibitors, have available targets for small subsets of TNBC patients, leading to partial responses and relatively low overall efficacy. Immuno-oncolytic viruses (OVs) have recently become significant in the pursuit of effective treatments for TNBC. OVs generally share the ability to ignore the heterogeneous nature of TNBC cells and allow infection throughout a treated tumor. Recent genetic engineering has allowed for the enhancement of efficacy against certain tumor types while avoiding the most common side effects in non-cancerous tissues. In this review, TNBC is described in order to address the challenges it presents to potential treatments. The OVs currently described preclinically and in various stages of clinical trials are also summarized, as are their strategies to enhance therapeutic potential. Breast cancer (BC) is the most diagnosed cancer in the United States for women, and is also the number two killer among cancers in women ,2. The b2+), G-protein-coupled receptor (GPCR), and extracellular-related kinase (ERK 1 and 2)), epithelial–mesenchymal transition (EMT), and Wnt/β-catenin. The MSL subtype also has a reduced expression of genes for cell proliferation. The IM subtype has an elevated expression of pathways involving immune cell signaling, cytokine signaling, antigen processing/presentation, and immune signal transduction. Finally, the LAR subtype has a heavily elevated expression of pathways related to hormone regulation, particularly with androgen receptor signaling (which can be approximately nine times as much as that of other TNBC subtypes) [The BL1 and BL2 subtypes differ in that BL1 has elevated gene expression for DNA damage response, proliferation signaling, and cell cycle checkpoint loss, whereas BL2 has elevated growth factor (GF) signaling, glycolysis/gluconeogenesis signaling, and myoepithelial surface receptors. This suggests that cells of the BL1/2 subtypes may be of basal or myoepithelial origin. The M subtype has elevated expression of cell motility pathways, receptor interaction with the extracellular matrix (ECM), and cell differentiation pathways. The MSL subtype shares expression with the M subtype, but it also uniquely expresses pathways related to GF signaling , epidermal growth factor receptor (EGFR), calcium (Caubtypes) . These eubtypes) ,11.There have been a few theories to describe how heterogeneity in TNBC is established and maintained. The two main concepts are the cancer stem cell (CSC) hypothesis and the clonal evolution model (CEM). Other concepts, such as the deregulation of adult mammary stem cells (aMSCs), are described as contributing to developing TNBC in its initial stages ,13,14,15The division of aMSCs gives rise to progenitors that further differentiate into basal or luminal progenitor cells. Under normal conditions, if basal or luminal progenitor cells are made, they further develop into mature basal or luminal cells; however, when the development of luminal progenitors is disrupted, cancer cells may develop. This is believed to occur via the improper regulation of Wnt/β-catenin pathways, transcription factors (TFs), such as Snail, and embryonic stem cell (ESC) markers . Combinations of genetic alterations to these pathways can make the progenitor cells de-differentiate and return to a proliferative state, which, when combined with other mutations, may result in TNBC .The CSC model proposes that there is a hierarchy within cancers of tumorigenic cancer cells and their non-tumorigenic progeny. Within these subpopulations, the CSCs are hypothesized to be the driving forces behind the growth of a tumor, its progression, resistance to certain treatments, and metastasis ,17,18,19The CEM presumes that there is one original clone that becomes cancerous, which then divides and, as carcinogenesis progresses, the cell as well as its subsequent progeny accumulate mutations in each cycle of clonal expansion, which results in the heterogeneity of the tumor. This model also assumes that there is no hierarchy among the cells and that, instead, natural selection determines which clones are successful and continue to divide. The cells that cannot undergo division are maintained as part of the tumor or will senesce, eventually being eliminated via necrosis ,14. TumoAmong the approximately 268,600 new cases of BC in the United States in 2019, about 10–20% of these were TNBC . EstimatIn the development and progression of carcinogenesis, epithelial–mesenchymal transition (EMT) has been repeatedly shown to be important in changing cell profile, metastasis, and even tumor recurrence ,25,26. TAnother factor that contributes to the difficulty in treating TNBCs is the high degree of variance among the mutations acquired. The most common mutations are to p53 (up to 80%), followed by phosphatase and tensin homolog (PTEN) 35%) as well as inositol-polyphosphate-4-phosphatase type 2 INPP4B, 30%) [% 30,31,,31,32. S% as wellNeoplastic cells must compete for resources with nearby healthy