Amyloid light chain (AL) amyloidosis is among the more common and more severe of the amyloidoses usually involving the slow proliferation of a bone-marrow-residing plasma cell (PC) clone and the secretion of unstable immunoglobulin-free light chains (FLC) that infiltrate peripheral tissues and result in detrimental end-organ damage. Disease presentation is rather vague, and the hallmark of treatment is early diagnosis before irreversible end-organ damage. Once diagnosed, treatment decision is transplant-driven whereby ~20% of patients are eligible for autologous stem cell transplantation (ASCT) with or without bortezomib-based induction. In the setting of ASCT-ineligibility, bortezomib plays a central role in upfront treatment with the recent addition of daratumumab to the current emerging standard of care. In general, management of AL amyloidosis is aimed at achieving deep, durable responses with very close monitoring for early detection of relapse/refractory disease. This article provides a comprehensive review of the management of patients with AL amyloidosis including goals of therapy, current treatment guidelines in the setting of both ASCT-eligibility and ineligibility, treatment response monitoring recommendations, toxicity management, and treatment of relapse/refractory disease. Even though rarely due to a nonplasma cell B-cell close, AL amyloidosis is usually a plasma cell (PC) disorder whereby a small, slowly proliferating, bone-marrow-residing PC clone, secretes unstable immunoglobulin light chains. Those amyloidogenic free light chains (FLCs) can then infiltrate peripheral organs resulting in organ dysfunction and ultimately, failure. Organs involved usually include kidneys, heart, gastrointestinal (GI) tract, liver, and nervous system whereby undoubtedly cardiac involvement is the main driver of disease prognosis and mortality2. Given that more than 69% of patients already have more than one organ involved at the time of diagnosis3, it becomes vital to not only diagnose AL amyloidosis early, but to also effectively control plasma cell dyscrasias and thus halt escalating organ damage.Systemic amyloidoses constitute disorders of diverse etiologies involving the synthesis and abnormal extracellular deposition of misfolded proteins in various organs with resultant damage. Among the different amyloidoses, amyloid light chain (AL), previously called “primary”, amyloidosis is among the more common and more severe, affecting ~10 per million per yearOwing to toxicity, autologous stem cell transplantation (ASCT) remains the standard of care and first-line treatment in a small proportion of patients. Importantly, the outcome of ASCT-ineligible patients has improved by the introduction of new first-line agents, specifically bortezomib. Currently, active research involving second-line novel treatments and anti-CD38 antibodies offers new hope. Nonetheless, the main hallmark of management remains early recognition and initiation of treatment before the occurrence of irreversible organ damage.5. Age  or an autosomal dominant amino acid substitution (mutant) result in fibrillogenesis whereby TTR dissociates into intermediates that misassemble into amyloid fibrils and deposit in end-organs, most notably the heart6. While AL amyloidosis is treated with chemotherapy and transplant, TTR cardiac amyloidosis is treated with targeted therapy such as tafamidis7.It becomes vital to differentiate AL amyloidosis from transthyretin (TTR) amyloidosis, another disease of increasing prevalence, due to the difference in management. TTR is a liver-synthesized thyroxine and vitamin A transporter9 , nephrotic range proteinuria, organomegaly due to amyloid deposition , peripheral neuropathy, and constitutional symptoms  Fig. . By the 9 Fig. . Once su. 9 Fig. .Fig. 1Mo14  as an additional marker for disease burden13. One point was attributed to each of TnT ≥ 0.025 ng/mL, NT-proBNP ≥ 1800 pg/mL, and dFLC ≥ 18 mg/dL, thus classifying disease into four stages, I–IV according to a total score of 0–3, respectively13. Recently, the BU group derived a new staging system that correlates with the Mayo 2004 system, but utilizes BNP instead14. A BNP threshold >81 pg/mL best identified cardiac involvement and thus correlated with the Mayo 2004 staging system (κ = 0.854)14. Three stages were thus developed based on a BNP > 81 pg/mL and troponin I (TnI) > 0.1 ng/mL, whereby disease was classified into stages I, II, and III when both markers were lower than prespecified thresholds, only one was elevated or both were elevated, respectively14. Furthermore, stage III was further divided to include stage IIIb in the event of a BNP > 700 pg/mL14. This allows for centers without access to NT-proBNP or TnT to accurately stage AL amyloidosis. It is evident that the aforementioned models are very successful in dividing patients into distinct survival groups when examining the 12-year survival curves for the same patient dataset10.There are four staging models that utilize cardiac soluble biomarkers—Mayo 2004 model, European 2015 modification of Mayo 2004 model, Mayo 2012 model, and Boston University (BU) modelWhen choosing the appropriate treatment approach, potential organ impairment needs to be considered. While the younger and fitter patient can tolerate more intense therapy, the frailer one has poor treatment tolerance and is, as such, at an increased risk of early mortality. The determination of frailty in amyloidosis is not accompanied by specific frailty scores, but short-term survival can be used as a surrogate to estimate frailty. Age and the number, type and extent of organ involvement and to a lesser extent, pre-existing comorbidities, are the main determinants of frailty in this patient population.15.The Mayo Clinic investigators looked at the effect of age, among other factors, on survival in a cohort of 592 patients with mass-spectrometry-verified AL amyloidosis between 2008 and 2015. The cohort was followed up for 8 years and the median overall survival (OS) was 44 months. Dividing the patients into two age groups, <65 years and ≥65 years, it was evident that the younger patients had a much better survival than those aged ≥65 yearsp < 0.001)15. However, it is not just the number of organs involved in the disease, but also the type of organs involved that determine survival. For instance, comparing survival in patients with isolated cardiac involvement to those with cardiac plus multi-organ involvement, yields very similar survival curves between the two groups (p = 0.51)15. On the other hand, comparing survival in patients with isolated renal involvement to those with renal plus multi-organ involvement, yields significantly different survival curves (p < 0.001) whereby isolated renal involvement infers a survival advantage compared to the latter. As such, cardiac involvement is therefore the most important prognostic factor in AL amyloidosis whereby when present, irrespective of other organ involvement, influences survival. As previously discussed, several powerful staging models build on cardiac involvement to classify disease and aid treatment decisions.The effect of the number of organs involved on survival was investigated in the same cohort. There was a significant decrease in survival as the number of organs involved increased  accounting for 44%, followed by hyperdiploidy (43%)16. A statistically significant relationship was noted between several abnormalities and increased plasma cell (PC) burden, including gain (+) 5p/5q (p = 0.025), del13q (0.009), +11q (p < 0.001), and hyperdiploidy (p < 0.001)16. In multivariable analysis, hyperdiploidy was confirmed a significant poor prognostic factor16. In the setting of cardiac involvement, hyperdiploidy was also associated with worsening progression-free survival (PFS) (p = 0.0497) and OS (p = 0.006)16. Similarly, del13q was associated with cardiac involvement but did not appear to impact survival16. In addition, the overall presence of t did not carry any prognostic value in terms of PFS and OS, although on further stratification, patients harboring isolated t had relatively worse PFS relative to those without any cytogenetic abnormalities16. Conversely, the absence of any cytogenetic abnormalities was associated with improved PFS and OS 16. This becomes important as well when choosing therapy. For instance, patients with +1q were found to have a better response to treatment with daratumumab, a monoclonal receiving increased attention in the field16. As will be further discussed, patients harboring t are more likely to have disease resistant to bortezomib-based regimens and are more likely to respond to venetoclax18.Another important determinant of outcome and treatment response is cytogenetic profile. A recent retrospective chart review of 140 AL amyloidosis patients explored the most frequent cytogenetic abnormalities and their impact on survival19. Approximately 20% of patients are likely eligible for this procedure. For those ineligible, a cautious approach is necessary, involving standard intensity and, for the frailer patients, low-intensity therapies  remains the standard of care in low-risk patients22. Typically, potentially reversible contraindications are assessed before deeming a patient ineligible for ASCT. Otherwise, the presence of one exclusion criteria, specifically pertaining to cardiopulmonary or renal status could suffice in making a patient ASCT-ineligible. The most common eligibility criteria for ASCT are9:Age < 70 yearsEastern Cooperative Oncology Group (ECOG) performance status < 2NYHA Class < III (New York Heart Association classification of the extent of heart failure)Left ventricular ejection fraction (LVEF) > 45%Systolic blood pressure (SBP) ≥ 100 mmHgTnT < 0.06 ng/mL or high-sensitivity (hs)-TnT < 75 ng/mLNT-proBNP < 5000 ng/LCreatinine clearance (CrCl) > 50 ml/min Bilirubin < 2 mg/dLDiffusion capacity of the lungs for carbon monoxide (DLCO) > 50%No more than two organs significantly involvedThe eligibility criteria for ASCT have evolved over the years and with the emergence of novel agents24. A study carried out between July 1994 and December 2008 compared HDM (200 mg/m2) to a reduced dose (100–140 mg/m2) in 421 AL amyloidosis patients. The results demonstrated a significantly higher CR rate in the high-dose group 24. Similarly, between January 2000 and August 2015, a study of 457 patients using the same melphalan conditioning-dose levels, showed a significantly higher CR rate in the high-dose group 23. In both studies, high-dose melphalan was associated with lower transplant-related mortality (TRM) 24. The median PFS and OS were also significantly longer in the high-dose groups in both studies.The choice of therapy for ASCT-eligible patients has evolved over time and has been a matter of perspective for many years. The advantages of stem cell transplantation are high response rate and response durability whereas the disadvantages are the short-term morbidity and mortality associated with it, which can be quite significant, and the fact that this therapy can only be offered to a minority of select patients. Nonetheless, complete response (CR) rates achieved with ASCT remain higher than those