To describe the characteristics and find out risk factors of COVID-19 patients infected with different categories of bacteria.Case-control.We conducted a retrospective study including 129 COVID-19 patients admitted to a tertiary hospital between October 13, 2022 and December 31, 2022. Patients’ data were collected from the hospital information system. Patients were classified as having or not having confirmed secondary bacterial infections, or gram-positive and gram-negative bacterial infections for analysis. Categories and sources of isolated bacteria, characteristics of the patients, and the risk factors for developing secondary bacterial infections were analyzed.p < 0.001), invasive therapy , and previous antibiotics use  were independent risk factors of secondary bacterial infections in COVID-19 patients. Ceftriaxone/cefotaxime use  was associated with gram-positive bacterial infections while age over 70 , invasive therapy , and carbapenems use  were associated with gram-negative bacterial infections.Gram-negative bacteria accounted for the majority of secondary bacterial infections of the included patients. Critical type of COVID-19 (OR = 12.98, 95%CI 3.43∼49.18, Critical patients with invasive therapy and previous antibiotics use should be cautious with secondary bacterial infections. Third-generation cephalosporins and carbapenems should be used carefully because both are risk factors for gram-positive or gram-negative bacterial infections. Patients with viral infections are predisposed to secondary bacterial infections, which are defined as infections occurred more than 48 hours after hospitalization. Recent meta-analysis showed that the prevalence rate of secondary bacterial infections was 18.4%. However, antibiotics were still empirically prescribed for 62% of COVID-19 patients, which was far surpassing the rate of bacterial infections. The decision to use antibiotics for patients with COVID-19 is often considered as a prophylactic therapy in order to prevent potential secondary bacterial infections. However, there is currently insufficient evidence supporting such prophylactic use of antibiotics. On the other hand, distinguishing between viral and bacterial infections, especially for patients with severe and critical conditions, can be challenging due to similarities in chest images, false negative or false positive results in laboratory tests, and time lags in microbial culture. These factors may contribute to the overuse and misuse of antibiotics for COVID-19 patients.The COVID-19 pandemic continues to constitute a public health issue, as pronounced by the World Health Organization. and antibiotic exposure was an independent risk factor for development of antibiotic-resistant bacterial secondary infection. It can be concluded that irrational antibiotic use could lead to worse outcomes in COVID-19 patients. Thus, only by administering the correct antibiotics to COVID-19 patients with the risk of secondary bacterial infections could benefits be obtained.High probability of antibiotic use during COVID-19 pandemic prompts concerns about selection of antibiotic-resistant bacteria. A retrospective study found that multi-drug-resistant pathogens were only detected in the antibiotic-exposed group of COVID-19 patients, However, knowing only the risk factors of secondary bacterial infection seems not enough, because this would result in broad-coverage of potential bacteria in COVID-19 patients with meropenem, piperacillin/tazolbactam, and ceftriaxone. Unfortunately, broad-spectrum antibiotic use contributed to a rapid spread of drug-resistant bacteria, which has no good for the prognosis of COVID-19 patients. So it is still needed to understand whether different conditions can lead to secondary infections caused by different bacteria in COVID-19 patients for the purpose of using antibiotics with a focused target.To find out the risk factors of bacterial infections in COVID-19 patients is important. So far, risk factors of bacterial infection in COVID-19 have been studied in several articles. Male sex, diabetes, hematological disease, mechanical ventilation, invasive devices, combination of antibiotics, and glucocorticoid treatment were found to be related to secondary bacterial infections in different articles.Based on these facts, we plan to further analyze risk factors associated with secondary infections caused by different bacteria of COVID-19 patients, in order to provide more information on better antibiotic use with COVID-19 patients.We retrospectively reviewed patients admitted to a tertiary hospital in Wuhan, Hubei Province, China between October 1, 2020 and December 31, 2022. The inclusion and exclusion criteria were as follows:Inclusion criteria: (1) with confirmed diagnosis of COVID-19 by polymerase chain reaction testing before admission and (2) with clear bacterial cultural results before discharge. Exclusion criteria: (1) positive bacterial cultural results from applications <48 hours after admission (2) with only one positive bacterial cultural result that was suspicious of contamination (3) with records of clear diagnosis of bacterial infections before admission. Finally, 129 patients were included, and we performed a 1:2 case-control study with 43 patients with confirmed secondary bacterial infections assigned to the case group and 86 assigned to the control group. and included invasive ventilators , vascular devices , renal replacement therapy, and indwelling urinary catheters.Data on baseline characteristics were collected from the hospital information system. Therapy records and medication records only before the (first positive) microbial culture application were included for analysis. Therapy was classified into noninvasive and invasive procedures. The definition of invasive procedure was previously describedp-values of <0.05.Categorical variables were presented as frequencies with percentages and Chi-square method was used to test the significance. Kolmogorov–Smirnov test and Shapiro–Wilk test were used to test the normality of continuous variables according to the sample size . Levene test was used to detect the homogeneity of variance. Continuous variables were then presented as mean±SD (standard deviation) or medians with interquartile range based on the normality. T-test was used for significance test of continuous variables, while Mann–Whitney U-test was used when the criteria of T-test was not satisfied. To assess the association between risk factors and bacterial infections, multivariate logistic regression models were used, and factors included in the model were based on the results of univariate significance test. All analyses were considered significant at two-tailed The average time between the first positive bacterial cultural result and the occurrence of first COVID-19 symptoms as well as the admission time were 32.07 ± 17.32 days and 18.95 ± 12.66 days, respectively (data were not shown). In total, 57 strains of bacteria were found in the 43 patients as described in Table In COVID-19, patients with confirmed bacterial infections, blood level of neutrophils, CRP, ferritin, IL-1β, IL-2R, IL-6, and TNF-α were higher than the upper limits of the normal values with significant differences, while lymphocyte was significantly lower. Though not statistically significant, WBC, IL-8, and IL-10 seemed higher in the bacterial-infected patients, too. The median value of procalcitonin (PCT) was slightly lower than the upper limit of the normal value, but no statistical difference was found. Details are shown in Table Though not significant, patients with bacterial infections seemed older than those without a confirmed bacterial infection (mean age 68.81 ± 11.32 vs 65.77 ± 10.94). There were no significant differences in sex, smoking history, and comorbidities between the two groups. Clinical type of COVID-19 showed statistical differences in subgroup analysis of total bacterial infections, gram-positive bacterial infections, and gram-negative bacterial infections, with a higher proportion of critical type in the infection group. As for therapy and medication, invasive ventilators, invasive vascular devices, renal replacement therapy, indwelling urinary catheters, antibiotics, and glucocorticoids were found with significant differences, which were more common in the infection group. There was also a significant difference in the median duration of glucocorticoid between the two groups (4 vs 0). Treating with noninvasive ventilators and using tocilizumab were not significantly different when comparing the two groups. For specific antibiotics, treating with ceftriaxone/cefotaxime, ceftazidime/cefoperazone sulbactam, carbapenems, glycopeptides, and linezolid were more common in patients with secondary bacterial infections, while differences of other antibiotics were not statistically significant.For COVID-19 patients infected with gram-positive bacteria, demographic characteristics of age, sex, smoking history, and comorbidities were found with no significance. Compared with using noninvasive ventilators, invasive ventilators were more common in the infected patients. Besides, there were significant differences in invasive vascular devices, and indwelling urinary catheter use between the two groups, which had larger proportions in the infected patients. However, renal replacement therapy was found without significant difference when comparing the two groups. For medication, glucocorticoid, overall antibiotics use as well as tocilizumab were all found with no significant differences between the two groups, though patients with confirmed bacterial infections had higher frequencies of using these three medications. In terms of antibiotic categories, third-generation cephalosporins including ceftriaxone, cefotaxime, ceftazidime, and cefoperazone sulbactam, as well as carbapenems, were found more frequent in the infected patients, while the other antibiotics were found not statistically different between the two groups.On the other hand, characteristics of patients infected with or without gram-negative bacteria varied. Ages were significantly higher in patients infected with gram-negative bacteria when compared with those without confirmed gram-negative bacteria infections. The rest demographic characteristics, including sex, smoking history, and comorbidities, were not significantly different between the two groups. Similarly, more invasive ventilators, invasive vascular devices, and urinary catheters were found in patients infected with gram-negative bacteria than those without infection of such bacteria. However, frequencies of renal replacement therapy were not significantly different between the two groups. For medications, overall antibiotic use and using tocilizumab were also found with no differences between the two groups. Differently, using glucocorticoid was found more frequently in patients with bacterial infections and the median duration of glucocorticoid was higher 3.5 vs 0) in the case group. Also, in terms of antibiotic categories, carbapenems and glycopeptides were more common in patients with gram-negative bacterial infections, which was different from the results between patients with and without gram-positive bacterial infections. Details are shown in Table .5 vs 0 ip < 0.001). Invasive therapy was also found as a risk factor for overall bacterial infections . Similarly, using antibiotics was associated with overall bacterial infections , while duration of glucocorticoid over 3 days was not . See Figure For overall secondary bacterial infections, clinical type of COVID-19, invasive therapy, using antibiotics, and duration of glucocorticoid over 3 days were included in the regression model. Critical type was associated with overall secondary bacterial infections . In terms of antibiotics categories, using ceftriaxone/cefotaxime was associated with developing gram-positive bacterial infections , while using ceftazidime/cefoperazone sulbactam and carbapenems were not . See Figure For gram-positive bacterial infections, clinical type and invasive therapy were not found with significant association with the infections (p = 0.039). Likely, invasive therapy was also associated with gram-negative bacterial infections . In