cells; however, these neoplastic clones also often compete amongst themselves. This is because different mutations acquired in these neoplasms impact replication and resource acquisition rates. It has been shown that neoplastic TNBC cell clones injected into opposite flanks of mice and rats can inhibit the growth of one another or that one set of clones can completely inhibit the other ,41. Neop−/PR− cells can utilize auto and paracrine self-regulation pathways to synthesize their own GFs more frequently than BC cells that express ER and/or PR [Furthermore, data acquired from studying cocultures of different BC cell lines in vitro show that certain interactions between cells can impact growth, both positively and negatively. MDA-MB-231 cells coincubated with supernatants of MCF-7 cells increased proliferation over time, whereas coincubation with BT-474 cells or simply other MDA-MB-231 cells hampered growth [nd/or PR . Therefond/or PR  and terand/or PR  or even nd/or PR  based onDue to the extreme heterogeneity of TNBC, chemotherapy with anthracyclines or taxanes remains the standard for the treatment of TNBC. There are a few mechanistic targets for therapy such as DNA repair, cell proliferation, and p53 . Patient®) combined with nanoparticle albumin-bound paclitaxel  in patients with metastatic TNBC where ≥1% of the tumor area is positive for immune cells expressing programmed cell death-ligand 1 (PD-L1) [However, in March 2019, the US FDA granted accelerated approval for an immunotherapy regimen for patients with BC. The treatment includes the use of atezolizumab (Tecentriq (PD-L1) ,59. Atez (PD-L1) . Nab-pac (PD-L1) ,64,65,66 (PD-L1) . When th (PD-L1) . Even thwt) viruses or genetically engineered via the deletion of viral genes and/or the addition of immuno-modulatory genes for use as therapeutics against cancer. These viruses enter a tumor and ultimately lyse the cancer cells in a targeted manner, aiming to leave non-cancerous cells alone. Some OVs bear genetic inserts that induce changes within a cell, to the TME, or to immune cells within a host. Many of these viruses may also be modified via deletions to accomplish things such as decreased pathogenicity or increased tumor preference/selectivity [These are a class of viruses that have either been selected as unmodified wild-type , and that there were no observable neurotropic effects, the results provided strong evidence that viruses were capable of cancer treatment. Further work still needed to be performed with potential OVs in order for them to become viable anticancer agents, as it was clear that many viruses were not suitable for therapeutic use in their natural forms.Other animal viruses were also explored, such as the avian plague virus (AVP), which was tested due to the belief that it would be less pathogenic to humans than many of the viruses used in the past were. A common side effect was strong neurotropism that would inevitably kill the host, even after destroying transplanted tumor cells . The hypPathogenicity and neurotropism concerns with most OVs led the field to focus on decreasing tropism for non-cancerous tissues. An early review addressed the future of OV development . One queIt took almost 30 years to see genetic engineering used for OVs, even though the principle was being used for gene editing therapies in other diseases ,111. TheSince then, the field of oncolytic virology has rapidly expanded, with the advent of genome sequencing to specifically target and remove pathogenicity genes , add in Much research is being carried out in order to study several different OV platforms for the potential treatment of TNBC. OVs being studied in BCs not classified as TNBC will not be described here, though recent reviews of these viruses are available ,116. BelMastadenovirus and the family Adenoviridae. At this moment in time, the largest number of OV recombinants from any viral classification that have been tested against TNBC are adenovirus strains. A type 5 adenovirus expressing green fluorescent protein (GFP) and the human telomerase reverse transcriptase gene (hTERT), OBP-401, was tested for infectivity in specialized MDA-MB-231 cells that had shown higher metastatic potential than their parental lines [Adenoviruses (ADV) are non-enveloped dsDNA viruses in the genus al lines ,118. In al lines . It was 8) and significantly prolonged survival in a metastatic tumor model, while also reducing metastases compared to control animals.Another adenovirus, P55-HTERT-HRE-TRAIL, expresses TNF-related apoptosis-inducing ligand (TRAIL) with expression specificity to tumor cells driven by hTERT and hypoxia response element (HRE) promoters . In vitrCombinatorial therapies and new approaches to the modulation of gene expression within infected tumors are also being tested in ADV platforms against TNBC. An oncolytic ADV was tested with an adjuvant therapeutic temozolomide, which is converted into an alkylating agent under physiological conditions. The