possible with any other treatment regimen but data from two major groups show that CR rates after ASCT are conditioning-dose dependent25. When categorized according to initial treatment modality, ASCT treatment versus standard-intensity therapies, 58% of ASCT patients were treatment-free at 10 years compared with only 36% in the non-ASCT group (Δ 22%)25. As such, long‐term survivors are increasingly seen in AL amyloidosis and ASCT is associated with a more durable response compared with standard-intensity therapies.Despite the deeper responses achieved with ASCT compared to other treatment modalities, it is important to assess the durability of such a response. A recent study looking into the need for subsequent or second-line therapy after ASCT in 186 patients highlighted that in those who had survived for at least 10 years post diagnosis, 47% remained treatment-free26. It compared high-dose standard-intensity therapy  with HDM and ASCT rescue. The non-ASCT arm had an improved survival (p = 0.04)26. Although HDM followed by ASCT had been in use for more than a decade by then, some of the participating centers in the study had just started adopting HDM-ASCT as a treatment strategy, thus contributing to the study’s limitation. In addition, patient selection criteria were not well established. For example, of the 50 patients randomized to the HDM-ASCT arm, only 37 (74%) went on to receive ASCT. This translated into a high day + 100 TRM of 24% in this group26. Most of the remaining patients went on to have an early death26. Along the same lines, a systematic review of 12 studies also found no advantage of ASCT over conventional chemotherapy in improving OS in AL amyloidosis27. However, the evidence was weak, and the authors cautioned that further research was needed27.That said, a randomized controlled trial in 100 patients performed between 2000 and 2005 by the Intergroupe Francophone du Myélome (IFM) reported evidence that argued against ASCT for AL amyloidosis28. At 30 days, the TRM rates were 10%, 7%, and 4% for these time periods, respectively, and at 100 days 19%, 11%, and 4%, respectively, (p < 0.01)28. Also, centers performing more than four transplantations per year for AL amyloidosis had superior survival outcomes28. This data highlight the importance of center experience in achieving better outcomes in this patient population but also explain one of the potential limitations of the IFM study—relatively earlier time of investigation.In North America between 1995 and 2012, a randomized, multicenter study of 1536 AL amyloidosis patients at 134 centers, demonstrated that the rate of early mortality after ASCT at 30 days and 100 days progressively declined over successive time periods 29. All patients (n = 415) who received ASCT within 12 months of diagnosis were included and were divided according to baseline bone marrow plasmacytosis (BMPC) level into two groups 29. Patients with a low tumor burden (BMPC ≤ 10%) did not show any difference in response rate irrespective of induction chemotherapy29. For patients with BMPC > 10%, the results were very different though, whereby the overall response rate (ORR) was significantly better with a near doubling of the complete response (CR) rate in patients who had received induction therapy pre-ASCT 29. Afrough et al. highlighted the improved survival with pre-ASCT induction as well in 128 patients comparing conventional induction with melphalan or steroids to novel agents such as the thalidomide-analogues, the immunomodulatory imide drugs (IMiDs), and proteosome inhibitors (PI) whereby hematological, but not organ response, was significantly higher with IMiD/PI-based regimens30. Induction with IMiD/PI-based regimens was also found to be a significant positive predictor of OS30.Another controversial aspect of ASCT in AL amyloidosis is whether or not induction chemotherapy is required. Hwa et al. investigated the impact of induction chemotherapy on the response following transplantn = 27) achieved hematological response (HR) with 63% CR and 37% very good partial response (VGPR) 6 months post-ASCT31. The cohort of patients was followed up for an extended median of 77 months whereby renal and cardiac responses occurred in 65% and 88%, respectively, at 5-years post-ASCT31. Median OS and PFS were not yet reached highlighting the durability of response with the incorporation of bortezomib32. The phase II HOVON 104 trial (NTR3220) investigated the use of four cycles of Vd induction treatment, followed by HDM-ASCT33. The overall HR after induction was 80% including 20% CR and 38% VGPR with improvement 6 months post-ASCT to overall HR of 86% with 46% CR and 26% VGPR. The trial confirmed the efficacy of Vd and demonstrated the amplified outcome post-ASCT. However, due to treatment-related toxicity and disease characteristics, primary endpoint could not be reached33.Multiple clinical trials have also looked into the role of bortezomib or bortezomib-based regimens in both induction and conditioning. A prospective, single-arm trial (NCT01083316) used bortezomib-dexamethasone (Vd) for induction followed by bortezomib-HDM for conditioning whereby 100%  genetics (uncommon in amyloidosis patients), such as those previously discussed, or patients with very high BMPC at diagnosis, which resembles multiple myeloma (MM) with amyloidosis.9.Given the rapid progression of amyloidosis and the fact that diagnosis and subsequent treatment are a race against the clock, it is imperative that patients do not suffer further deterioration in organ function. As such, induction chemotherapy with bortezomib-based regimen should be considered for stem cell eligible patients, particularly with BMPC > 10% or in the event of a foreseeable delay of >1 month in ASCT35 During stem cell mobilization, patients can experience various toxicities including tachyarrhythmias, thromboembolic events, weight gain (due to fluid retention), bleeding, acute kidney injury, hypertensive crises, or hypotension. To minimize risk of toxicity, it is recommended to use granulocyte-colony-stimulating factor (G-CSF) without cyclophosphamide given the increased cardiac morbidity, significantly higher number of apheresis required, increased hospitalizations and increased toxicity associated with the latter36. The recommended dose of G-CSF is 10–16 μg/kg/day, either as one dose or divided into two doses, 3 days before stem cell collection for an optimal total of at least 5 × 106 CD34+ cells/kg37. The general recommendation though, is to split the dose. If patients fail stem cell mobilization, plerixafor is a well-tolerated adjuvant36.The first stage in the management of toxicity in relation to ASCT is during stem cell mobilization and collection whereby low serum albumin, elevated NT-proBNP and increased septal thickening were found to be important risk factors for toxicity38. On adjustment for cardiac (Mayo) stage, NSVT was actually not found to impact peritransplant mortality and pretransplant NSVT is therefore not an exclusion criterion38. As such, ~50% of patients will experience arrhythmias following ASCT, most of which will be low grade but with grade 3/5 arrhythmias accounting for ~10%39. It remains unclear whether primary AICD (Automatic Implantable Cardioverter Defibrillator) implantation will be of benefit, even among those who are at high-risk of sudden cardiac death and so, decisions should be case specific.The major ASCT toxicities remain cardiac arrhythmias, worsening HF, syncope, and end-stage renal disease (ESRD). Twenty-five percent of patients who have had pretransplant 24-h Holter monitoring will have evidence of nonsustained ventricular tachycardia (NSVT), a condition associated with an inferior short-term (6 months) survival40. Multiple factors contribute to the heart failure risk during peritransplant in this population including electrolyte imbalance, medications, fluid overload, arrhythmia, temporary cessation of cardiac medications, sepsis, and exacerbation of organ dysfunction during transplant. It is worth noting that standard heart failure treatments such as beta blockers, calcium channel blockers and/or ACE inhibitors, cannot be used for worsening heart failure management in this patient population and so should be avoided41.Worsening heart failure is another toxicity that may be encountered during transplantation and about 5% of patients will have a reduction in EF of >10%, which increases the 100-day mortality42. Dialysis was required by 72 (18%) patients. Eight patients started dialysis >30 days prior to ASCT (Group II), 36 started ±30 days after ASCT (Group III) and 28 initiated dialysis >1 month after ASCT (Group IV) whereby patients who were never dialyzed were assigned to Group I42. Median OS was not reached in Groups I and II but was 7 months in Group III and 48.5 months in Group IV (p < 0.001)42. TRM was observed in 44.4% of the patients in Group III, 6-fold higher than the next highest group with a TRM of only 3.6% (p < 0.001)42. The most common causes of TRM were cardiac and sepsis. In the multivariate analysis, only hypoalbuminemia  and estimated glomerular filtration rate (eGFR) <40 mL/min/1.73 m2 (p < 0.001) were independently associated with starting dialysis within 30 days of ASCT42. The risk of dialysis increased exponentially with increasing the number of risk factors present. If none or 1 factor was present, risk of dialysis was 2% and 10%, respectively42. Nonetheless, the presence of both, hypoalbuminemia and eGFR <40 mL/min resulted in a 44% risk of dialysis. As such, screening with serum albumin and eGFR may reduce the risk42. Unfortunately, these two risk factors are also risk factors for progression to ESRD as part of the natural progression of AL amyloidosis. Thus, the decision on whether to proceed to ASCT is a difficult one. It is important to note though, that patients are likely to experience improved renal function as the disease burden decreases post induction, then making them eligible for subsequent treatment with ASCT.Another important toxicity that could complicate ASCT is ESRD. In a study examining the association between acute renal failure and mortality in AL amyloidosis during ASCT, the medical records of 408 ASCT patients between 1996 and 2010 were examinedIn summary, HDM-ASCT is a durable and reliable treatment option that allows for deep and sustainable responses in a minority of select, eligible patients which could be preceded by bortezomib-based induction therapy when BMPC > 10% or a foreseeable delay in ASCT is expected. Despite its consistent results, treatment with ASCT is associated with a handful of detrimental adverse events including arrhythmias, worsening HF and progression to ESRD, with the introduction of novel agents, such as bortezomib, resulting in improved results and offering an alternate treatment modality.p < 0.001)43.The survival pattern for transplant ineligible patients has been improving over time. Trends in presentation, management and outcome among 1551 newly diagnosed AL amyloidosis patients between 2000 and 2014 were evaluated and when stratified into three eras , survival improved significantly over time with OS rates of 25%, 46%, and 47%, respectively, , a change largely