terms of medications, using carbapenems was a risk factor of the infections , while clinical type, glucocorticoid, and glycopeptides were not independent risk factors in the regression model. See Figure For gram-negative bacterial infections, we found that patients with ages over 70 were at a risk of developing gram-negative bacterial infection  of serum PCT from the hospital laboratory as the comparison instead of statistical results of COVID-19 patients without bacterial infections. The reason was that we were unable to determine at which time point the laboratory test results could be used as a reference in the control group. Secondly, it was reported that serum interferon-gamma synthesis increase could inhibit PCT secretion, especially in respiratory viral infections. As COVID-19 is a viral disease, we could reasonably explain the relatively low PCT levels in our study based on this result. But we still must acknowledge that more data on PCT of COVID-19 patients with confirmed bacterial infections are needed. In terms of other biomarkers, lymphopenia was found in the bacterial infectious group, which was within up to 85% of severe COVID-19 patients and the decrease in lymphocytes could be explained by more severe conditions of patients who were with secondary bacterial infections. We also found an increase in neutrophils. Neutrophilia was associated with high-inflammatory status and cytokine storm, and the latter is part of the pathogenesis of COVID-19, which is common in patients with severe COVID-19. Of course, the increase in neutrophils could also be caused by secondary bacterial infection. Collectively, when neutrophils are elevated in patients with COVID-19, poor outcomes and possible bacterial infections should be considered. As for cytokines, IL-1β, IL-2R, IL-6, and TNF-α were found with significant increases in patients with confirmed secondary infections, which was consistent with Lee’s research. Interestingly, there were differences in cytokine changes between COVID-19 and other viral respiratory infections such as influenza. It was reported that responses to interferon I/II pathways were more associated with the influenza whereas pathways for the response to TNF-α or IL-1β were more prominent in COVID-19, besides IL-1β was also amongst the identified strongest markers of ventilator-associated pneumonia, and our results aligned with these conclusions.Laboratory findings are often used for aiding in diagnosis of bacterial infections and as a guide to initiate antibiotic therapy. In our study, we found that most inflammatory indicators increased, which was consistent with other studies. In our study, we identified critical COVID-19 type, previous antibiotics use, and invasive therapy were the three risk factors of overall bacterial infections. In a retrospective study of 201 COVID-19 patients, Iacovelli et al found exposure to antibiotic therapy in last 30 days  was an independent risk factor for new-onset superinfections development, and this result was consistent with ours. Invasive therapies including invasive ventilators, blood devices, and urinary catheters were often used in critical COVID-19 patients. Retrospective studies have shown that in COVID-19 patients, especially those who were admitted into ICUs, longer duration of invasive mechanical ventilation, blood devices, and indwelling urinary catheters were associated with higher bacterial superinfection rates. These results along with ours make two suggestions, the first is that strict antibiotic stewardship should be carried out for COVID-19 patients to reduce the risks of secondary bacterial infections, and the second is that for those patients who need invasive therapies, secondary bacterial infections must be cautious with.It is always interesting to understand the risk factors of secondary bacterial infections in COVID-19 patients, and it is also with great significance of antibiotic rational use for physicians and pharmacists since reports have confirmed that secondary bacterial infections were a dependent factor of in-hospital mortality in COVID-19 patients. In vitro experiments have described this mechanism. It was shown that ceftriaxone application with mice led to a reduction of the mucus-associated microbiota layer and segregation of Enterococcus faecium from the intestinal wall, thus leading to more frequent infection of the bacteria. As for gram-negative bacterial infections, we found previous carbapenems use was an independent risk factor for these infections. The result was also consistent with other studies. In a meta-analysis, Karlijn et al showed that carbapenems use increased the risk with a fold of 4.71 as the second factor of carbapenem-resistant Enterobacteriaceae infections. In another systematic review by Zaira et al, the authors analyzed 92 articles and found that 82.6% of the studies reported previous carbapenems use was associated with multiple-drug-resistant (MDR) gram-negative bacterial infections. Since most of the isolated gram-negative bacteria in our COVID-19 samples were MDR bacteria (data were not shown), we could see from these results that in COVID-19 patients, carbapenems should be carefully used since this kind of antibiotic was an independent risk factor for infections caused by gram-negative bacteria, especially MDR or carbapenem-resistant species.In the subgroup analysis based on bacterial species, we found specific antibiotic categories were risk factors for different bacterial infections. For gram-positive bacteria, ceftriaxone/cefotaxime increased the infection rates. This could be explained as a selection of microbiome with disruption of colonization and proliferation of gram-positive bacteria since majority isolation were Enterococcus faecium and MRSA, which were not sensitive to third-generation cephalosporins.Limitations existed in this study. First, this was a single-center study, and the sample size of patients with gram-positive bacterial infections was relatively small, so we were not sure whether this would affect the results of gram-positive bacterial infections. Second, it was common for physicians to give antibiotics to patients under severe or critical conditions even without very clear evidence of bacterial infections. We included some patients who had previously received antibiotics and were classified as the control because they did not have a clear diagnosis of bacterial infection and did not have positive bacterial culture results after admission, and this probably brought some bias. More work is still needed.In summary, in this retrospective study, we’ve described the microbiology and clinical characteristics of COVID-19 patients with confirmed secondary bacterial infections. Critical patients with invasive therapy and previous antibiotics use should be cautious with secondary bacterial infections. Third-generation cephalosporins and carbapenems should be used carefully because both are risk factors for gram-positive or gram-negative bacterial infections.


In general, aggressive PTCs showed distinctive clinical and morphological features. Among adverse prognostic parameters, the presence of necrosis and an increased mitotic index were associated with shorter disease-free and overall survivals. Other parameters associated with shorter disease-free or overall survivals include a lack of tumor capsule, the presence of vascular invasion, tumor-infiltrating lymphocytes, fibrosclerotic changes, age > 55 years, and a high pTN stage. Various pathways were differentially regulated in non-aggressive as compared to aggressive PTC, including the DNA damage repair, the MAPK, and the RAS pathways. In particular, the hedgehog pathway was differentially de-regulated in aggressive PTC as compared to non-aggressive PTC cases, being WNT10A and GLI3 genes significantly up- and down-regulated in aggressive PTC and GSK3B up-regulated in non-aggressive PTC cases. In conclusion, our study revealed specific molecular signatures and morphological features in aggressive PTC that may be useful to predict more aggressive behavior in a subset of PTC patients. These findings may be useful when developing novel, tailored treatment options for these patients.Papillary thyroid carcinoma (PTC) is considered an indolent neoplasm but it may demonstrate aggressive behavior. We aimed to identify clinical and pathological characteristics and molecular signatures associated with aggressive forms of PTCs. We selected 43 aggressive PTC cases based on the presence of metastases at the time of diagnosis, the development of distant metastasis during follow-up, and/or biochemical recurrence, and 43 PTC patients that were disease-free upon follow-up, matching them according to age, sex, pT, and pN parameters. Twenty-four pairs  and 6 normal thyroid tissues were studied using targeted mRNA screening of cancer-associated genes employing NanoString nCounterThe online version contains supplementary material available at 10.1007/s12022-023-09769-x. Papillary thyroid carcinoma (PTC) derives from follicular cells and is the most common thyroid malignancy, comprising more than 80% of all malignant thyroid tumors –3. PTC iBRAF p.V600E mutation is the most common genomic alteration in PTC (found in approximately 70% of cases). It is currently considered that the BRAF mutation by itself is not a high-risk factor, but it has a strong adverse prognostic impact in association with TERT promoter mutations [NRAS, KRAS, and HRAS are also common [TP53, PIK3CA, and AKT1 [TERT promoter mutations (C228T and C250T) are present in more advanced PTCs and are strongly associated with a higher risk of distant metastases, recurrence, and cancer-related mortality [In terms of the impact of the molecular signature on prognosis, various studies have attempted to better characterize the genetic make-up of these neoplasms. The utations , 11. Muto common , especiao common , but lacand AKT1 , 15. In ortality , 16.Apart from genomic alterations, some studies investigated gene or microRNA expression profiling in PTC. However, only a few studies were designed with the specific purpose of characterizing PTCs with aggressive clinical features. The most recent data are obtained by analysis of publicly available datasets  but are affected by the lack of a detailed characterization of cases and by an unbalanced selection of cases with or without aggressive features –22.® technology in a series of well-characterized clinically aggressive PTC, matched with non-aggressive PTC cases, to identify molecular biomarkers potentially predictive of an aggressive clinical course that might be integrated with the current pathological characterization. A summary of the study design and main results is illustrated in Fig. We therefore aimed to test the profiles of gene expression through the NanoString nCounterA group of 43 cases with aggressive clinical behavior, defined based on the presence of metastasis at diagnosis, development of distant metastases during follow-up, and/or biochemical recurrence (> 2 ng/ml levels of thyroglobulin after TSH stimulation), was selected from a large series of PTC undergoing thyroidectomy from 2002 to 2017 at “Città della Salute e della Scienza” Hospital , San Luigi Gonzaga Hospital , and Mauriziano Hospital . Six cases had a biochemical recurrence, only. Thirty-seven cases had metachronous metastases. These latter cases included one case with synchronous lung metastases, only, four cases with metachronous multiorgan dissemination , and 32 cases with metachronous lymph node involvement, with or without infiltration of soft tissues in the cervical neck. These cases were matched for age, sex, and T and N parameters with 43 controls, undergoing surgery in the same period, which resulted in free of disease during the clinical follow-up . Characteristics of lymph node metastases in both aggressive and non-aggressive cases are reported in Supplementary Table 2. Five cases (four in the aggressive PTC group and one in the non-aggressive PTC group) met the criteria for high-grade differentiated thyroid carcinomas because of the presence of coagulative necrosis. One case had also a mitotic index of 5 in 2 mm2. However, due to their limited number, these cases were not further considered as a