combination was shown to enhance viral replication and oncolytic effect in TNBC cells in vitro . Long no+ T cells infiltrating tumors treated with the chimeric ADV, demonstrating that Rantes expression was helping to direct the chemotaxis of immune cells towards the tumor.Finally, a chimeric ADV has also been constructed to be tested as an oncolytic against TNBC. A chimeric virus between type 5 ADV and type 11b ADV was produced and the cytokine Rantes  was inserted into the E3 region of the viral genome . The chiSimplexvirus, family Alphaherpesvirinae. Canerpaturev (C-REV) is an oncolytic mutant of HSV that alone has antitumor efficacy against TNBC. It has also shown a promising oncolytic effect when combined with 5-fluorouracil (5-FU) and S-1  against TNBC in vivo in a bilateral 4T1 tumor model [+ T cells and their interferons, and decreasing the number of myeloid-derived suppressor cells (MDSCs). Reduced MDSC numbers are important as these cells suppress T cell activation, which would directly inhibit the antitumor effects driven by the immune response [Herpes simplex virus (HSV) is a dsDNA virus from the genus or model . S-1 metor model . The antresponse ,129,130.Talimogene laherparepvec  is another HSV-1 mutant that has GMCSF inserted and deletes viral ICP34.5 as well as ICP47. The deletion of ICP34.5 is to decrease pathogenicity and make the virus tumor-selective . ICP47 i3 and 104) demonstrated a significant reduction in tumor volume when compared to mock treated control tumors.The original study detailing a chimeric Orthopoxvirus (dsDNA virus), CF33, describes the genome of this OV derived from nine different parental poxviruses: cowpox virus (Brighton strain), raccoonpox virus (Herman strain), rabbitpox virus (Utrecht strain), and six different vaccinia virus (VV) strains  . These vThese results prompted another preclinical study where CF33 was modified to CF33-hNIS-anti-PD-L1 . CF33-hN3 PFU of CF189. Contralateral tumor sites on these mice were also controlled, even when the virus was not injected directly into those tumors, indicating the virus can travel to distal sites and exert antitumor effects.Another chimeric poxvirus has been developed and studied against TNBC. This chimeric virus, CF189, was created using a coinfection of cow kidney epithelial cells with orf virus strain NZ2 and the TJS strain of pseudocowpox virus . CF189 cYatapoxvirus and family Poxviridae. It is of similar genomic and particle size to VV , and causes mild febrile illness that is limited to humans and monkeys [Tanapoxvirus is a dsDNA virus from the genus  monkeys ,139,140. monkeys . The fir monkeys ,142; the monkeys ,144,145. monkeys .Orthopoxvirus in the family Poxviridae. It is a prominent poxvirus in many cancer studies due to its large genome, which can handle many genetic inserts simultaneously, the fact that it replicates quickly, and due to it having a known safety profile. GLV-1h153 is an oncolytic VV that expresses hNIS. The natural expression of hNIS exists in tissues outside of the thyroid, including breast tissues; therefore, its overexpression in some cancers serves as a potential target, including in TNBC [VV is a dsDNA  in TNBC ,146. Thi in TNBC . A simil in TNBC . DNA-basTogaviridae. Initial studies showed that this virus had the ability to selectively kill cells lacking the antiviral zinc finger protein (ZAP) in human cancers. Oncolytic activity was reported to be mediated through stress to the endoplasmic reticulum leading to apoptosis [M1 is a strain of the Getah-like alphavirus that was derived initially from mosquitos and is non-pathogenic in normal mice or rat tissues. It also has no known disease-causing capability in humans. It is a positive-sense RNA virus from the family poptosis . Later, poptosis . In thispoptosis . This stpoptosis .Enterovirus in the family Picornaviridae. A coxsackievirus B3 (CVB3) platform was used to generate a recombinant virus that expressed multiple targeting sequences (TSs) of microRNAs (miRNAs) to reduce off-target toxicities when used in vivo. The initial TSs selected were mouse miR-145 and miR-143 as they are significantly downregulated in tumor cells to assist in proliferation and cell division. Multiple copies of each were inserted into the 5′UTR of the CVB3 genome. Initial testing showed safety concerns by day 12 following intraperitoneal (IP) injections of recombinant miR-CVB3 [6 when compared to an untreated control. The recombinant virus also significantly increased survival after 28 days compared to both untreated and wtCVB3 groups; however, intravenous (IV) administration failed to yield similar results to IT administration [Coxsackieviruses are positive-sense RNA viruses from the genus miR-CVB3 . FurthermiR-CVB3 . The newstration .Vesiculovirus. Mutations were made to amino acid L123W in the M protein and Q242R in the G protein for attenuation in normal cells, while improving replication in cancer cells. This virus was also altered to include GFP [The