driven by improved VGPR rates in the non-ASCT population43. In one study by Palladini et al., 0.22 mg/kg of melphalan plus 40 mg/day of dexamethasone were given on days 1–4 in a 28-day cycle44. The study proved that this combination resulted in high response rates, with a 67% HR including 33% CR. Responses were durable, and the median time to response was 4.5 months44. This makes MDex an effective regimen, which can be used when bortezomib is contraindicated or is unavailable. However, when dealing with elderly patients or those with pre-existing severe renal or cardiac involvement, a lower dose of dexamethasone should be considered  due to the toxicities and increased fluid retention associated with dexamethasone.The main first-line options for treatment of transplant ineligible patients are (1) bortezomib-based regimens: Vd, cyclophosphamide–bortezomib–dexamethasone (CyBorD), and bortezomib-melphalan-dexamethasone (BMDex), or 2) melphalan-dexamethasone (MDex)  analysis ranged from 21 to 25%46. Cardiac response varied from 17 to 38% and renal response, 15 to 44%46. The median OS across these studies was >4 years and the median follow-up period ranged from 25 to 32 months46. The phase III trial (NCT01277016) comparing MDex to BMDex in newly diagnosed AL patients reported very exciting data in 109 patients recruited in Europe and Australia 46. Although there was no difference in the CR and partial response (PR) rates, or in cardiac and renal response rates, at the end of treatment after a median of five cycles, the overall HR rate was significantly better in the BMDex arm compared with the MDex arm  whereas VGPR or CR was achieved in 64% of the patients on the BMDex arm versus 39% of those on the MDex arm46. This translated into improvements with BMDex over MDex in PFS and OS with a 2-fold decrease in mortality 46.Multiple studies have looked into the use of bortezomib-based regimens upfront2) and uptitrating as tolerable can be considered when dealing with patients with cardiac involvement. Patients should be monitored carefully for side effects and symptoms including regular monitoring for cardiac biomarkers, especially in the setting of high-risk disease. In the emergence of neuropathy or other major toxicity, the dose of bortezomib can be reduced or discontinued.With respect to toxicity management, it is important to avoid bortezomib in the setting of pre-existing neuropathy or to opt for an attenuated dose regimen. Subcutaneous administration is recommended at weekly dosing; twice weekly dosing is not recommended and is likely to result in termination of therapy. Because bortezomib can be cardiotoxic to this patient population, initiating at a lower dose  and VGPR/CR (79% versus 49%) with better cardiac (42% versus 22%) and renal (54% versus 27%) responses and prolonged PFS. Thus, the study concluded that the addition of daratumumab to CyBorD results in both, deeper and more rapid, HR47. The preliminary ANDROMEDA results are thus promising and favorably comparable to ASCT.The recent ANDROMEDA phase III trial (NCT03201965) compared CyBorD to daratumumab-CyBorD in patients with newly diagnosed AL amyloidosis. Patients received weekly daratumumab in cycles 1 and 2, every 2 weeks in cycles 3–6, and every 4 weeks thereafter up to 2 years. The safety run-in of the study showed an overall HR rate of 96% without any new safety concerns beyond that already demonstrated for daratumumab in multiple myeloma (MM) and CyBorD48. CAEL-101 is an AL amyloid fibril reactive IgG1 antibody aimed at potentially clearing amyloid deposits48. The study enrolled 13 patients  in a 3 + 3 dose escalation safety study whereby the maximum dose tolerated was 1000 mg/m2 weekly for 4 weeks and then every other week. Patients were receiving CyBorD in tandem48. Of the six patients receiving the maximum dose, it was well tolerated with the exception of three significant adverse events 48. Seven patients were evaluated for organ response and 2 met criteria in the 500 mg/m2 cohort48. Given the potential for early organ response, CAEL-101 could prove groundbreaking, awaiting two recently initiated phase III trials employing the maximum dose of CAEL-101 at 1000 mg/m2.A phase II, open-label, dose selection study (NCT04304144) evaluated the safety and tolerability of CAEL-101 in AL amyloidosis to provide a recommended dose of CAEL-101 to be given in combination with CyBorD for a planned randomized study in Mayo stage IIIa and IIIb patients49 , negative urine protein electrophoresis and immunofixation (UPEP/IFE) and normal FLC ratio (FLCr); VGPR with a dFLC <40 mg/L; PR with dFLC decrease >50%; and no response (NR) with a dFLC less than that achieved in PR49. With the availability of highly effective antiplasma cell therapy, the criteria requirement of normal FLCr was further clarified to include abnormal FLCr inverted in favor of the nonamyloidogenic FLC50. As such, an abnormal FLCr does not preclude achieving CR when the uninvolved (uFLC) is greater than the involved FLC (iFLC)50.Palladini et al. identified the criteria for both, hematological and cardiac responses to treatment offering surrogate end points for clinical trials49 Fig. . The ana51. In patients with renal failure where the free light chains are cleared by the kidneys, it is sometimes hard to assess the response and a bone marrow biopsy may help to see if a deep remission has been achieved.In assessing treatment effectiveness, the target response for most patients should be at least a VGPR. In those with a low presenting dFLC of 20–50 mg/L, a target response should be a dFLC <10 mg/L. If a patient has a serum M-spike (true for ~50% of patients), this can help in assessing response, especially in those with a low level of circulating light chains where assessing response becomes very challenging. A retrospective analysis of 716 patients, 73% of which had a measurable M-spike before ASCT, found a measurable M-spike prior to ASCT an independent negative predictor of OS and PFSThat said, it is empirical to determine when a shift in gears and initiation of new therapy is warranted. There are three occasions when this should be considered: (1) if there is no change in serum iFLC after the first cycle, (2) if a patient does not achieve a PR after 2–3 cycles, and (3) if treatment is not tolerated.45. The study looked at three levels of dFLC 45. The median OS was not reached in patients with the first two levels and was 53 months in those with dFLC of >40 mg/L (log-rank p < 0.0001)45. The median TNT was not reached in the lowest level of dFLC, and was 38 and 13 months in the 10–40 mg/L and >40 mg/L groups, respectively (log-rank p < 0.0001)45. Cardiac responses were better in those with stringent dFLC responses (61%) compared with lesser responses 45. The authors concluded that a stringent dFLC response predicted prolonged TNT and impressive organ responses45. Similarly, in another study of patients who achieved at least a VGPR, an iFLC < 20 mg/L was associated with significantly better OS and PFS (p < 0.001)52. Using a more stringent dFLC of 5 mg/L, there appeared to be a further significant improvement in PFS (p < 0.001) but not in OS (p = 0.11)52. In summary, as well as using the HR criteria, a further understanding of the depth of response in a patient is possible if low levels of iFLC and dFLC are achieved.Increasing data are emerging about the depth of response irrespective of the HR criteria. The UK prospective study on upfront bortezomib therapy reported outcomes in the largest AL amyloidosis cohort treated with upfront bortezomib and explored the impact of post-treatment dFLC < 10 mg/L (“stringent dFLC response”) and portrayed that patients with stringent dFLC responses had significantly better OS and time-to-next treatment (TNT) compared to those with weaker responses-553. The MRD negative rate among patients in CR was 75% relative to 50% in patients achieving VGPR. Patients with MRD-negativity were more likely to have achieve cardiac response  and improved 1-year PFS after a median follow-up of 14 months 53. Similarly, Palladini et al. evaluated MRD by NGF at a minimum sensitivity of 10−5 in 92 AL amyloidosis patients who have achieved CR54. Fifty-four percent had detectable MRD. Undetectable MRD was associated with higher rates of renal and cardiac responses 54. Even more importantly, hematological progression was more frequent in the setting of persistent MRD , highlighting an important population who would benefit from further treatment54.It is important to emphasize that complete HR does not necessarily translate into organ response. As such, increasing attention is now being channeled to minimal residual disease (MRD). Sidana et al. evaluated MRD in 44 AL amyloidosis patients using MRD by next-generation flow-cytometry (NGF) at a minimum sensitivity of 1058. In a recent French study including 15 patients (median age 60 years) treated with daratumumab, ORR was 86% including 43% CR and 14% VGPR59. The most common side effect was infections due to induced hypoglobulinemia59. Thus, daratumumab has proven an excellent agent for AL amyloidosis resulting in deep remissions, high response rates and low toxicity. Nonetheless, the long-term results of daratumumab treatment and length of remission are not yet known.Upon recognizing patients who were not able to attain a satisfactory response, it becomes vital to initiate second-line therapy as soon as possible. Currently, the recommendation for second-line therapy for relapsed/refractory AL amyloidosis (RRAL) is daratumumab or daratumumab-based therapy Fig. . This isBeyond daratumumab, the recommendations for third-line treatment of RRAL depend on whether the patient had a hematological relapse ≥2 years since the last therapy, in which case, the physician should consider repeating the original therapy. If the patient is not bortezomib refractory, then a bortezomib-based regimen is the preferred choice . On the other hand, if the patient is bortezomib refractory, then pomalidomide-dexamethasone (pom-dex) or lenalidomide-dexamethasone (len-dex) can be used. Importantly, ASCT can be considered in the relapsed setting, either as first ASCT or as a second transplant.60, and (2) a rise in NT-proBNP/BNP. To avoid or manage this type of toxicity, we recommend a low starting dose of lenalidomide of 5–15 mg/day with the lower dose reserved for elderly patients or patients with baseline cardiac involvement or elevated creatinine. The dose can then be adjusted according to tolerability. One should observe carefully for fluid retention by weighing patients daily and measuring cardiac biomarkers as well as monitoring kidney function.IMiDs are generally not well tolerated by AL amyloidosis patients. Whereby thalidomide is known for its high toxicity, lenalidomide comes with concerns too. The two main toxicity concerns with lenalidomide are (1) lenalidomide-related renal deterioration which, in a study of 41 patients, occurred in 66% and was only reversible in about half of them61. Median time to response was 1.9 months and the median duration of response was 19 months 61. Similarly, a phase 1/2 trial of pom-dex reported an ORR of 50% in 24 evaluable patients62. The median time to best