separate group but necrosis and mitotic index were considered individually for the sake of statistical analyses.Representative hematoxylin and eosin (H&E) stained slides were re-evaluated in all cases enrolled in the study by three of us . The following parameters were re-assessed and recorded: histological subtype, TNM stage according to AJCC 8th edition, tumor diameter, uni- or multifocal presentation , presence of tumor capsule, presence and extent of microscopic extrathyroidal invasion, presence of angioinvasion, status of surgical margins, presence of necrosis, presence of tumor-infiltrating lymphocytes (TILs), and presence of fibro-sclerotic changes. TILs were coded as absent, present “non-brisk,” and present “brisk” according to standard evaluation methods applied in breast cancer . For staA pool of thyroid tissues devoid of macroscopically recognizable alterations from patients operated on due to follicular nodular disease was used as the baseline for gene expression testing and consisted of 3 female  and 3 male patients .Before the study started, all cases were de-identified and coded by a pathology staff member not involved in the study, and all data were accessed anonymously. The study was approved by the local Ethical Committee  and conducted in accordance with the principles set out in the Declaration of Helsinki. Considering the retrospective nature of this research protocol and that it had no impact on patients’ care, no specific written informed consent was required.For molecular testing, 24 pairs of aggressive and their non-aggressive match were analyzed by means of gene expression profiling  along with the pool of 6 samples of non-neoplastic thyroid tissue. In this sub-series, the aggressive cases were also matched with non-aggressive cases for the histological subtype.For RNA extraction, two 10-μm-thick formalin-fixed and paraffin-embedded (FFPE) tissue sections were obtained from each tissue block and collected in sterile Eppendorf tubes by means of manual microdissection. The same procedure was adopted for normal thyroid tissues, making sure that only healthy/unaltered parenchymal areas were dissected. RNA isolation was performed using the FFPE RNA Isolation Kit , according to the manufacturer’s protocols. Total RNA concentration was assessed using a NanoDrop spectrophotometer .® technology was used to detail the transcriptomic cancer-associated landscape in PTC. Three hundred nanograms of total RNA from each sample were hybridized to the nCounter PanCancer Pathways Panel, according to the manufacturer’s instructions . The nCounter PanCancer Pathways panel includes 730 genes from 13 canonical pathways and 40 selected reference genes. The analyses were set up according to the protocol provided by the manufacturer. Expression data were normalized and analyzed with the nSolver Analysis Software (version 4.0.62). For background correction, the mean count of negative controls plus two times the standard deviation was subtracted from the counts of each gene. The means of the supplied positive controls and the geometric mean of the housekeeping genes were used to normalize the measured expression values. Both positive and negative controls were included in the panel, according to the manufacturer’s instructions. Additionally, the advanced analysis module (version 2.0.134) was used to perform differential expression analyses. Briefly, by the differential expression analysis tool , for each gene, a single linear regression was fit using all selected covariates to predict expression. Volcano plots were generated to display each gene’s -log10  and log2 fold change with the selected covariate. Highly statistically significant genes fell at the top of the plot above the horizontal lines, and highly differentially expressed genes fell to either side. Horizontal lines indicated various p-value thresholds.NanoString nCounterp-value < 0.05 was considered significant.All analyses were performed using Stata/MP 15.0 Statistical Software . The differences in the distribution of clinical-pathological variables were analyzed using parametric and non-parametric tests . Overall survival (OS) was calculated from the surgical excision date of the primary tumor to the date of death or last check-up. The disease-free survival (DFS) was calculated from the date of surgical excision of the primary tumor to the date of the first relapse or last check-up. Survival curves were plotted using the Kaplan-Meier method, and the statistical comparisons were performed using the log-rank test. The Cox regression analyses were carried out on DFS and OS to calculate HRs and 95% CIs for the different study groups. Cox proportional hazards regression model was used in multivariate survival analysis to assess the independent role of parameters significant at univariate analysis. All statistical tests were two sided. A p = 0.031). The tumor capsule was absent in more than half of aggressive cases  and in only 17/43 (39.5%) of non-aggressive ones (p = 0.015). Furthermore, aggressive PTCs had more frequent TILs  as compared to the non-aggressive group  (p = 0.001). Only 10/43 (23.3%) non-aggressive PTCs had extensive fibro-sclerotic changes, as compared to 21/43 (48.8%) aggressive cases (p = 0.047). Although not statistically significant, angioinvasion was observed more commonly in aggressive  as compared to non-aggressive cases  (p = 0.077). Of note, at follow-up, 11/43 (25.6%) patients with aggressive PTC died (8 of their PTC and 3 due to other causes), as compared to 4/43 (9.3%) patients with non-aggressive PTC who died of other causes, all not related to their PTC (p = 0.047).As shown in Table p = 0.019), TILs (p = 0.001), necrosis (p = 0.050), and higher mitotic counts (p = 0.001) were all correlated with an adverse prognosis. Extensive fibro-sclerotic changes were also associated with shorter DFS . In terms of OS, tumor diameter larger than 4 cm (p = 0.004), presence of necrosis (p < 0.001), and higher mitotic index (p = 0.001) were parameters correlated with adverse prognosis in terms of OS. However, none of the parameters significant in univariate analyses showed independent statistical significance in multivariate analyses.By means of Cox univariate analyses Table , in termp = 0.038)  Fig. .Fig. 4KaThe clinical and pathological characteristics of the 48 patients analyzed for gene expression profiling are summarized in Supplementary Table PKMYT1 and WNT10A) and one downregulated (GLI3)  were significantly upregulated in the group of non-aggressive cases as compared to the aggressive ones  that were upregulated in non-aggressive PTC cases were found differentially expressed in aggressive PTC , whereas several other genes were found to be de-regulated in non-aggressive cases   and a more favorable outcome –27. As aAnother feature associated with an aggressive clinical course in PTC was the presence of fibro-sclerotic tissue within the tumor. Our findings are supported by a few previous studies , 29 but Finally, increased mitotic activity and the presence of necrosis were identified as strong adverse prognostic indicators, both in disease-free and overall survival analyses, although their distribution was not statistically different comparing aggressive and non-aggressive PTC cases. This latter finding is strongly supportive of the concept of the high-grade differentiated thyroid cancer group, as a specific prognostic category within differentiated thyroid cancers and is in line with the novel category proposed by the fifth edition of the WHO book on endocrine tumors, which embeds aggressive differentiated carcinomas and poorly differentiated carcinomas in a single group named high-grade thyroid carcinomas , 34.® PanCancer Pathways Panel to explore different canonical pathways in correlation with PTC aggressiveness. Our study falls in the scarce literature aimed at testing gene expression profiles as determinants of aggressiveness in PTC. The weakness of our approach is mostly based on the targeted approach , the lack of genomic data of the cases investigated, and the lack of RNA validation using an alternative approach or protein analysis by means of immunohistochemistry. By contrast, the strengths of our study are the robustness of the nanostring technology and the study design in terms of case selection. In fact, as compared to similar studies, we designed a comparative study on aggressive and non-aggressive PTC cases based on a strongly balanced and matched selection. A recent study based on TCGA data identified a specific signature of immune-related genes  associated with prognosis in PTC, but using data from a series of nearly 500 PTC cases with 3.2% of patients, only, died of disease and less than 2% of cases with distant metastases [FN1, ITGA3, and MET and downregulated TPO and TG.A second part of our study was focused on the definition of gene expression profiles in a subset of aggressive and non-aggressive PTC cases, selected from the pathological series and matched also for the histological subtype. Despite other studies employed NanoString nCounter technology to explore the molecular make-up of thyroid malignances –37, to ttastases . Anothertastases . In thisWNT10A and GLI3 were significantly up- and down-regulated in aggressive PTC, GSK3B was upregulated in non-aggressive PTC cases.Our study clearly shows that clinically aggressive and non-aggressive PTC cases in our series consistently share a gene expression signature as compared to normal thyroid tissue. In terms of pathway analysis, up-regulation of the RAS, MAPK, and PI3K pathways was consistent in both groups, although with an enrichment of de-regulated genes in the non-aggressive PTC group. However, a larger number of genes was deregulated in non-aggressive PTC as compared to normal tissues than those deregulated in aggressive PTC cases. The larger set of de-regulated genes depicted in non-aggressive PTC cases shows a more significant upregulation of genes belonging to the DNA damage repair and JAK/STAT pathways and a downregulation of genes belonging to the WNT pathway. Interestingly, the hedgehog pathway was differentially de-regulated in aggressive PTC as compared to non-aggressive PTC cases. In fact, whereas GSK3B expression has been described to inhibit the progression of several cancer models, such as, among others, lung [PKMYT1 was significantly upregulated in aggressive PTC cases, only. Although no data are available on the role of this gene in thyroid cancer, PKMYT1 has been shown to exert an important effect on tumor immunity and progression in several cancer models [These findings suggest that these genes are likely involved in molecular mechanisms of aggressiveness. In fact, WNT10A/beta-catenin pathway activation has been demonstrated to promote cellular characteristics of aggressiveness, such as proliferation and migration in thyroid cancer . Data onrs, lung , gastricrs, lung , and heprs, lung  cancers.r models .In conclusion, we demonstrate that clinically aggressive PTC is characterized by peculiar pathological features, including an increased TILs infiltration and a more prevalent occurrence of intratumoral fibrosis, supporting the value of a careful reporting of these parameters in the PTC diagnostic work-up. Moreover, although non-aggressive PTC in our series showed a larger number of deregulated genes as compared to aggressive PTC cases, the existence of a set of differentially regulated genes in the two groups suggests that different molecular mechanisms are active in promoting clinical aggressiveness in PTC with special reference to the impairment of the hedgehog pathway. Although limited by the relative small sample size and biases in case selection, these results have a prime impact highlighting the need to evaluate the stromal component as part of the diagnostic workup for PTC, with special reference to the presence of fibrosclerotic changes, a pathological characteristic that is currently not considered in standardized pathology reporting for thyroid cancer . MoreoveSupplementary file1 (PDF 1144 KB)Supplementary file2 (DOCX 18 KB)Supplementary file3 (DOCX 19 KB)Supplementary file4 (XLSX 18 KB)Supplementary file5 (XLSX 14 KB)Below is the link to the electronic supplementary material.