oncolytic Maraba virus, MG1, is a genetically modified rhabdovirus (negative-sense RNA) originating from the vesicular stomatitis virus serogroup in the genus lude GFP . After slude GFP . MG1 sholude GFP . Furtherlude GFP .Morbillivirus in the family Paramyxoviridae. One oncolytic measles virus recombinant has been modified to include the proapoptotic gene BNiP3 in humans, rMV-BNiP3, which can act on BCL-2 family proteins and E1B 19 kDa proteins from adenoviruses [Measles virus (MV) is a negative-sense RNA virus from the genus oviruses . This viAnother set of measles viruses was generated in order to target the urokinase cell surface receptor (uPAR), which is overexpressed in both mouse and human cancers . One virOrthorubulavirus in the family Paramyxoviridae. Samples of mumps virus used in the clinical trials described earlier in this review were obtained with the goal of continuing its development as an OV [7 median tissue culture infectious doses (TCID50), both as single-dose and multi-dose administrations. Conclusions drawn from this study support the idea of using MuV-UC for translation into clinical studies in the future.Mumps virus is an enveloped, negative-sense RNA virus from the genus as an OV . Since iAvian orthoavulavirus 1 from the Avulavirinae subfamily and the Paramyoviridae family. A lentogenic strain of NDV, called LaSota, was used to determine the oncolytic nature and efficacy of this virus in combination with doxorubicin in TNBC. The in vitro results showed that this NDV strain could replicate in and cause the cytolysis of 4T1 cancer cells in a dose-dependent manner [NDV is a negative-sense RNA strain of t manner . When teOrthoreovirus genus, subfamily Spinareovirinae. A reassortant r2Reovirus was genetically engineered to include nine segments from the T1L strain (serotype 1 Lang) and one segment from the T3D strain (serotype 3 Dearing) of reoviruses [Reoviruses are segmented dsRNA viruses in the oviruses . The r2Roviruses . The r2Roviruses . In a dioviruses . Both r2Oncolytic viruses that have reached human clinical trials for patients with TNBC are described below. A number of different approaches are being tested, including viruses being used as a monotherapy or combined with adjuvant chemotherapies, radiotherapy, or immunotherapies. A recent review of all available human trials involving OVs as of 2020 summarized the results and discussed the conclusions drawn from these studies . Many tr®) (NCT03004183). ADV/HSV-tk + valacyclovir has shown an increased antitumor activity of NK cells, the stimulation of T-cells, and promoted lymphocyte infiltration into treated tumors in a previous human trial for patients with recurrent ovarian cancer [ADV/HSV-tk is being tested in a phase II trial, with the objective being to test the efficacy as well as safety of stereotactic body radiation therapy (SBRT) together with the administration of ADV/HSV-tk and valacyclovir prior to treatment with pembrolizumab (Keytrudan cancer . The addn cancer . Pembrol11 adenovirus particles (NCT05076760).A different adenovirus, MEM-288, is being tested in a phase I dose escalation study as a monotherapy against numerous solid tumors, including TNBC. MEM-288 carries human interferon beta and recombinant, chimeric CD40 (MEM40). The overall goal of this study is to determine the maximum tolerated dose within a 3-week period and its safety in patients. The secondary objectives include the determination of the recommended dosage for a phase II trial (where MEM-288 will be combined with an ICI), efficacy, progression-free survival, response duration, and the presence as well as nature of the antitumor responses elicited. Dosages in the preliminary trial will reach as high as 1 × 10+ tumor-infiltrating T cells. This increased CD8+ cell population was significantly higher in RCB0-1 patients than RCB2-3 patients. Responses for patients with >1% PD-L1+ cells were positively correlated with treatment outcome, though not significant. A key conclusion from this study was that immunotherapy agents should be administered as closely as possible to the adjuvant chemotherapeutic to help take advantage of the activation of immune cells rather than waiting for residual disease or planning for sequential administrations after the chemotherapy regimen.T-vec has entered a phase I clinical trial (NCT04185311) for patients with TNBC, where the goal of the trial is to study the OV together with iplilmumab (an anti-CTLA-4 antibody) and nivolumab (an anti PD-1 antibody) prior to an attempted surgical resection. T-vec is to be injected intratumorally (IT) on three separate days  with four nivolumab treatments as well as two ipilimumab treatments over the course of the study. In a different phase II study with T-vec, it was administered IT and combined with paclitaxel, followed by doxorubicin, cyclophosphamide, and surgery in patients with stage 2–3 TNBC. This study has been completed, with an analysis having been carried out of 37 of the 40 