ORR was three cycles, and median duration of ORR was 15 months62. With a median follow-up of 17.1 months, median OS had not yet been reached, and median event-free survival (EFS) was 17.8 months62. A phase 2 trial of pom-dex rescue treatment in 28 patients previously exposed to alkylators, PIs, and lenalidomide demonstrated a hematological ORR in 68% and a VGPR/CR in 29%, as well as improved survival with median time to response of 1 month63. Hematologic ORRs were also rapid, with half of responding patients doing so within 2 months63. Pomalidomide dose reduction was required in two out of three of those studies due to toxicity whereby patients experienced grade 3–4 myelosuppression (26–45%), fatigue (18%), pneumonia (11–21%), renal failure (3–7.5%), and arrhythmias (0–21%)63. It is important to note that AL amyloidosis patients treated with pomalidomide frequently experience increases in proBNP/BNP without clinical congestive heart failure.Pomalidomide is slightly better tolerated than lenalidomide and several studies have demonstrated rapid responses (median 1–3 months) and improved survival with pom-dex. A study of 33 patients with a median follow-up of 28 months highlighted the activity of the pom-dex combination even in the setting of having failed lenalidomide and bortezomib whereby the confirmed overall HR rate and PFS were 48% and 14%, respectivelyn = 85) to physician choice 64. TOURMALINE-AL1 is the first phase III trial in patients with RRAL to show a significant clinical outcome improvement. Despite the trial not meeting the first primary endpoint of hematologic ORR, treatment with ixa-dex resulted in an improved CR rate (26% versus 19%) and duration of overall HR (46.5 months versus 20.2 months) when compared to physician’s choice of treatment64. Although there was no difference between OS and overall PFS, treatment with ixa-dex increased hematologic/vital organ PFS and decreased time to vital organ deterioration/death, time to treatment failure, and time to subsequent therapy64. Considering that the duration of treatment in the ixa-dex arm was twice that of the physician’s choice arm (median 11.7 months versus 4.9 months), continuous ixa-dex was generally well tolerated64. When stratified by prior PI exposure, hematologic ORR was 63% versus 50% for Ixa-dex versus physician choice in PI-naïve patients and 41% versus 51% in PI-exposed65. Thus, ORR was higher for Ixa-dex versus physician choice in PI-naïve patients and lower in PI-exposed 65. Based on these results, ixa-dex may be considered a new option for patients with RRAL, given sensitivity to bortezomib. Ixazomib was also studied in the all-oral combination, ixazomib, cyclophosphamide, and dexamethasone in a phase II trial66. The study included 35 patients with newly diagnosed, biopsy-proven AL amyloidosis but excluded patients with severe organ involvement 66. The overall HR was 57% including CR in 4%, VGPR in 26%, and PR in 17%66. Median PFS and OS have not been reached, four patients had disease progression and six have died66. Thus far, this points to modest activity in the all-oral regimen awaiting further results.PIs other than bortezomib also play a role. A few studies have looked at treatment of AL amyloidosis with the second-generation PIs, ixazomib and carfilzomib. The phase III randomized controlled trial TOURMALINE-AL1 (NCT01659658) has recently published its results. It compared the combination of ixazomib and dexamethasone 67. HR rates were promising in this bortezomib-exposed population, including PI-refractory patients. The ORR and ≥VGPR were 54% and 39%, respectively, and although these are encouraging results, cardiac toxicity was experienced by 36% 67.On the other hand, carfilzomib is a challenging agent due to the high incidence of cardiac involvement with AL amyloidosis. It is known that up to 10% of MM patients treated with carfilzomib experience cardiac toxicity. It is also an intravenous infusion and patients require hydration, which can aggravate patients predisposed to congestive heart failure. Results of an investigator-initiated, multicenter, phase I/II study of carfilzomib in AL amyloidosis (NCT01789242) showed that carfilzomib monotherapy was feasible and effective in 28 RRAL patients17, and patients with this translocation are less likely to respond to a bortezomib-based regimen18. There is limited experience with venetoclax in AL amyloidosis but in 2018, Leung et al. reported on perhaps the first patient with AL amyloidosis to be successfully treated with venetoclax after his disease markers plateaued on CyBorD18. Sidiqi et al. have recently demonstrated efficacy and safety of venetoclax in 12 RRAL patients treated with venetoclax between January 2017 and May 201968. Patients had a median of two prior lines and all but 1 had t68. The dose used was 400–800 mg/d; 7 in combination with dexamethasone68. Eight patients were evaluable for response and the response rate was 87% comprising 3 with CR and 4 with VGPR68. The median time to best response was 3.4 months and median follow-up was 11.5 months, but two patients progressed at 4 and 5 months from initiation of therapy68. Toxicity was minimal and there were no reports of tumor lysis syndrome. Notably, in this cohort no deaths have been observed so far. Albeit a retrospective case series with a small sample size, this study suggests high efficacy and good tolerability of venetoclax monotherapy and combination therapy in patients harboring t.Venetoclax is an oral, small-molecule B-cell lymphoma 2 (BCL-2) inhibitor that induces cellular apoptosis, with encouraging activity in chronic lymphoid leukemia (CLL), non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), and a subset of MM patients whose clonal plasma cells harbor t and/or overexpress BCL-2. Venetoclax may be useful in the management of AL amyloidosis for two reasons: t is present in ~50% of AL amyloidosis patients71. Milani et al. reported on 122 patients, 36 of whom had IgM amyloidosis69. With a median time to response of 3 months, the ORR to bendamustine in the whole cohort was 35%69. In the 12 patients with IgM-AL amyloidosis, this was achieved in 58% of subjects (21% with ≥VGPR)69. The median follow-up of living patients was 31 months  and severe adverse effects were observed in 26%69. Lentzsch et al. conducted a phase II, multicenter trial (NCT01222260) to assess the efficacy and safety of bendamustine with dexamethasone in 31 patients with persistent or progressive AL amyloidosis after ≥1 prior therapy70. Dose reduction occurred in 31% of patients and 57% of patients achieved a PR or better 70. The median OS was 18.2 months and the median PFS was 11.3 months70. Side effects were commonly myelosuppression, fatigue, and nausea/vomiting70. Manwani et al. reported outcomes in 27 patients identified from the UK National Amyloidosis Centre database (5 of which were RRAL) treated with bendamustine and rituximab showing an ORR of 59% 71. The median follow-up was 18 months and the median PFS was 34 months. Side effects comprised: GI symptoms, fatigue, cytopenias, and neutropenic fever71.Bendamustine is another agent to consider although the response rates are not very high in the AL amyloidosis population. However, for those with an IgM amyloidosis or a lymphoplasmacytic histology, in combination with rituximab, response rates are much better, and survival is improved even in heavily pretreated patients. This is highlighted by three recently published studies72. The median age was 70 and prior therapies included PI (89%), high-dose therapy followed by ASCT (47%), IMiDs (28%) and an anti-CD38 monoclonal antibody (6%)72. Nineteen patients discontinued treatment whereby the most common reasons were adverse events, disease progression, suboptimal response and COVID-19 concerns72. The median duration of therapy for the 17 patients currently on treatment is 11.8 months72. The overall HR was 77% with 3% CR, 54% VGPR, and 20% PR72. The majority of drug-related side effects were grade I/II infusion-related (50%), grade I anemia (25% and lymphopenia (22%)72. Isatuximab thus has promising efficacy with a side effect profile similar to other anti-CD38 monoclonal antibodies72.Finally, another anti-CD38 monoclonal antibody, isatuximab is an IgG1k monoclonal antibody with high-affinity binding to CD38 expressed on plasma cells, which has proven efficacious in MM as a single agent or in combination. The phase II trial SWOG S1702 (NCT03499808) investigates the role of isatuximab. It included 36 patients with RRAL who have received at least one previous line of therapyThe presentation of AL amyloidosis could be rather deceitful due to its mimicry of various conditions. The hallmark of disease management is early diagnosis, ideally during the routine monitoring phase of high-risk patients before the onset of symptoms and irreversible organ damage. The goal of therapy in this challenging disease is at least a hematological VGPR (dFLC > 50 mg/L). Treatment should be guided by ASCT-eligibility given the robust and durable response attainable with transplant. This could be preceded by induction chemotherapy with bortezomib-based regimens especially in the setting of high disease burden or foreseeable delay in ASCT. In the setting of ASCT-ineligibility, upfront treatment with bortezomib-based regimens, now with the incorporation of daratumumab is standard. In the setting of relapse, the best response appears to be with the use of daratumumab though multiple emerging data on other novel agents including ixazomib, isatuximab, and pomalidomide are quite promising and remarkable. As we await the results of the multiple ongoing trials including the recently initiated trials involving the anti-fibril, CAEL-101, the field is still in dire need of research involving higher risk patients.