Hepatocellular carcinoma (HCC) recurrence after liver transplantation (LT) results in a relevant worsening of the prognosis of transplanted subjects. Pre-transplant HCC characteristics are the main determinants of the risk of recurrence and various tools exist to estimate recurrence risk. Once recurrence has occurred, the pattern of recurrence greatly influences whether the patient is a candidate for curative treatment and consequently significantly impacts prognosis. We reviewed different predictive models of post-transplant recurrence, the role of immunosuppression, and the efficacy and feasibility of various therapeutic approaches.Hepatocellular carcinoma (HCC) is a growing indication for liver transplantation (LT). Careful candidate selection is a prerequisite to keep post-LT recurrence rates within acceptable percentages. In the pre-LT period, various types of locoregional treatments and/or systemic therapies can be used for bridging or downstaging purposes. In this context, one of the factors limiting the possibility of treatment is the degree of functional liver impairment. In the LT subject, no widely accepted indications are available to guide treatment of disease recurrence and heterogeneity exists between transplant centers. Improved liver function post LT makes multiple therapeutic strategies theoretically feasible, but patient management is complicated by the need to adjust immunosuppressive therapy and to assess potential toxicities and drug–drug interactions. Finally, there is controversy and uncertainty about the use of recently introduced immunotherapeutic drugs, mainly due to the risk of organ rejection. In this paper, we will review the most recent available literature on the management of post-transplant HCC recurrence, discussing evidence and controversies. Liver transplantation (LT) is the ideal curative treatment for hepatocellular carcinoma (HCC) as it can remove not only liver cancer but also resolve liver cirrhosis, which is the most important cause of hepatocarcinogenesis .In patients with significantly impaired liver function, LT can be the only viable curative approach for HCC.The prerequisite for the use of LT as a therapy for HCC is that the neoplasm must be localized exclusively in the liver, as the presence of extrahepatic localization would be accompanied by a universal post-LT HCC recurrence.Pre-LT imaging techniques can identify macroscopic metastases but do not guarantee the exclusion of microscopic metastases. To prevent post-transplant recurrence, various selection criteria for LT candidates have been proposed over the years with the intent to reduce the risk of misrecognized micro-metastases. The use of such criteria allows for the recurrence rate to be maintained within acceptable rates.In this narrative review, we will review the main scientific evidence regarding selection criteria for transplant candidates, risk factors for recurrence, and treatment of HCC recurrence. In conducting the review, a literature search in the PubMed database was performed by two of the authors (GM and MSL) independently. Papers published up to 31 August 2023 and concerning selection criteria for transplantation, risk factors for recurrence, post-transplantation surveillance, and recurrence treatments were considered. Papers were selected based on title and abstract and, if deemed relevant based on the authors’ arbitrary judgment for the conduct of the review, the full text was retrieved and fully evaluated.In 1996, Mazzaferro et al. first established morphological criteria for successful transplantation for HCC, proposing what later became known as the “Milan criteria”. Transplantable subjects must present with a single lesion with a maximum diameter ≤ 5 cm or up to three separate lesions no larger than 3 cm, along with no evidence of gross vascular invasion and no regional nodal or distant metastases. Patients who satisfied these criteria showed a 4-year survival rate of 75%, similar to expected survival rates for patients undergoing transplantation for cirrhosis without HCC. In the original Mazzaferro study, only 8% of enrolled patients experienced HCC recurrence within 4 years ,3.Over the years, less restrictive criteria have been proposed with comparable results to the Milan criteria. In 2001, the University of California, San Francisco (UCSF) proposed new extended criteria which are fulfilled if the patient has a single nodule up to 6.5 cm in maximum diameter or three nodules with a total diameter of up to 8 cm. These criteria led to a favorable 5-year patient survival rate of 75.2% and a post-LT recurrence rate of 11.4% [In 2009, Mazzaferro et al. proposed the so-called “up-to-seven” criteria. Using these criteria, subjects are considered for OLT if the sum of the number of nodules and the diameter (in cm) of the largest nodule does not exceed seven  Table 1Table 1.Several studies have evaluated the reliability of the up-to-seven criteria as inclusion criteria, showing post-LT survival rates roughly overlapping with patients meeting the Milan criteria ,7.All the mentioned criteria are based on the morphological characteristics of the HCC and are built on the pre-LT accuracy of liver imaging techniques such as contrast-enhanced computed tomography or magnetic resonance imaging, which can be affected by heterogeneity and suboptimal sensitivity ,9,10.For patients with a tumor stage beyond morphologic criteria, the possibility of tumor stage reduction in order to fall within morphologic criteria  using various treatments  has been explored with good results ,12,13. DThe introduction of morphologic criteria resulted in a marked improvement in LT outcomes for HCC compared with the time before the introduction of the Milan criteria ,16,17. TSince Mazzaferro’s original criteria, macroscopic vascular invasion has been considered an unacceptable risk factor for post-transplant recurrence. Over the years, how microvascular invasion is also associated with a high rate of recurrence has come to be understood ,7.Encouraging post-LT results have recently been reported for patients with complete regression of macroscopic vascular invasion after downstaging protocols including chemoembolization, radioembolization, or surgery. Indeed, recurrence rates are found to be acceptable (11%) in subjects presenting low alpha-fetoprotein (AFP) (<10 ng/mL) and complete vascular invasion regression at pre-transplant imaging. However, prospective validation is needed, and caution is required in considering this category of patients for transplantation .The diagnosis of HCC in cirrhotic individuals is often based on radiological criteria in accordance with current guidelines, and liver biopsy is reserved for cases with nontypical imaging ,23 so inMore recently, the Toronto extended criteria have been proposed, which also consider preoperative histology. Using these criteria, liver transplantation could be offered to subjects with any number and size of hepatic nodules if they have no extrahepatic localization (including macrovascular invasion), no poorly differentiated neoplasm at preoperative histology of the larger nodule, and no cancer-related symptoms .Serum alpha-fetoprotein (AFP) can also be considered a surrogate marker of HCC differentiation and vascular invasion , and theIn 2018, Mazzaferro et Al. proposed the so-called Metroticket 2.0 model, which was constructed to predict post-transplant HCC-related specific death by considering AFP as a surrogate marker of microvascular invasion and morphological characteristics of HCC .Also, in Toronto cohorts, high AFP levels at listing and at the time of transplant were associated with worse post-transplant outcomes, both in “Milan in” subjects and in subjects transplanted according to the extended Toronto criteria. AFP levels and trends over time appeared as relevant prognostic indicators, and a cut-off of 500 ng/mL was proposed as indicative of a poor prognosis .In 2012, Duvoux et al. proposed and validated a model incorporating AFP, tumor size, and nodule number to predict post-transplant HCC recurrence: a cut-off of 2 was identified for the selection of patients with an acceptable risk of recurrence. In patients exceeding Milan criteria, a score ≤ 2 identifies subjects with low AFP values (<100 ng/mL) with an acceptable risk of 5-year post-transplant recurrence (14.4 ± 5.3%). On the contrary, in subjects within Milan criteria, a score higher than 2 identifies subjects with high AFP values (>1000 ng/mL) with an unacceptable risk of 5-year post-transplant recurrence (37.1 ± 8.9%)  Table 2Table 2. Post-LT HCC recurrence is among the leading causes of death in subjects transplanted for this indication. Emerging data and expert consensus support post-transplant HCC surveillance, as early diagnosis and aggressive treatment have been demonstrated to improve survival outcomes ,39,40.Time to recurrence can be highly variable. HCC recurrence is an infrequent cause of death in the first year after LT (5.3% of deaths in the first year) . A peak Considering the very high impact of HCC recurrence on post-transplant prognosis, many of the criteria and scores for selecting transplant candidates were constructed with both post-transplant survival and HCC recurrence as outcomes. As previously mentioned, the HCC recurrence rate is largely dependent on the characteristics of the pre-transplant tumor. Using Milan criteria, recurrence has been observed in 5.7%–16% of cases, and this has become the benchmark for all the subsequent proposed criteria ,47,48.Data coming from analysis of the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) registry confirmed that some pre-transplant factors are strong predictors of recurrence, including the presence of extrahepatic or lymph nodal spread , poor tumor differentiation (OR 2.8), micro- and macrovascular invasion , explant TNM stage T4 or T3 , downstaging from a stage > than T2 (OR 1.8), and high AFP values (OR 1.2) .In a systematic review published in 2015, de’ Angelis et al. evaluated 61 heterogeneous studies. The observed overall mean recurrence rate was 16%, and the median time from LT to recurrence was 13 months (range 2–132 months). Most observed recurrences (67%) were extrahepatic. Interestingly, 51% of patients included in the study were transplanted beyond Milan criteria according to explant pathology . Also, ip ≤ 0.001, τ2 = 0.01) with a non-statistically significant downward trend over the years. Geographical area and ethnicity have been shown to significantly influence the recurrence rate, ranging between 10% in South American studies and 21% in Asian studies, with Asian and African American men showing higher recurrence rates compared with Caucasians [In a recently published systematic review and meta-analysis of 125 studies including 55.333 patients, the pooled reported recurrence rate was 17%  [Tumor histology can provide useful information in predicting the risk of recurrence. Lasagni et al. analyzed a group of 70 patients with HCC recurrence and compared them with a matched cohort of patients without recurrence and validated their observations in an independent cohort. Patients experiencing HCC recurrence showed significantly increased expression of angiopoietin-2 in tumor