enrolled patients’ biopsies and trial outcome results (NCT02779855) . The pri8 plaque-forming units (PFU)/4 mL every 2 weeks, for up to 10 total doses. No dose-limiting toxicity was observed, and the side effects were deemed to be mild, grade 1–2 [Additionally, another HSV-1, called ONCR-177, is being tested in a phase I trial against a multitude of cancers, including TNBC (NCT04348916). ONCR-177 expresses five different transgenes: interleukin 12 (IL-12), C-C motif chemokine 4 , FMS-like tyrosine kinase 3 ligand extracellular domain (FLT3LG ECD), and anti-PD-1 as well as anti-CTLA-4 antibodies. It also does not have γ34.5 deleted, as is the case in most other HSV recombinant OVs. Previous work used miRNA silencing to allow the functional copy of ICP34.5 to exist in the viral genome and be inactivated at discretion; however, in cases where the interferon response may still function properly within a cancer cell, ONCR-177 would not be attenuated in these cells . The goarade 1–2 .Pelareorep (Reolysin) is a naturally occurring Dearing strain of reovirus serotype 3, and is currently in two different clinical trials for humans. The first is an early phase I clinical trial that includes the use of three other drugs, in addition to the oncolytic, in patients with various types of BC. In addition to pelareoreop, atezolizumab will also be used for patients with TNBC, administered on day 3 of treatment. The goal of this study is to determine the safety profile and elicitation of antitumor responses by the OV in early stage BC (NCT04102618). The second is a phase II clinical trial combining pelareorep with an ICI monoclonal antibody (retifanlimab) in late-stage TNBC (including metastatic TNBC) and other BCs (NCT04445844). The goal is to test the efficacy and safety of the treatment regimen, as well as whether the PD-L1 expression levels pre-, during, and post-treatment are indicators of treatment outcomes. In both cases, the OV is delivered IV, and the virus will be administered a total of four times in the planned 28-day increments .A phase I/IIa multicenter trial using BT-001, an oncolytic vaccinia virus encoding recombinant human anti-CTLA-4 and GMCSF, is being tested both alone and in combination with pembrolizumab. The phase Ia trial is exploring the dose escalation for BT-001 via repeated IT injections in many different advanced solid tumors with or without metastases as a monotherapy. Phase Ib will have BT-001 administered IV in combination with pembrolizumab in the same types of tumors. Finally, in the phase IIa trial, BT-001 will be administered IT while pembrolizumab is used IV (NCT04725331).11 capable of being tolerated in the treatment of multiple myeloma as long as the viral uptake was relatively slow [A measles virus expressing hNIS (MV-NIS) is currently in a phase I clinical trial treating a variety of cancer types from squamous cell head and neck cancer to metastatic TNBC (NCT01846091). The primary objective of the trial is to determine how well the virus is tolerated in these patients and to assess the safety profile of the virus when administered IT. The study also aims to look for efficacy in treating the target cancers as well as tracking the expression of NIS throughout cancerous tissues using SPECT imaging . MV-NIS ely slow . MV-NIS ely slow .Helicobacter pylori and was tested for safety in mice prior to clinical assessment [Another phase I trial using a different recombinant, MV-s-NAP, is recruiting patients with metastatic breast cancers (NCT04521764). The primary objectives of this trial are to determine the overall safety profile, MTD, and cumulative immune responses to the virus and tumor following IT injection of the virus. Virus administration is set to occur up to three additional times following the initial injection, provided that there are no signs of unacceptable toxicities or disease progression. This virus recombinant expresses neutrophil-activating protein (NAP) from sessment . MV-s-NAThis review summarizes the cellular biology of TNBC and the unique challenges that it presents to treatment. A brief history of OVs and their current studies in the pursuit of treating TNBC were also described. The combination of engineered tumor selectivity or replication preference, safety, low toxicity, and efficacy in vivo against a cancer type that makes conventional treatment extremely difficult would deem OVs as logical choices for further and continued investigation. Due to the extreme heterogeneity of TNBCs, many standard therapies have fallen short of achieving similar treatment success as compared to other BCs; all types except TNBC have seen increased 5-year survival rates over the past 20 years. The use of OVs with or without combinatorial drugs or radiotherapy has been taken to the clinic as a potential future treatment modality for TNBC and many other cancer types. Combinatorial regimens, such as the use of HDACis to enhance oncolytic HSV entry and replication within tumor cells , show th