Sepsis is a global health emergency, which is caused by various sources of infection that lead to changes in gene expression, protein-coding, and metabolism. Advancements in “omics” technologies have provided valuable tools to unravel the mechanisms involved in the pathogenesis of this disease. In this study, we performed shotgun mass spectrometry in peripheral blood mononuclear cells (PBMC) from septic patients (N=24) and healthy controls (N=9) and combined these results with two public microarray leukocytes datasets. Through combination of transcriptome and proteome profiling, we identified 170 co‐differentially expressed genes/proteins. Among these, 122 genes/proteins displayed the same expression trend. Ingenuity Pathway Analysis revealed pathways related to lymphocyte functions with decreased status, and defense processes that were predicted to be strongly increased. Protein-protein interaction network analyses revealed two densely connected regions, which mainly included down‐regulated genes/proteins that were related to the transcription of RNA, translation of proteins, and mitochondrial translation. Additionally, we identified one module comprising of up‐regulated genes/proteins, which were mainly related to low-density neutrophils (LDNs). LDNs were reported in sepsis and in COVID-19. Changes in gene expression level were validated using quantitative real-time PCR in PBMCs from patients with sepsis. To further support that the source of the upregulated module of genes/proteins found in our results were derived from LDNs, we identified an increase of this population by flow cytometry in PBMC samples obtained from the same cohort of septic patients included in the proteomic analysis. This study provides new insights into a reprioritization of biological functions in response to sepsis that involved a transcriptional and translational shutdown of genes/proteins, with exception of a set of genes/proteins related to LDNs and host‐defense system. In the past few decades, sepsis has gained immense attention as a global health emergency that affects millions of people worldwide. Clinically, it is defined as a life‐threatening organ dysfunction caused by the dysregulated host response to infection . DespiteThe complex pathophysiology of sepsis is largely dependent on the host’s immune response against microbial infections , 4. The In the last decades, the development and advancement of “omics” technologies revolutionized the field of sepsis research. These technologies ensured a better understanding of disease pathogenesis , 8 and eIn the present study, concepts of network biology were employed to evaluate the systemic host response in patients with sepsis. The study aimed to utilize PPIN analysis to gain better insights into the interconnections present between the genes/proteins with highly altered expression, and thus unravel the functional modules involved in the biological processes associated with clinical sepsis. For this purpose, we performed shotgun mass spectrometry in a cohort of septic patients combined with two publicly microarray dataset to identify co-differentially expressed genes/proteins and overrepresented pathways. Results were validated by qPCR and flow-cytometry.Two publicly available microarray datasets GSE65682 (SD1) and E-MTAB-5273 (SD2) were obtained from the Gene Expression Omnibus and Arrayexpress, respectively. These datasets were selected from other datasets with reference to the following criteria: i) dataset providing data information about gender and age; ii) the presence of corresponding controls in the same dataset ; iii) available processed data and iv) samples collected within 24 h after ICU admission. The exclusion criteria include: i) animal and pediatric studies and ii) datasets with small number of samples  < −0.26 or > 0.26 significance thresholds.We read the processed data into the R environment (v. 4.0.2). First, the transcripts were collapsed to unique genes by calculating the mean expression of transcripts from the same gene locus using tidyverse and dplyr packages. The annotation file used to map the probes to genes was previously published in Khan, Perlee . Comparihttp://www.interactivenn.net/) was used to overlap the lists of differentially expressed genes (DEGs) from the two different datasets. Then, a set of DEGs was selected based on those overlapping and following the same direction of expression (up-regulated or down-regulated). In addition, we selected only genes classified as “protein-coding” for further analysis.The InteractiVenn tool  and the volume was adjusted with 50 mM HEPES (pH 7.5) to a protein concentration of 1 µg.µL-1. The proteins were digested with trypsin  at a 1:100 enzyme:protein ratio at 37°C overnight, and the digestion was stopped by the addition of 10 µL of 5% trifluoroacetic acid. The samples were then desalted using C18 tips, vacuum-dried, and stored at −80°C. Before the analysis, the peptides were suspended at 0.1% formic acid to a final concentration of 2 µg.µL-1.PBMC were isolated from blood samples using the Ficoll gradient method  and stored in liquid nitrogen before use. After thawing, the protein extracts were obtained by lysis in 7 M urea, 2 M Thiourea, and 200 mM Dithiothreitol (DTT) with the Protease Inhibitor Cocktail . After centrifugation at 13,000 E (m/z range of 50–2000 and set up in the resolution mode. Peptide ions were fragmented by collision-induced dissociation (CID), in which collision energies were alternated between low (4 eV) and high (ramped from 17 to 60 eV) for precursor ion and fragment ions, respectively, using the scan time of 1.0 s. The ESI source was operated in the positive mode with a capillary voltage of 3.0 kV, block temperature of 100°C, and cone voltage of 40 V. The column temperature was set at 55°C, and the samples were maintained in an autosampler at 10°C. For lock mass correction, a [Glu1]-Fibrinopeptide B solution  was infused through the reference sprayer at 2 μL/min and sampled every 60 s for external calibration  (Waters) coupled to the Synapt G2 HDMS Mass Spectrometer (Waters). Samples (10 µg) were loaded into a trap column  at 8 μL/min of phase A  for 5 min. Then, the mixture of trapped peptides was eluted in an analytical column  with a gradient of 7%–35% of Phase B (0.1% formic acid in acetonitrile) over 60 min at a flow rate of 3 µL/min. The MS data were acquired on data-independent mode (DIA) using ion mobility separation UDMSE . in the ibration .Homo sapiens sequences from the UniprotKB/Swissprot . The database was combined with a list of 259 most common contaminants as well as examined with all their reverse sequences.Label-free quantification was performed in Progenesis QI for proteomics  as previously reported , 24. BriSearch parameters were set as follows: carbamidomethyl cysteine residues were selected as fixed modification and N-terminal Acetylation and methionine oxidation as variable modifications. Ion account was selected as the peptide identification method with the search tolerance parameters set as automatic for peptide and fragment tolerance. The minimum ion match requirements for protein identifications were set to at least one fragment per peptide, five fragments per protein, and two peptides per protein. Up to two missed cleavage sites were allowed for trypsin digestion. A maximum peptide false discovery rate (FDR) of 1% was set at the peptide level. Proteins were quantified by the average signal intensity of the three most intense tryptic peptides of each protein .one of which was unique). The resulting table was then imported and analyzed using the Perseus software (v. 1.6.15.0)  . Brieflyr > 0.37 , 28.p-value was calculated using Fischer’s exact test and adjusted for multiple comparisons by BH-adjusted p-values. We used the z-score >2 (pathway increased) or z-score <−2 (pathway decreased) with a B–H p-value <0.05 as the significance cutoff for our analysis. Then, we performed the comparative analysis in order to compare the similarity and difference among the enriched pathways and diseases or functions resulting from genes/proteins within each dataset.We performed the canonical pathway enrichment analysis in the ingenuity pathway analysis  in DEGs and DEPs separately to determine the most significantly affected pathways and the predicted activation state. To gain additional insights, we performed the diseases and functions analyses in IPA that displayed the predicted diseases or biological functions. The statistical significance https://david.ncifcrf.gov/conversion.jsp) and then and secondly, employed the InteractiVenn tool (http://www.interactivenn.net/) to uncover overlapping genes and protein products. Throughout this analysis we matched for directionality of expression patterns. Based in Shapiro–Wilk test we calculated the Spearman’s rank correlation coefficient as a measure of the strength of the relationship between messenger RNAs (mRNAs) and protein expression levels. For this purpose, we used the R environment (v. 4.0.2) for statistical was done by ggpubr (v. 0.4.0) and visualization by ggplot2 (v. 3.3.3).We analyzed the list of DEGs and DEPs looking for co-differentially expressed genes/proteins in the two lists. First, we converted the gene symbol to Entrez Gene IDs using the DAVID mapping service , and the representation of the log2FC variation was created using the Omics Visualizer app (v. 1.3.0)  for the . 1.3.0) . To grou. 1.3.0) .In order to expand the information from the modules, we created a global PPIN using all DEGs and DEPs, with the same interaction criteria as described above. In this case, we performed a multiscale community analysis to group the proteins. We used the “CyCommunityDetection” app. with the following criteria: Algorithm: OSLOM, weight column: STRING score, random number seed: 1, and all other settings at default. Then, we performed functional enrichment on the hierarchy network using g: Profiler as the algorithm .-∆∆Ct method. The syntaxin-5 (STX5) gene was used as endogenous control , treated with DNAse I (Qiagen), and quantified in Nanodrop . Next, 500 ng of total RNA per sample was used for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor . The real-time polymerase chain reaction was performed in the 7500 Real-Time PCR System  with the gene-specific primers listed in  control , 33.PBMC from septic patients and healthy volunteers were thawed. Then, five hundred thousand cells were transferred to polystyrene tubes and stained with CD66b FITC clone G10F5 (BD Biosciences), CD45 BV421 clone HI30 (BD Biosciences) and CD16 PE clone 3G8 (BD Biosciences); 7-Amino-Actinomycin D  was included to assess cell viability. Samples were incubated for 15 min in the dark at room temperature. After washing, the cells were suspended in a buffer solution  and analyzed by flow cytometry using a LSR FORTESSA (BD Biosciences). Data analysis was performed in the FlowJo software . A fluorescence minus one (FMO) control was included to delimit the CD16 populations. Gate-selection strategy is described in t-test was used to compare the normally distributed continuous variables while Mann–Whitney U-test and Kruskal-wallis (followed by the post hoc Dunn multiple comparisons test) were used to compare non-normally distributed variables. P-values < 0.05 were considered statistically significant.Statistical analysis was performed with Graph Pad Prism 9 . The normality distribution of variables was evaluated by the Shapiro–Wilk test. Student’s z‐score algorithm predicted the decrease of eight pathways and increase of 10 pathways. Additionally, IPA analysis predicted 14 diseases or functional annotations that were found to be decreased. Most of these were involved in the proliferation, activation, and homeostasis of lymphocytes  were analyzed, and a total of 2699 DEGs, including 1151 up-regulated and 1548 down-regulated, were selected on the basis of the Venn diagram analysis  samples obtained from 24 septic patients, and resulted in the identification of 2839 proteins, which are presented in a volcano plot –36. We iz‐score < −2, while six CPs showed z‐score > 2  for the expression of these 170 genes/proteins in the transcriptome and proteome profiles  analyses identified seven densely connected regions (modules) that were related to different biological functions. In particular, one module was related to neutrophil degranulation and antimicrobial response, with most genes/proteins up‐regulated. This module included genes/proteins associated with neutrophil collagenase (MMP8), neutrophil subset marker (OLFM4), neutrophil elastase (ELANE), molecules expressed in