endothelium but not in the hepatocyte at explant pathology. Univariate analysis identified BMI, log AFP at transplant, endothelial angiopoietin-2 expression, Milan criteria, Metroticket AFP score, and the AFP model as significantly associated with recurrence, while in multivariate analysis angiopoietin-2 expression was the only independent factor associated with recurrence  .Recently, six histological features of explant tissues were examined in a database of 380 patients undergoing LT for HCC: tumor region, normal liver tissue, portal area, fibrous tissue, hemorrhagic and necrotic tissue, and immune cells. Using artificial intelligence techniques with neural networks, all of these features, except for normal liver tissue, were used to create a Deep Pathomics Score (DPS) with the aim of predicting the risk of recurrence. DPS was able to discriminate between subjects with a low risk of recurrence  and high risk of recurrence . Among the evaluated histological aspects, immune cells were the strongest prognostic determinant of recurrence in multivariate analysis  . This waAs previously reported, AFP levels at the time of transplant are a strong predictor of HCC recurrence, but this remains a suboptimal biomarker. Des-gamma-carboxyprothrombin (DCP) and AFP bound to Lens culinaris agglutinin (AFP-L3) have been recently proposed as promising biomarkers. In a prospective cohort of 285 patients transplanted according to Milan criteria, AFP-L3 and DCP resulted in the strongest predictors of HCC recurrence, and a combination of both (AFP-L3 ≥ 15% and DCP ≥ 7.5) predicted 61.1% of observed recurrence  Figure .Evidence-based recommendations for HCC post-LT surveillance protocols are lacking to date as there are no adequately powered studies evaluating the best frequency, duration, and effects on LT outcomes ,55, evenDespite the absence of solid scientific evidence supporting the cost-effectiveness of post-transplant surveillance for HCC recurrence, there is broad agreement that periodic contrast imaging with CT or MRI should be performed in the post-LT setting and that periodic AFP should be obtained, particularly in subjects with high AFP values before LT. In subjects undergoing liver resection for HCC, current guidelines suggest indefinite cross-sectional imaging examinations of the abdomen and chest plus serum AFP every 3–6 months . In the In this perspective, explant pathology can be useful in providing an estimate of the risk of HCC recurrence and therefore guide post-transplant surveillance intervals, reducing unnecessary radiation exposure. This is also crucial as suboptimal concordance data between pre-transplant imaging staging and pathological findings have been reported over time  , althougIn 2017, the RETREAT score (Risk Estimation of Tumor Recurrence After Transplant) was proposed to stratify the risk of HCC recurrence in subjects transplanted with HCC meeting Milan criteria at pre-LT imaging. The 1061 patients included in the study were divided into a development cohort (721 subjects) and a validation cohort (341 subjects). A score including the presence of microvascular invasion at explant (present vs. absent), the sum of the largest diameter of viable tumors, the number of viable tumors at explant, and AFP levels at transplant was constructed in the development cohort and validated in the validation cohort. RETREAT score ranges from 0 to 8 and can stratify the risk of recurrence between very low risk  to extremely high risk  . The scoMore recently, a new score based on the number of nodules, the size of the largest nodule, the presence of microvascular invasion, nuclear grade, and the last pre-LT AFP value—namely the Recurrence Risk Reassessment (R3)-AFP score—was tested and validated with a large multicentric cohort. This score ranges between 0 and 10 and stratifies patients into categories of very low risk of recurrence , low risk , high risk , and very high risk . This score showed superimposable performance with the RETREAT score in both the test and the validation cohort . This scThe mainstay of post-LT immunosuppression in the early post-transplant period is the use of calcineurin inhibitors (CNIs) such as cyclosporine and tacrolimus. However, these drugs affect the immune system’s response and cancer immunosurveillance, thus promoting cancer cell survival and increasing the risk of HCC recurrence in a dose-dependent manner ,64,65,66Mammalian target of rapamycin inhibitors (mTORi), such as sirolimus and everolimus, exhibit anti-tumoral growth effects (anti-proliferation and anti-angiogenetic) ,68,69,70On this basis, the current recommendation is to taper immunosuppression to the lowest effective dose for protection against graft rejection and reconsider immunosuppressive therapy regimens by combining or switching to an mTOR and decreasing the dosage of CNIs .Patients with post-LT HCC recurrence should be promptly treated. As in the pre-transplant setting, curative treatments when feasible provide the best results in terms of long-term survival ,81,82,83In 2015, Sapisochin et al. proposed a prognostic score for HCC recurrence using three variables independently associated with poor prognosis in a multicentre cohort of patients with HCC recurrence. The three variables considered were the following: being non-amenable to curative-intent treatments (surgery or ablation), early recurrence (<1 year), and AFP values of ≥100 ng/mL at the time of recurrence. Patients with a 0 score (none of the considered variables) showed 5-year survival of ∼50% . The scoIn many of the papers that have analyzed the treatment of post-transplant recurrence, surgical treatments  and percutaneous or laparoscopic ablative treatments are considered together, both because of the small sample size and because, in the case of small nodules (up to 2–3 cm in maximum diameter), the results of the two approaches are comparable.In the UCLA series, the majority of patients experiencing post-LT HCC recurrence (59.9%) received non-surgical therapy, 23.3% received surgery , and 18% received only supportive treatments. Surgical treatments achieved the best results in terms of survival, especially in the case of graft surgical resection for intrahepatic oligo-recurrence . In this study, the patients were followed with a surveillance protocol including serum alpha AFP and axial contrast-enhanced imaging every 6 months post LT. Despite this intensive surveillance program, HCC recurrence was often not suitable for curative treatments because of the advanced stage or because of technical issues. Interestingly, the authors found that the pattern of tumor recurrence , rather than explant pathology, was the main factor affecting post-recurrence survival . SimilarExtrahepatic recurrence should not be a reason for exclusion from surgical treatments, as various studies have shown a survival advantage in subjects undergoing resection for such recurrences ,86,87,88As in the previously cited series, Fernandez-Sevilla et al. reported significantly longer survival in patients undergoing surgical resection compared to that of non-resected patients (35 vs. 15 months). Most resected patients received surgery for extrahepatic sites (2 intrahepatic vs. 20 extrahepatic), and patients with extrahepatic unilocular recurrence presented the best results after resection. In contrast, the intrahepatic location of the recurrence was found to be significantly associated with worse outcomes (HR 1.8), together with AFP level > 100 ng/mL at relapse (HR 2.01) and multifocal recurrence  .Among extrahepatic recurrences, multiple experiences are reported in the literature about pulmonary recurrences with satisfactory outcomes for treated patients ,91,92,93However, it must be emphasized that the surgical treatment of post-LT recurrent HCC poses some issues to be kept in mind. First of all, transplanted subjects are at higher risk of peri-operative infective complications due to immunosuppression. Furthermore, as concerns the resection of hepatic or perihepatic recurrences, the possible occurrence of post-surgical adhesions should be considered, which can be particularly challenging at the hilar site and, in general, near surgical anastomoses. In recent years, liver resections using minimally invasive surgical techniques have been increasingly performed, both for primary and recurrent HCC . The useIn patients who are not candidates for surgical resection or in the case of small recurrent lesions, locoregional techniques are viable alternatives. Among these, ablation has shown the best results. Ablation can be performed with two major techniques: radiofrequency ablation (RFA) and microwave ablation (MWA).Percutaneous or laparoscopic RFA is ideal for small tumors deeply located in the liver parenchyma and with limited anatomical relationships to vessels and other abdominal organs. In general, it is well known that RFA provides equivalent local disease control compared with surgical resection for nodules up to 3 cm in diameter. In these patients with initial intrahepatic post-LT recurrence, the less invasive nature of such procedures is an additional advantage over surgery due to lower morbidity and the lower risk of complications, although comparison studies in this setting are limited ,104,105.In an Asian cohort of patients with post-transplant HCC recurrence, the authors found no significant differences in the 5-year overall survival of patients treated with RFA or surgical resection, but not statistically significant worse disease-free survival was observed in RFA-treated subjects . It should be noted that in the treatment algorithm used in this study, RFA was used exclusively in cases of contraindication to surgery, and given the small number of patients, balancing for site and type of recurrence could not be performed .Although numerically limited, there are published experiences in the literature about the use of the MWA ablation technique, either percutaneously or laparoscopically, for post-transplant recurrences, with some of these experiences describing encouraging results ,108.In a small retrospective series of 11 patients with post-LT recurrence, MWA ablation was well tolerated and only 3 of 11 patients exhibited tumor progression after treatment; the 2-year survival rate was 15.3% and the mean survival time was 17.3 months .No specific studies have evaluated this technique in post-LT recurrence in comparison with RFA or surgery.Trans-arterial chemoembolization (TACE) is widely used in pre-transplant settings both as a bridge treatment and in downstaging protocols. In the case of post-LT recurrence, it is feasible, but results seem to be worse than those achieved using surgery or ablation.Ko et al. reported no complications in a group of 28 patients treated with one or more cycles of TACE for recurrent HCC after living donor liver transplantation. A large percentage of treated subjects experienced a reduction in target tumor volume of 25% or more; however, 92.9% of patients experienced intra- or extrahepatic recurrence in the first 6 months after the procedure. The 1-, 3-, and 5-year survival rates after TACE were 47.9%, 6.0%, and 0%, respectively. It should be noted, however, that the enrolled population is numerically limited and