unstimulated circulating neutrophils (GCA), azurophilic granule proteins (PRTN3 and AZU1), and neutrophil granule proteins .Further, these 170 co-differentially expressed genes/proteins were employed in the construction of sepsis PPIN using the STRING database The present assessment identified three modules that were related to the transfer of information from DNA to mRNA and protein synthesis. In particular, Module 2 mainly included genes/proteins related to mRNA splicing, spliceosome, and ribonucleoprotein complex assembly, whereas Module 3 was associated with rRNA modification, ribosome biogenesis, and RNA metabolic process. These two modules largely included down‐regulated genes/proteins. Module 6 included genes/proteins linked to protein targeting to ER, peptide metabolic processes, and translational initiation. These modules included genes/proteins that are known to play a pivotal role in pre‐mRNA 3′‐end formation (CPSF1), splicing , enhancing mRNA stability and translation (NAT10), and ribosomal subunit biogenesis . Additionally, this set also included an RNA helicase (DDX24) and transcription factor (BTF3). Importantly, all these genes/proteins followed the same expression pattern in both transcriptomic and proteomic profiles. In contrast, signal peptidase complex Subunit 1 (SPCS1), polyadenylate-binding protein 1 (PABPC1), and ribosomal proteins subunits (RPS21 and RPS2) were found to be down‐regulated at transcriptomics level, whereas proteomics profile displayed an up‐regulated expression of these molecules. Additionally, three modules were also found to be related to metabolism , granulopoiesis (MPO), and neutrophil apoptosis (MNDA). Additionally, the set also included neutrophil‐specific matrix metalloproteinase, namely MMP25 and MMP9 . BesidesInterestingly, the present analyses reported down‐regulation of the components of the community that were annotated for functions related to transcription and translation, including the metabolism of RNA, mRNA splicing, rRNA biogenesis, epigenetic regulation of gene expression, and 55S mitochondrial ribosome. These communities included 15 eukaryotic initiation factors (eIFs). Among these, 13 eIFs were found to be down‐regulated. These communities also included 25 components of ribosomal subunits (22 down‐regulated) and 24 mitochondrial ribosomal proteins (19 down‐regulated). As well as include exosome complex components proteins, WD-repeat proteins, nucleolar proteins, small nuclear ribonucleoproteins, serine/arginine‐rich splicing factors, pre‐mRNA processing factors, and RNA polymerase subunits. Additionally, the study revealed alteration of carbon metabolism.RPL11 , RPS21 , NCBP2 (nuclear cap binding protein subunit 2), and MRRF  were found to be significantly reduced in the sepsis group as compared to the control group . In contrast, a significant increase in the transcription of CLEC4D (C-type lectin domain family 4 member D), GCA , and ELANE  was reported in the sepsis group as compared to the control. These results were consistent with the findings of the transcriptomic data analysis. Nevertheless, two genes, EIF2AK2  and ADAM8  did not follow the same expression trend. No significant differences were recorded for the expression of GTF2F1 , POLR2H , GFM1 , and MRPS34  between patients and control groups.To confirm and validate the aforementioned changes in gene expression, we performed quantitative real-time PCR (qPCR) for PBMCs from patients with sepsis. Thirteen genes were selected based on the results obtained from MCL analyses and/or multiscale community detection and their roles in transcription (mRNA metabolism), translation , and immune response. As illustrated in The results for proteomics analysis observed in module 1 , 39, tubint phenotype)  and reactive oxygen species (ROS) in macrophages and Natural Killer (NK) cells were found to be increased. These results are in concordance with previous studies published from our laboratory, which reported that white blood cells of septic patients were characterized by increased NO and ROS production and preserved phagocytic activity , 45. In Comparison and correlation of the proteome and transcriptome profiles were performed to investigate which genes/proteins are co-differentially expressed in sepsis. The study revealed co‐differential expression of 170 genes/proteins, with 122 genes/proteins displaying the same expression trends in both transcriptome and proteome profiles. The low to moderate correlation could be attributed to a variety of factors, including translation efficiency, alternative splicing, mRNA stability, folding, assembly, transport and localization, secretion, and degradation –50. SincCLEC4D, GCA, and ELANE in patients with sepsis.The results for MCL analyses emphasized that these co‐differentially expressed genes/proteins played a central role in clinical sepsis. Among the seven modules identified using MCL analyses, Module 1 was found to be associated with neutrophil degranulation and antimicrobial humoral response. In the multiscale community analyses we also highlight the up-regulation of genes/proteins that were related to “Azurophil granule lumen” and “Immune System”. These findings were in concordance with the results for qPCR analysis, which revealed a significant increase in the transcription levels of Int to CD16hi expression  and transcriptomics analysis (z‐score = −2.40 and −log10 FDR = 2.91). This pathway plays a crucial key role in global and specific mRNA translation processes. In particular, the phosphorylation of eIF2 is known to inhibit eIF2B, which is further related to translational initiation  and E-MTAB-5273 (EBI ArrayExpress). Mass spectrometry data were deposited to the ProteomeXchange Consortium The studies involving human participants were reviewed and approved by University Ethical Committee (CEP/UNIFESP 1171/2017). The patients/participants provided their written informed consent to participate in this study.GL, Data analysis, curation and visualization, methodology, writing and review. BF, Proteomic samples preparation, qPCR experiments, flow cytometry analysis, writing and review. RS, Supervision, conceptualization, writing and review. AT and EN, performed the mass spectrometry-based data acquisition, processed the mass spectrometry data and review. MC, Flow cytometry data acquisition, analysis and review. TP and BS, Methodology and review. EC-N, Bioinformatics platform support for analysis and review. MA, LA, and FF, contributed to the design of the study, the selection, enrollment, and monitoring of patients. All authors contributed to the article and approved the submitted version.This work was funded by FAPESP (2017/21052-0) to RS. GL has a scholarship from FAPESP (2019/20532-3). BF has a scholarship from FAPESP (2016/13855-2).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Prunus mume (PM) is an essential traditional Chinese medicine for the adjuvant treatment of solid tumors, including CRC. In the current study, we utilize means of network pharmacology, molecular docking, and multilayer experimental verification to research mechanism. The five bioactive compounds and a total of eight critical differentially expressed genes are screened out using the bioinformatics approaches of Cytoscape software, String database, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathways, and molecular docking. RelA has been proven to be highly expressed in CRC. Experiments in vitro have shown that kaempferol, the main active component of PM, dramatically inhibited the growth, migration, and invasion of CRC cells, and experiments in vivo have shown that PM effectively delays CRC formation and improves the survival cycle of mice. Further analysis shows that PM inhibits the CRC progression by down-regulating the expression level of RelA, Bax, caspase 3, caspase 9, and EGFR in CRC. PM and its extract are potentially effective therapeutics for the treatment of CRC via the RelA/nuclear factor κB signaling pathway.Colorectal cancer (CRC) is one of the most pervasive cancers in the human disease spectrum worldwide, ranked the second most common cause of cancer death by the end of 2020.  Prunus mume (PM) and differentially expressed genes (DEGs) of colorectal cancer (CRC) screening by intelligent network pharmacology and molecular docking.1. Components of PM-DEGs.2. Bioinformatics approaches of Cytoscape software, String database, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathways, and molecular docking applying to select the critical genes of RelA proved to be highly expressed in CRC in human CRC tissue in vitro and in vivo.3. PM in the treatment of CRC.4. First report on the comprehensive study to explore the molecular mechanism of Colorectal cancer (CRC) without obvious symptoms expresses early the main symptoms of occult or overt rectal bleeding, anemia, change in defecation habits, or abdominal pain in an advanced period . CRC hadPrunus mume (PM) can be guaranteed. PM has the function of inhibiting a variety of inflammatory factors, anti-inflammation and antioxidation, repairing gastrointestinal tract, and regulating gastrointestinal movement and intestinal secretion  is natural medicine, including plants, animals, and minerals. As most TCM are plants, TCM is also called CHM . CHM andecretion . PM treadementia . Mumefurammation . PM inhis in CRC . Isoqueres HaCaT . HoweverRelA/P65 is a member of the nuclear factor κB (NF-κB) transcription factor family, which is a class of proteins with specific DNA-binding activity that regulates a variety of functional target genes  website and the TCM Integrative Database (www.megabionet.org/tcmid/). The bioavailability (OB) >30% and drug similarity (DL) >0.18, main pharmacokinetic parameters, were used to screen the active components of PM using the pharmacokinetic indexes of absorption, distribution, metabolism, and excretion (ADME) (https://tcmsp-e.com/).We searched the critical word “n (ADME) . The OB n (ADME) . The comHomo sapiens,” “normal intestinal tissue,” and “colorectal cancer,” were used as keywords in the GEO database. Genes expressed differently between CRC tissues and normal tissues were sorted out and classified into functional groups with high expression and low expression . The obtained gene list was imported into the Uniprot database (http://www.uniprot.org/) to bring the name and gene ID . In the network diagram, nodes represent the active components or gene/proteins of PM, and interactions between nodes are represented by lines.Drugs’ active ingredients, the corresponding target genes of active ingredients, and disease screening target genes form the building “drug–disease” common target genes together, which build plum-visual networks of CRC target genes by Cytoscape 3.7.0 (https://string-db.org/) to study the target protein interaction relationship, also known as protein–protein interaction (PPI). All targets were limited to the species H. sapiens. In the PPI diagram, each solid circle depicts a gene, and the middle of the circle represents the protein structure. The circles are connected by different-color lines, and the strength of the interaction relationship is indicated by the number of connecting lines. At the same time, the bioinformatics technology was used to verify the protein interaction relationship again and draw the PPI interaction graph.The “drug–disease” target genes were imported into the STRING biological database (http://www.bioconductor.org/), Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to extract the biological functions of target proteins and cancer-related pathways. PM plays an anti-CRC role by regulating the biological functions of target proteins or related signaling pathways. At the same time, the histogram and bubble graph are drawn to visualize the data.By bioinformatics technology, with its programming packets and Bioconductor high-throughput genomic data analysis (PM and their related targets for CRC treatment can explain the mechanism of action and binding activity between the active components and target proteins to some extent. Compound structures in SDF format downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) were converted to a mol2 format file using Chem 3D software. The PDB format structures of BAX (PDB ID: 4S0O), BCL2 (PDB ID: 4LXD), CASP3 (PDB ID: 4PS0), CCND1 (PDB ID: 2W96), EGFR (PDB ID: 4I24), MAPK8 (PDB ID: 2XRW), RELA (PDB ID: 3QXY), and VEGFA (PDB ID: 1MKK) were downloaded from the RCSB Database (HTTPS://www.rcsb.org/), using Pymol software to remove solvent molecules and ligands, using AutoDock Tools 1.5.6 software to hydrogenate, calculate charge, assign atomic types, and so on and saving them as PDBQT format. Finally, Autodock Vina 1.1.2 was run for molecular docking, and Discovery Studio 2020 was used to analyze the conformation of docking visually. According to the data of intermolecular binding energy, hydrophilic and hydrophobic interaction, and electrostatic interaction, the score was given comprehensively, and then the intermolecular interaction was verified by next experiments.Using molecular docking technology to study the active components of The experiments involving human tissues have been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans and uniform requirements for manuscripts submitted to biomedical journals. All animal experiments should be carried out in accordance with the National Institutes of Health guide for the Care and Use of Laboratory animals . All animal studies complied with the ARRIVE guidelines. All of the following tissue and animal experiments have passed the ethical review of Huazhong University of Science and Technology and Wuhan Union Hospital.PM was purchased from Chengdu Effa Biological Technology Co., Ltd. . Certificate of analysis is shown in PM was dried and extracted in 75% ethanol in a shake incubator  overnight at room temperature and filtered with Whatman Quantitative Filter Paper. The extracts of PM (EPM) were concentrated using a rotary vacuum evaporator .Plant extract takes plants as raw materials, and according to the needs of the final product extracted, it is obtained and concentrated through the physical and chemical extraction and separation process. A product formed by one or more of the active ingredients in a substance without changing the structure of the active ingredients. Wumei is extracted on a proportion, which is the powder made after the extraction and concentration of plants raw materials. The means of proportion is the mathematical proportion of the amount of raw materials before extraction and the product after extraction and concentration. Proportional extracts generally do not have a very specific composition and content. Wumei was extracted from 10 kg of raw material and condensed into 1 kg of powder product. 2). Cell lines were authenticated by short tandem repeat analysis using the GenePrint 10 System at the Australian Genome Research Facility and confirmed to be Mycoplasma-free using the Lookout Mycoplasma PCR Detection Kit.The CRC cell lines of HCT116 and Lovo were purchased from the American Type Culture Collection . The culture media was Dulbecco modified eagle medium, supplemented with 10% fetal bovine serum. The cells were put in an incubator . When the cells reached 70%–80% confluence, we performed siRNA transfection using Lipofectamine® RNAiMAX Transfection Reagen  strictly under the manufacturer’s instructions.The overexpression plasmid transfection of PM 200 mg/kg group): except for the above operation, the mice were given 200 mg/kg PM three times a week  by intragastric administration for 9 weeks; group 3 (PM 400 mg/kg group): except for the above operation, the mice were given 400 mg/kg PM three times a week  by intragastric administration for 9 weeks. All mice were killed by cervical dislocation in week 10, and the number of macroscopic colonic tumors and the length of colorectum of the four groups were analyzed. Animal experiments were conducted following internationally recognized experimental animal management standards and approved by the Animal Ethics Committee of Huazhong University of Science and Technology  with ad libitum access to water and standard rodent diet.Sixty C57BL/6 wild-type age-matched (8–9 weeks) male mice  were used for this study. Forty-five mice were injected intraperitoneally (i.p.) with 12 mg/kg azoxymethane (AOM) and 3% dextran sulfate sodium (DSS). Starting at week 2, the mice were given 3% DSS three times a week  by intragastric administration for 1 week, followed by regular water for 2 weeks and repeated three cycles for 9 weeks. The rest of the 15 mice were injected i.p. with only 0.9% normal saline in week 1 served as untreated healthy controls. The AOM-injected mice were further subdivided into three groups of 15 mice. Group 1 (model group): Except for the above operation, the mice were given a regular diet; group 2 . Then, the cells were treated with various concentrations of kaempferol . After treatment for 6, 12, 24, and 48 h; 10 μL of CCK-8 reagent was added to each well and then cultured for 2 h. All experiments were performed in triplicate. The absorbance was analyzed at 450 nm using a microplate reader  using wells without cells as blanks. The proliferation of cells was expressed by absorbance.Cell viability was analyzed by Cell Counting Kit-8 , according to the manufacturer’s protocols. Cells were seeded and cultured at a density of 5 × 10RELA , Bax , Bcl2 , caspase 3 , cleaved caspase 3 , EGFR , CCND1, and GAPDH  and then incubated with goat anti-rabbit secondary antibodies  for 2 h at 37°C  purified and assessed qualitatively by Western blot (WB) analysis. Total protein extracts (30 µg) were shifted to 12% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels to separate the proteins by different molecular weights and transferred onto nitrocellulose membranes for antigen–antibody reaction. The membranes were then blocked with 5% skimmed milk in TBST for 1 h and mixed with primary antibodies against  at 37°C .RELA (mouse), Bax (mouse), Bcl2 (mouse), caspase 3 (mouse), cleaved caspase 3 (mouse), EGFR (mouse), CCND1(mouse), GAPDH (human), RELA (human), Bax (human) and Bcl2 (human) were executed using specific primers and the QuantiTest SYBR-Green polymerase chain reaction (PCR) kit  on an Applied Biosystems 7500 real-time PCR machine . The GAPDH acted as a normalization control for all of the mRNAs listed above. The primers for qRT-PCR are shown in Total RNA was extracted from the cancer tissues, matched normal tissues and cell lines. Total RNA was prepared using Trizol (Takara), and the cDNAs were generated by PrimeScript™ RT reagent kit according to the manufacturer’s instructions. The relative mRNA expression of GAPDH (mouse), RELA  overnight at 4°C and then incubated with goat anti-rabbit secondary antibodies . 3,3′-Diaminobenzidine was used as chromogen. The sections were then dyed using hematoxylin and mounted. RELA expression was evaluated qualitatively by two independent laboratory technicians.Formalin-fixed tissues were embedded in paraffin and sliced. The 4-mm-thick sections were then incubated with monoclonal antibodies against p < 0.05).Based on three independent experiments, data are expressed as mean ± SEM, and statistical analyses were performed using SPSS 18.0 software, R software version 3.5.0 , and GraphPad Prism version 8.0.1 . Significant differences were considered if the probability of the difference was <5 in 100  in CRC were obtained from TCMSP (https://old.tcmsp-e.com/tcmsp.php) and GEO (https://www.ncbi.nlm.nih.gov/geo/) databases. The 40 known components and 720 drug target indexes of PM were obtained by TCMSP database retrieval. A total of eight compounds were obtained from the TCMSP database under the screening conditions of OB ≥30% and DL ≥0.18 (https://pubchem.ncbi.nlm.nih.gov/) (Data of DL ≥0.18 . The corih.gov/) . DifferePM targets and 8,981 CRC DGEs were comapped, common targets were obtained through the Venny 2.1 website, and Venn diagrams were drawn , Mol000422 (kaempferol), Mol000358 (β-sitosterol), and MOL000449 (stigmasterol). This study speculated that these active ingredients might be the fundamental constituents for PM treatment of CRC.A total of 170 re drawn . Throughet genes . As showPM target–CRC DGE network. PM-CRC target genes were imported, the species selected was H. sapiens with a combined score of ≥0.4, and the network diagram of protein interaction was obtained , CASP3, CASP9, BAX, BCL2, CCND1, and CDKNIA were screened as relatively high-level targets  and inhibited tumor formations in macroscopic perspective  score was significantly decreased (p < 0.01) (p < 0.01) (p < 0.01)  , weight  < 0.01) , and sur < 0.01)  in the P changes . WesternRelA was a potential target gene of kaempferol, one of the active components of PM, for the treatment of ulcerative colitis  in CRC were identified. Simultaneously, we detected the bioactive components  and main target proteins related to CRC in PM using the TCMSP database. Subsequently, we further screened essential genes and core-dependent pathways through the PPI network, GO analysis, and KEGG analysis. Ultimately, the eight genes  with the highest differential expression within the constraints and the apoptosis-associated pathway were identified. Then, we performed molecular fitting scoring and molecular docking for the eight genes and five bioactive components. The outcomes indicated that RELA and kaempferol were genes and bioactive compounds, respectively, with the best affinity and reasonable degree in PM for CRC treatment. The NF-κB transcription factors are known to consist of five homologous subunits , which are usually dimerized and controlled by NF-κB inhibitors (IκBs) in the cytoplasm (RelA/P50 dimer is one of the regulatory factors of NF-κB canonical pathways, which is closely related to inflammatory tumorigenia (RelA had been used as an evaluator to evaluate the role of NF-κB activation in various tumors, including lung adenocarcinoma (RelA/NF-κB plays a crucial role in colorectal tumor–related processes such as angiogenesis, cell proliferation, apoptosis, autophagy, and metastasis (PM inhibited the occurrence and growth of tumors by inhibiting the expression of RELA/NF-κB and the apoptosis-associated pathway.In this study, we found that ytoplasm . Nuclearorigenia . RelA haarcinoma , breast arcinoma , liver carcinoma , CRC (Liarcinoma , and so arcinoma . At the tastasis . NF-κB