very heterogeneous, with some patients having largely multinodular recurrence .p = 0.035) [Recently, better outcomes have been reported in an Asian retrospective cohort. In a group of 54 patients treated with TACE, the 1-, 2-, and 3-year overall survival rates were 44.71%, 42.2%, and 19.5%, respectively. Significantly better results were observed in a group of 52 propensity score-matched patients treated with TACE plus Sorafenib  .Better results have been reported by Zhou et al., who observed 1-, 2-,3-, and 4-year overall survival rates after OLT that were 85.7%, 56.3%, 37.6%, and 18.8%, respectively . In thisSystemic therapies, mainly tyrosine kinase inhibitors (TKIs), have been extensively studied in non-transplanted patients with advanced HCC ,113.In 2013, a retrospective case–control Italian study explored the use of Sorafenib in patients with post-LT HCC recurrence not eligible for surgery or locoregional techniques. Sorafenib provided better outcomes compared with the best supportive care, with post-recurrence survival of 21.3 months in treated subjects compared to 11.8 months in non-treated subjects . SorafenThe association of Sorafenib and mTORi has been evaluated in a retrospective study including 31 patients. Median overall survival after the initiation of mTORi + Sorafenib was 19.3 months. The time to disease progression after treatment initiation was 6.77 months. In this cohort, 14 patients presented a treatment response  .Recently, the safety of early Sorafenib administration for post-LT HCC recurrence was evaluated in a retrospective cohort including 50 patients. All the enrolled patients presented a Sorafenib-related adverse event, with 56% of them being grade 3–4. There was no significant difference in Sorafenib-related adverse events between patients taking mTORi or other immunosuppressive regimens, except for any grade of hand–foot syndrome. Interestingly the use of mTORi + Sorafenib was associated with an increase in overall survival in univariate analysis but not in multivariate analysis .Lenvatinib has also recently been used for the treatment of post-transplant HCC recurrence.A recent multicentre international retrospective study evaluating 45 Lenvatinib-treated subjects with post-LT recurrent HCC reported median progression-free survival of 7.6 months (95% CI: 5.3–9.8 months) and overall survival of 14.5 months (95% CI: 0.8–28.2 months) over a 12.9-month median follow-up. In total, 20% of patients presented a partial response, 68% stable disease, and 6.7% progressive disease during treatment. These results are comparable to those found in non-transplanted subjects. No effect on survival was found for the immunosuppressive regimen in this study. The majority of analyzed patients experienced an adverse event (97.8%) but the overall safety profile of the drug appeared satisfactory, with only 8.9% of subjects discontinuing the drug due to adverse events .Yang et al. analyzed a retrospective cohort of patients with post-LT recurrent HCC treated with various approaches. In the sub-group of patients treated with TKIs progressing during Sorafenib treatment, Sorafenib-tolerant subjects receiving Lenvatinib showed significantly longer survival when compared with patients shifted to Regorafenib or patients just stopping Sorafenib. Also, in this report, the use of mTORi was associated with better overall survival if compared with CNIs .p: <0.01) and the treatment was well tolerated, with 44% of patients receiving the full dose of the drug [Second-line systemic therapy has been evaluated in the post-LT setting. In two multicentre international studies, Iavarone et al. evaluated the efficacy and the safety of Regorafenib in patients with post-LT HCC recurrence tolerant to Sorafenib but progressing during treatment. In the first study, the safety of Regorafenib was evaluated in 28 subjects. All enrolled subjects presented at least one adverse event, with fatigue, weight loss, and dermatological reactions the most frequently observed . Severe adverse events occurred in 43% of subjects. During treatment, immunosuppressive drug adjustment was needed in five patients because of increased circulating levels of the immunosuppressive drugs. The median OS was 12.9 months with a median follow-up of 8.9 months (1.5–24.0) . In the the drug   has changed the scenario of systemic treatment for advanced HCC in non-transplanted patients ,122. TheData on the efficacy and safety of ICIs for the treatment of HCC recurrence in LT recipients are largely lacking, with the few existing examples in the literature represented by case reports, small case series, and single-centre experiences with contrasting results ranging from optimal responses to catastrophic events ,127,128.The time elapsed between liver transplantation and the administration of ICIs seems to have an influence on the development of liver allograft rejection, with higher risk in the early post-transplant period. The minor risk of adverse events in long-term transplanted patients could be due to transplant immunological tolerance and reduced immunosuppression needs ,132,133.Further and adequately designed studies are needed to better understand the efficacy and safety of these treatments in transplant recipients in general and in post-LT HCC recurrence in particular.Post-LT HCC recurrence dramatically worsens the prognosis of transplanted patients. Several pre-transplant factors have been identified as risk factors for recurrence, and specifically designed scores are available to predict the risk of recurrence and guide post-transplant surveillance and management. Immunosuppressive protocols may affect the risk of HCC recurrence post-LT but the best immunosuppressive schedule for patients transplanted for HCC is yet to be defined. Among treatments for HCC recurrence, surgery, whether for hepatic or extrahepatic recurrence, shows the best results and should be the preferred approach of treatment in selected patients with a limited burden of tumor recurrence. In more advanced cases, systemic therapy with TKIs demonstrates promising results in terms of improved patient survival.LT for HCC is a growing indication. In this context, the introduction of new biomarkers to clinical practice is needed to provide a deeper understanding of tumor biology, to allow for better patient selection, and to stratify post-LT recurrence risk.Prospective validation of predictive models is needed to implement customized surveillance programs, and specifically designed studies are needed to evaluate the cost-effectiveness of post-transplant surveillance protocols.The definition of personalized immunosuppressive schemes and drug levels with the measurement of specific biomarkers of immune system activity could help in reducing the risk of HCC recurrence and improve the management of recurrent cancer.The potential use of systemic adjuvant treatments and the use of ICIs could significantly change the prognosis and management of HCC recurrence post LT in the near future.


The therapeutic strategy for and the staging system of primary thyroid mucosa-associated lymphoid tissue (MALT) lymphoma are not established. We conducted a retrospective analysis to (i) establish the appropriate staging system and (ii) assess the role of curative thyroidectomy vs. involved-site radiation therapy (ISRT) after open biopsy in stage IE MALT lymphoma. The modified staging system allows us to distinguish between stages IE and IIE primary thyroid MALT lymphoma. Information about the side effects under ISRT may have limited informative value, but approximately one in three to four patients had radiation-induced permanent complications (mainly dry mouth). Curative thyroidectomy provides good prognoses, equivalent to that of ISRT after open biopsy; it avoids permanent dry mouth, shortens painful periods during treatment, and simplifies ultrasound follow-ups. In the NCCN guidelines, ISRT is the preferred initial therapy for limited-stage non-gastric MALT lymphoma. However, curative thyroidectomy alone can serve as the initial treatment equivalent to ISRT post-open-biopsy.p = 0.0092) and relapse-free survival (0.00113) were significantly better in stage IE vs. stage IIE under the Tokyo classification. No OB-ISRT and Surgery patients died, but three OB-ISRT patients relapsed. The incidence of permanent complications was 28% in OB-ISRT (mainly dry mouth) and 0% in Surgery (p = 0.027). The number of painkiller prescription days was significantly greater in OB-ISRT (p < 0.001). During follow-up, the rate of the new appearance/change of the low-density area in the thyroid gland was significantly higher in OB-ISRT (p = 0.031). Conclusions: The Tokyo classification allows an appropriate discrimination between stages IE and IIE MALT lymphoma. Surgery can provide a good prognosis in stage IE cases; it also avoids complications, shortens painful periods during treatment, and simplifies ultrasound follow-up.Purposes: To establish the appropriate staging system and assess the role of curative thyroidectomy alone (Surgery) vs. involved-site radiation therapy after open biopsy (OB-ISRT) in stage IE mucosa-associated lymphoid tissue (MALT) lymphoma. Methods: We examined the Tokyo Classification as a modified classification. This retrospective cohort study included 256 patients with thyroid MALT lymphoma; 137 underwent standard therapy  and were enrolled for the Tokyo classification. Sixty stage IE patients with the same diagnosis were examined to compare Surgery with OB-ISRT. Results: Overall survival ( Primary thyroid lymphoma (PTL) accounts for only 1–2% of extranodal lymphoma and <5% of thyroid malignancies . PTL is The therapeutic strategy of primary thyroid MALT lymphoma is not well-established, in part because of primary thyroid MALT lymphoma’s rarity. Treating PTL thus usually follows non-organ-specific guidelines, such as the U.S. National Comprehensive Cancer Network (NCCN) clinical practice guidelines for B-cell lymphomas , in whicNo staging system for primary thyroid MALT lymphoma exists. The Ann Arbor classification system was initially developed for staging Hodgkin’s disease but has some use in non-Hodgkin lymphoma . After tIto Hospital (Tokyo) specializes in the treatment of thyroid diseases and has long collected clinical data on PTL cases ,9,10,11.We conducted a retrospective cohort study of patients with limited-stage primary thyroid MALT lymphoma at Ito Hospital (Tokyo) between January 1990 and October 2021 after obtaining Institutional Ethics Board approval (no. 169). Patients with limited-stage MALT lymphoma were included for the modified staging system’s assessment. The preferred initial therapy for limited-stage non-gastric MALT lymphoma is OB-ISRT . Our excStage IE primary thyroid MALT lymphoma is defined as involvement of the thyroid alone; stage IIE is defined as the major site of involvement being in the thyroid gland with associated regional lymph node involvement . To divide limited-stage MALT lymphoma into clinical stage IE and stage IIE, the conventional staging systems, i.e., the Lugano classification  and the Moreover, the coexistence of Hashimoto’s disease makes it difficult to distinguish ‘involved’ from ‘inflamed’ lymph nodes. A minimal axial diameter of 10 mm is generally thought to be the most accurate size criterion for predicting lymph node metastasis . After wWe next compared surgery  alone with OB-ISRT in the stage IE patients defined using the Tokyo classification. Because a thyroidectomy without regional lymph node dissection is not curative for stage IIE, we did not include stage IIE patients in this analysis. Initially, at Ito Hospital, all patients who underwent a thyroidectomy for limited-stage primary thyroid MALT lymphoma also underwent post-operative radiotherapy. After 2012, we provided a therapeutic option: no ISRT after a curative thyroidectomy for stage IE MALT lymphoma.Given this background, the inclusion criteria were patients with stage IE MALT lymphoma who had undergone OB-ISRT or a thyroidectomy alone during the period between January 2012 and October 2021. The exclusion criteria were: (1) patients whose data concerning treatment complications could not be collected , and (2) patients who had another disease associated with lymphadenopathy (other than autoimmune thyroid disease [Graves’ disease and Hashimoto’s disease]).The patient demographics included age, sex, the coexistence of Hashimoto’s disease, hypothyroidism before treatment, and lesion location. We also analyzed variables pertaining to treatment-related complications, the new appearance or change of a low-density area (LDA) on the ultrasound post-treatment, the performance of a biopsy (Bx)/fine-needle aspiration (FNA) or scintigraphy due to the suspicion of recurrence, and cancers after treatment. These data were collected retrospectively from the medical records. Radiation-induced complications were graded using CTCAE ver. 5.0 .Most of the patients with ISRT underwent radiotherapy with 30 Gy at Ito Hospital, but some patients underwent ISRT at other institutions nearby. 2-test or Fisher’s exact test to compare nominal data, and we used the Kaplan–Meier method and stratified log-rank tests for the overall survival (OS) and relapse-free survival (RFS) rates. For all procedures, p-values < 0.05 were considered significant. The statistical analyses were conducted using STATA software ver. 15.0 .We used the χn = 59), and 60 patients underwent initial treatments other than ISRT after open biopsy . Thus, 137 patients were included in the analyses. The selection process is summarized in p = 0.0673), although the OS in stage IIE tended to be worse compared to that in stage IE. The RFS was also not significantly different between stages IE and IIE under the conventional staging systems .p = 0.0092). The 5- and 10-year RFS rates were 94.7% and 89.7% for stage IE and 74.1% and 69.8% for stage IIE, respectively  under the Tokyo classification. After the restaging using the Tokyo classification, no upstaging was detected, but downstaging from stage IIE to stage IE was observed in 17 patients (33.3%). No relapse was detected in these 17 cases with a median follow-up of 8.1 years.After the restaging using the Tokyo classification, the 5- and 10-year OS rates were 96.8% and 95.0% for stage IE, and 78.1% and 78.1% for stage IIE, respectively  and OB-ISRT (n = 46) groups. To assess the advantages/disadvantages of curative thyroidectomy for primary thyroid MALT lymphoma, we next compared surgery alone to OB-ISRT in the stage IE patients. Both radiation therapy and thyroidectomy have treatment-specific complications. For radiation therapy to the cervical area, well-known radiation-induced complications are dermatitis, oral mucositis, dry mouth, dysphagia, esophagitis/pharyngitis, and hoarseness. Possible surgical complications after thyroidectomy are hypoparathyroidism and recurrent laryngeal nerve paralysis (RLNP). Both treatments also pose a risk of thyroid dysfunction. Almost all of the cases of dermatitis and/or hoarseness were CTCAE grade 1 and did not require any specific treatment, but many of the cases of esophagitis/pharyngitis, dysphagia, dry mouth, and/or oral mucositis were CTCAE grade 2 and needed additional treatment. Most of the radiation-induced complications improved with time, but 28% of patients with OB-ISRT had permanent complications, most of which were dry mouth. One patient in the OB-ISRT group had post-operative bleeding and required re-operation after the open biopsy.p = 0.027). The number of painkiller prescription days was significantly greater in the OB-ISRT group vs. the Surgery group (p < 0.001).In the Surgery group, all cases of surgical complications were transient hypoparathyroidism (incidence rate: 36%), and no patients faced permanent hypoparathyroidism or post-operative RLNP. The incidence of permanent complications other than thyroid function was thus significantly higher in the OB-ISRT group compared to the Surgery group (p = 0.685). There were also no significant differences between the groups in terms of the incidence of new hypothyroidism or in the rate of patients whose levothyroxine dose was increased after treatment.Concerning complications associated with thyroid function, only 15% and 21% of the patients in the OB-ISRT and Surgery groups, respectively, were euthyroid after treatment (p = 0.031). However, there were no significant differences between the OB-ISRT and Surgery groups in the post-treatment performance of Bx/FNA (15% vs. 7%) or scintigraphy . After the initial treatment for lymphoma, three patients in the OB-ISRT group had cancers . During the follow-up (93.3% follow-up rate with an average follow-up of 5.12 years), none of the patients died, and three OB-ISRT patients had a relapse of MALT lymphoma (two in the thyroid gland and one in a lymph node) (The patients’ post-treatment courses are described in ph node) .The results of our analyses demonstrate that the modified staging system  is suitable for primary thyroid MALT lymphoma compared to the conventional staging systems (Ann Arbor and Lugano), which are non-organ-specific systems. Under the Tokyo classification, both curative thyroidectomy alone and OB-ISRT provided a satisfactory prognosis.Because the morphological and immunohistological findings are sometimes insufficient for a confident diagnosis in terms of regional lymph nodes, the pathological diagnosis of lymph node involvement in MALT lymphoma is more difficult than that of lymph node involvement in DLBCL. Moreover, FDG PET/CT has limited sensitivity in the assessment of MALT lymphoma because of the low metabolic rate of this malignancy and the high rate of concurrent Hashimoto’s disease . For theThere is currently no consensus as to which treatment is the best for stage IE primary thyroid MALT lymphoma. Our present analyses of stage IE patients with primary thyroid MALT lymphoma revealed that both curative thyroidectomy alone and OB-ISRT provided satisfactory overall and relapse-free survival. It is thus reasonable for physicians to present both treatments as therapeutic options to these patients.Because ISRT and thyroidectomy have different types of treatment-related complications, it is difficult to state which treatment is better in terms of complications. Nonetheless, our present findings demonstrate that thyroidectomy alone was superior in terms of permanent complications and pain duration compared to OB-ISRT. In this series, radiation-induced permanent complications (mainly dry mouth) occurred in approx. one in three to four patients. Most patients who have primary thyroid MALT lymphoma are in the seventh and eighth decades of life , and dryAlthough an appropriate radiation dose for primary thyroid MALT lymphoma has not been established, the NCCN guidelines recommend 24–30 Gy for MALT lymphoma . In a seIf a patient’s clinical features  and/or tThe potential advantages of a curative thyroidectomy are (i) easy ultrasound follow-up due to the removal of LDA(s) in the thyroid gland, (ii) a more definite removal of lymphoma within the thyroid gland, and (iii) the removal of the risk factor of lymphoma when a total thyroidectomy is performed. After ISRT, an LDA inside the thyroid gland does not disappear soon and remains in some cases. In such cases, careful observation of the lesions is required. For this reason, the removal of LDA(s) by a thyroidectomy provides the possibility of relieving both patients’ and physicians’ psychological stress caused by LDAs remaining after treatment. Moreover, a curative thyroidectomy may help rule out histological transformation. Rajamäki et al. reported that a rebiopsy based on a high SUVmax in FDG-PET/CT was valuable in detecting the transformation of follicular lymphoma , but it Despite our encouraging results, some study limitations should be noted: (i) We did not assess whether curative thyroidectomy following an open biopsy is an acceptable treatment. (ii) The data on radiation-induced complications were collected from the patients’ medical records and not questionnaires completed by the patients. (iii) We did not evaluate the appropriate radiation dose for each stage of primary thyroid MALT lymphoma.All of the patients in the Surgery group underwent one-stage surgery, i.e., a thyroidectomy, for diagnosis and treatment, and none of the patients underwent a thyroidectomy alone after a diagnosis by open biopsy. However, we feel that performing a thyroidectomy after a diagnosis by open biopsy is also an acceptable therapeutic option. In addition, an open biopsy exposes only the front side of the thyroid and does not expose the recurrent laryngeal nerve (RLN); a subsequent thyroidectomy can thus be performed without adhesion around the RLN . We thusThis was a retrospective study and did not use any questionnaires completed by the patients to collect the data on radiation-induced complications. Hospital records usually underestimate the true number of complications. As noted above, we could not assess the appropriate radiation dose with a good balance of reducing radiation-associated complications and avoiding relapse within the thyroid gland because few patients in this series received 24 Gy radiotherapy. Because ISRT is the preferred initial therapy in the NCCN guidelines, the present Surgery group was quite smaller than the OB-ISRT group .Our study results demonstrated that the modified staging system (the Tokyo classification) allowed us to distinguish between stage IE and stage IIE. Although our findings regarding ISRT side effects may have limited informative value due to this study’s retrospective nature, approx. one in three to four patients had radiation-induced permanent complications (mainly dry mouth) in this series. The use of the Tokyo classification revealed that (i) curative thyroidectomy provides a good prognosis that is equivalent to that provided by OB-ISRT, and it also avoids permanent dry mouth, shortens painful periods during treatment, and simplifies the ultrasound follow-up in stage IE patients; and (ii) approx. three in 10 stage IIE patients were reclassified as stage IE and would thus be able to choose thyroidectomy alone as a therapeutic option, although, in the NCCN guidelines, ISRT is the preferred initial therapy for limited-stage non-gastric MALT lymphoma. Further research is necessary to determine the precise potential implications of both curative thyroidectomy and the clinical usage of the Tokyo classification.


The primary outcome was time to ART attrition  and any difference in one-year retention in PMTCT and ART care between intervention and control facilities. Secondary outcomes were maternal viral suppression  and mother-to-child HIV transmission (MTCT) by ≥12 months post-partum. Analyses were done using Kaplan Meier and Cox regression (ART retention/attrition), generalized estimating equations  and random effects logistic regression (MTCT); reporting rates, proportions and 95% confidence intervals (CI). There were 1957 women in the peer-mother and 1384 in the control facilities who enrolled in routine PMTCT care during 2018–2019 and were followed for a median [interquartile range (IQR)] of 23  of 23 , 31 mont Use of lifelong anti-retroviral treatment (ART) for all pregnant and breastfeeding women with HIV regardless of immunologic or clinical disease status, also known as the WHO Option B+ recommendation of 2012 . BarrierPeer support services have been widely in use to mitigate barriers of uptake, adherence and outcomes in HIV and PMTCT services in low- and middle-income countries –12. The This study was approved by the ethical review boards of Muhimbili University of Health and Allied Sciences, ref. 2017-06-28/AEC/Vol.XII/83, and the Tanzania National Institute for Medical Research, ref. NIMR/HQ/R.8a/Vol.IX/2594. Informed consent was waived on the basis that the study used anonymized data from electronic routine healthcare records.st October 2015 and 30th September 2016 was identified  of Dar es Salaam, Tanzania. Dar es Salaam is the largest and main commercial city of Tanzania with a population of over five million people and an adult HIV prevalence of 4.3% per the 2016/17 Tanzania HIV impact survey , 26. Durentified  for the entified . RandomiIn Tanzania, PMTCT is integrated with ANC and provided under one roof as standard of care. All pregnant women found to be HIV positive at ANC, are enrolled in PMTCT care and started on lifelong ART on the day of HIV diagnosis, or, those with known HIV are encouraged to continue taking their ART. Since 2019, the default adult (including pregnant women) first-line ART regimen backbone in Tanzania changed to an integrase inhibitor-based regimen containing dolutegravir (DTG). Patients who were on the first-line ART containing non-nucleoside reverse transcriptase inhibitor (NNRTI) were gradually transitioned to DTG regimens from 2019 and onwards. Protease inhibitors (PIs) are backbone regimens for second-line ART. All PMTCT clients are followed up through monthly ANC-PMTCT visits for: clinical and laboratory monitoring; ART refills; counselling on adherence, infant feeding and Early Infant Diagnosis of HIV (EID); and other antenatal/ postnatal care services. As part of routine care, women who miss their scheduled PMTCT follow-up visit by three or more days are traced via phone call by a health care provider (HCP) and, when unsuccessful, a home visit by a community health worker (CHW). These women are declared as lost to follow-up after at least three unsuccessful tracking efforts over 90 days. Viral load monitoring is done at first ANC visit for women already on ART, and three months after starting ART for women new on ART, then six-monthly until cessation of breastfeeding, and thereafter once annually. Women with high viral loads (1000 or more copies per mL) are offered enhanced adherence counselling, repeat viral load testing after three months and are switched to second line ART upon persistent high viral load. At birth, infants born to women with HIV, i.e. HIV Exposed Infants (HEIs) are given daily nevirapine prophylaxis for six weeks, with addition of zidovudine for high risk HEI . At six weeks, HEI receive their first HIV test for EID using DNA-polymerase chain reaction (PCR), started on daily co-trimoxazole prophylaxis and enrolled on monthly mother-infant follow-up. Follow-up for the mother and infant in PMTCT care goes on for up to two years post-partum unless the infant is diagnosed with HIV infection, at which point the infant and mother are transitioned to routine ART care. Infants who are not HIV infected are discharged after a negative HIV antibody test by 18 months of age. All HIV patients in Tanzania, are issued national HIV care and treatment clinic (CTC) unique identification (ID) numbers, known as CTC-ID. The CTC-ID is used to register, record and manage ART/ PMTCT care of all HIV patients using health facility charts (CTC2 cards) and the national electronic CTC2 database, as well as identify/link patients across facilities upon transfer of care. The HEI are also issued unique IDs and registered in HEI cards, linked to their mother’s CTC-ID, which is used to record and manage their care including EID tests.The study population included all pregnant women with HIV , and later their infants, enrolling in routine PMTCT care at peer-mother and control facilities between 1st January 2018 to 31st December 2019, in total 1957 women in peer-mother facilities and 1384 women in control facilities (st July 2021 with a median [interquartile range (IQR)] follow up of 23  compared to control facilities [17.6 ], unadjusted HR (95%CI) = 0.82 . The attrition rate per 1000 person months in peer-mother versus control facilities had a more pronounced difference among ART naïve women  than ART experienced women .In the Kaplan Meier survival analysis, retention on ART in the first year of PMTCT care was significantly higher among women in peer-mother (78.0%) than control (73.6%) facilities, . The effA total of 1564 (79.9%) women in the peer-mother facilities and 1073 (77.5%) women in the control facilities had at least one viral load test done during the study follow-up period. Overall, viral suppression among all women with data was high but similar in both peer-mother and control facilities [92.0% versus 91.1%, p = 0.18). Viral suppression increased significantly across months from PMTCT enrolment in both groups ranging from 90.7% at 0–6 months to 93.8% at ≥25 months in peer-mother facilities and 88.8% at 0–6 months to 92.9% at ≥25 months in control facilities . HoweverOf note, from the 13–18 months timepoint to 19–24 months post PMTCT enrolment viral suppression dropped in both peer-mother facilities (from 94.2% to 91.4%) and control facilities (from 93.1% to 91.6%), A total of 20 out of 1022 infants in the peer-mother facilities and 9 out of 725 infants in the control facilities who had a confirmed HIV status by at least 12 months of age were diagnosed with HIV infection. This yielded low and similar un-adjusted MTCT risks in both groups: 2.2% (95%CI: 1.4 to 3.4) in the peer-mother and 1.5% (95%CI: 0.7 to 2.8) in the control facilities; p = 0.31. A majority of HIV positive infants in both the peer-mother (16 out of 20) and control facilities (6 out of 9) were diagnosed already by 2 months of age. In the adjusted multivariable random effects logistic regression, the risk of MTCT in the peer mother facilities compared to control facilities remained similar, adjusted OR (95%CI) = 1.87 .This cluster-randomized study examined the effect of integrating peer adherence and psychosocial support services to improve PMTCT outcomes in pregnant women on lifelong ART care in routine healthcare settings in Tanzania. We found that integration of peer-mother support services resulted in significantly lower ART attrition rate (14 versus 18 per 1000 person months) over three and a half years of follow-up and a modest but significantly higher one-year retention in PMTCT and ART care (78% versus 74%) in unadjusted analyses. Although the retention in PMTCT and ART care was still far from the ideal ≥ 90% even in the intervention facilities and not significant in adjusted analysis, this study provides useful insights regarding the potential of intensifying the role of peer-mothers in mitigating attrition from lifelong ART among women living with HIV during and after PMTCT care. Peer-mothers can be particularly complementary in overburdened, understaffed and resource constrained care settings that are struggling to retain women on ART care, especially post-partum, a common challenge as we move toward universal lifelong ART for all people living with HIV. Of note, the tendency towards higher benefits of the peer-mother intervention among ART naïve women and among those in early stages of their HIV disease, indicates that peer-mother support may be particularly important to mitigate retention barriers among newly diagnosed asymptomatic women who have a higher risk of poor retention [Our findings add to a growing body of evidence that reaffirms the contribution of peer-mother services in improving PMTCT outcomes. Only three previous publications (one cluster randomized and two cohort studies) were identified that quantitatively evaluated the effect of peer-mother services on ART retention, viral suppression and MTCT in the era of lifelong ART initiation during PMTCT , 23, 32.Our study found significantly higher viral suppression among the newly HIV-diagnosed ART naïve women in peer-mother (91%) versus control (88%) facilities. This implies that peer-mothers can support known challenges of newly HIV-diagnosed women in PMTCT care such as adjusting to one’s new HIV status, HIV anxiety and stigma, disclosure of HIV status, obtaining social support as well as adapting to consistent use of lifelong medication , 33, 34.Overall, among all women studied, we observed high and similar proportions of viral suppression  and significant positive trends of improved viral suppression over time in care in both arms. This may be attributed to successful roll-out of lifelong ART for all women in PMTCT care, transition to newer integrase inhibitor-based ART regimens from 2019, improved overall quality of care and ART adherence among women who remain in care, as reported earlier from a similar setting .Our study was directly affected by COVID19 that caused a temporary suspension of peer-mother services for at least six months due to safety concerns in response the first wave of the pandemic in Tanzania from March 2020. The COVID19 pandemic, and countries’ response to it, has had multiple health and social impacts including loss of lives, disruption of livelihoods and deterred access and/or uptake of health services in Tanzania and the world at large , 37. Of The three previous studies of peer-mother interventions in Africa mentioned above, reported mixed findings on virologic outcomes in PMTCT care. In the Malawian cohort study, the mentor mother intervention was associated with a higher (90%) viral suppression  than the expert clients (78%) intervention , indicatOn the MTCT outcome, the low and similar proportions of MTCT by 12 months among women in both peer-mother (2.2%) and control (1.5%) facilities observed in our study as well as in Malawi , is gratOur reliance on data from routine healthcare settings has several implications. In the absence of a robust evaluation of how peer-mothers adhered to the key components of the intervention, suboptimal implementation in some facilities is possible and would, if being the case, underestimate the true impact of the intervention. Furthermore, the attrition of peer-mothers over time as well as the suspension of their services due to COVID19, could also contribute to blunt the effect size of the intervention. Other concurrent routine care efforts to strengthen PMTCT and ART services—including transition to DTG regimens, improved couple HIV testing at ANC, and improved follow-up and tracking of ART patients—may have improved PMTCT outcomes overall, further diminishing the difference between control and peer-mother facilities. Overall, we believe that the routine care setting is a major strength of our study representing realities, easily transferable to other real-life PMTCT settings in sub-Saharan Africa. The fact that we relied entirely on routine care data for all our analyses minimises the risk of bias due to study procedures. Furthermore, missing data and incomplete follow-up, which inherently is a problem in real-life routine study designs, was non-differential across intervention and control facilities in our study  and therOur study contributes to a growing body of evidence on the effectiveness of peer-mother support services in improving ART retention among women in PMTCT care, as well as viral suppression among ART naïve women. Peer-mothers could play an important role to further optimize ART for life among women who are newly diagnosed with HIV, in early stages of HIV and among adolescents. These findings underscore the need for increased investment, strengthening and scale-up of peer-mother adherence support services in PMTCT care across high HIV-burden countries in Africa.S1 Table(DOCX)Click here for additional data file.