rtastasis . Iκβ-medtastasis . ConsequPM in the treatment of human CRC from the perspective of morphology and protein expression was further proven (RelA in human colon cancer tissues was indeed higher than that in normal tissues and paracancer tissues. Then we explored the potential mechanism of PM in the treatment of CRC, as it is well-known that inducing tumor cell apoptosis is a crucial therapeutic approach in the treatment of tumors. After applying kaempferol, apoptosis of HCT116 cells was significantly increased, and the expression of apoptosis-related proteins was increased.The feasibility of r proven , by veriRelA over with kaempferol treatment group showed that PM mainly affected apoptosis by inhibiting RelA. Our study showed that RelA overexpression could reduce the therapeutic effect of kaempferol, inhibit the expression of Bax, and promote the expression of Bcl2. Therefore, we can demonstrate that kaempferol, the bioactive component of PM, can promote the apoptosis of colon cancer cells by inhibiting RelA, thus playing a role in treating cancer. Simultaneously, we conducted experiments in mice to verify that PM improved the symptoms of CRC model mice and participated in regulating the expression of RelA and apoptosis-related proteins. PM extract inhibited the expression of RelA, Bcl2, caspase 3, and CCND1 and promoted apoptosis-related proteins Bax, cleaved caspase 3, and EGFR. These results were consistent with the predictions of network pharmacology. In addition, the binding force between a drug and its target protein is an important indicator to evaluate its mechanism of action on disease (RelA had the best docking effect. These results confirmed that PM extract is mainly involved in inhibiting CRC through apoptosis-related pathways, especially the NF-κB pathway in which RelA is engaged.The comparison results between the kaempferol treatment group and the  disease . We invePM, β-sitosterol, kaempferol, stigmasterol, methyl arachidonate, and quercetin showed strong pharmacological effects. β-Sitosterol, kaempferol, and stigmasterol had obvious antioxidation, anti-inflammatory, antitumor, immunoregulation, and metabolic regulation activities (PM and its bioactive ingredients to inhibit the occurrence and development of CRC.CHM was also verified as a regulator to improve the function of the immune system and inhibit tumor progression . Many sctivities . β-Sitostivities . In termtivities . Kaempfetivities . Stigmastivities . Quercettivities . TherefoPM promotes colorectal cell apoptosis and inhibits the development of CRC mainly by inhibiting the expression of RelA. Among the five bioactive ingredients associated with CRC in PM, kaempferol and RelA had the highest molecular docking level. We also verified the effect of kaempferol on RelA and CRC cells. However, there are also limitations. First, PM is a natural botanical medicine containing many components, so pharmaceutical composition identification should be conducted. Second, other bioactive ingredients of PM should also be verified by experiments. Finally, it is necessary to further explore the direct regulation of bioactive ingredients on RelA.Combined with the results of network pharmacology, molecular docking, and experimental verification of cell and animal and human tissue samples at three levels,


To tackle the long-standing opioid misuse problem, Myanmar introduced the methadone maintenance treatment (MMT) program in 2006, starting with 260 clients. Since then, the program has been expanded across different geographical sites in the country. This study was done in 2017 to explore the treatment satisfaction of the clients towards the MMT program.A total of 210 clients with a minimum of six-month treatment history enrolled in five MMT program sites across Myanmar were recruited through stratified random sampling. Administering the Verona Service Satisfaction Scale for Methadone-Treatment (VSSS-MT), this study assessed the satisfactory responses under three categories viz., 1) clinic staff professional skills; 2) basic drug intervention; 3) specific intervention .n = 186) of the respondents were satisfied with the current MMT program. Specifically, 89.5% (n = 187) were satisfied with the clinic staff professional skills category, 91.9% (n = 192) with the basic program intervention and 74.6% (n = 156) with specific interventions. Among the respondents, treatment satisfaction with the MMT program was higher (p < 0.05) in those (i) with a higher quality of life score in physical, psychological, social and environmental categories, ii) who were satisfied with their current marital and leisure status, and those iii) who consumed alcohol. Results from stepwise binary logistic regression showed alcohol consumption and physical health status had a significant association with MMT treatment satisfaction.The majority  for the specific interventions category compared with 89.5 and 91.9% respectively for staff and basic drug management categories highlights the need for improvement in this category for overall enhancement of the MMT program. The Global Health Observatory data from the World Health Organization (WHO) had highlighted that in 2014, 45% of the 147 responding countries had methadone therapy chiefly for OUD detoxification purposes . The WorMyanmar, a country that accounted for 7% of the world’s opium production , continu. Given the rapid expansion of the MMT program, several validated tools have been developed to assess clients’ treatment satisfaction with the MMT program. Commonly used tools include the Client Satisfaction Questionnaire (CSQ-8) [It has been pharmacologically shown that the MMT program is effective as it is associated with decreased opioid use . Improve (CSQ-8) , the Ser (CSQ-8)  and the  (CSQ-8) . SatisfaA recent situational analysis indicated that MMT programs are bound to have several limitations or barriers. They are inconvenient operational hours, long travel distance to MMT sites, lengthy registration process, coping with the continuous drug use issue among clients, long induction period, and a lack of confidentiality . In view1) to explore clients’ overall satisfaction towards the program in general;2) to assess clients’ satisfaction towards a) professional skills of the clinic staff; b) basic drug intervention (Methadone administration); c) specific intervention such as individual rehabilitation and psychotherapy,3) to find out if any of clients’ characteristics are associated with their satisfactory response.Two hundred and ten clients enrolled in the MMT program across different geographical provinces in Myanmar were recruited for this study. We used the stratified random sampling approach to recruit a quota sample of 42 respondents from each of the following locations across Myanmar; Yangon Region, Mandalay Region, Lashio (from Shan State), Kawlin (from Sagaing Region), and Mohnyin (from Kachin State). Surveys were conducted through face-to-face interviews by trained researchers at the targeted MMT facilities. Respondents were assured that this study would not collect their personally identifiable information and all the study data would be kept confidential. Each interview session lasted 25 to 30 min. All the respondents gave informed consent and were compensated for their time.The inclusion criteria for the study were as follows; a) Age 18 years and above, b) self-reported as a methadone client currently enrolled in a formal MMT program in Myanmar, and c) at least a minimum of six-month methadone treatment history. We believe six-month treatment history would be long enough to provide us with a better picture of the client’s satisfaction with the MMT program and ancillary services.Those observed to be in an incoherent state of mind and those who were reluctant to participate voluntarily were excluded from the study.There were full cooperation and participation from the methadone clients who were selected for participation in the survey. Drop-in-centres managers and methadone clinic doctors explained to the client the initial information session before the survey. During the interview visit, the research team also explained the importance of this survey and the potential benefit of the survey results in the operation of the methadone programme in different areas.We collected the study data from May 2017 to July 2017. A semi-structured questionnaire was used to elicit respondents’ socio-demographic characteristics such as current age, gender, employment status, MMT treatment history, HIV status, illicit drug use history, and service satisfaction with the MMT program. We also used the Verona Service Satisfaction Scale for Methadone Treatment (VSSS-MT) , the AddAll the respondents were required to undergo a supervised urine drug test for the following substances; Methadone, morphine, cannabis, methamphetamine, amphetamine, and benzodiazepine. All the urine specimens were appropriately discarded at the end of the interviews to avoid legal consequences.p < 0.05. Next, we compared the mean scores of certain parameters (VSSS-MT scores) of interest with a t-test. We ran the Binary logistic regression for identifying the predictors to the outcome “VSSS-MT score” at p < 0.05 with confounding variables controlled. The Cox regression model for HIV infection was used to explore the relationship between treatment satisfaction and HIV status.We analysed all the study data with Stata 14.0 software. First, to identify the association between VSSS-MT Treatment satisfaction and categorical variables of patient characteristics, a Chi-squared test was performed for two tailed-test at a significance level of The study had been approved by the Human Ethics and Research Committee of Universiti Sains Malaysia and then, Myanmar Ministry of Health and Sports, Department of Medical Research (No: Ethics/DMR/2017/057) [n = 210) were males and only 1.4% were females. The mean age was 33.3 years . All clients took their daily methadone supply from MMT clinics, and 83% (n = 173/210) were in their first-time treatment of MMT. The average daily methadone dose was 83 mg , and the average duration was 28 months . Almost two thirds  received ≤80 mg/day dosage, while 76 (37%) received a higher dosage of more than 80 mg/ day. The average Body Mass Index (BMI) was 20.5 . Regarding education status, 75.6%, (n = 159/ 210) had primary to high school education, 21.43% had a college education, and only six clients lacked formal education. Almost half of the respondents (46.4%) were single or divorced, while 40.58% were married.In this study, the majority (98.6%) of clients  were employed, and only 8.57% were unemployed. Twenty-nine respondents worked as outreach workers or peer educators for people with substance use problems.n = 186) of clients were highly satisfied with their MMT program, there were variations in the ratings among the three categories: professional skill items  was rated 3.89 out of 5, basic intervention items, 3.83 and specific intervention items 3.42. This highlights that among three categories of methadone service that captured treatment satisfaction, specific intervention category scored the lowest.Treatment satisfaction rating scores varied from study site to site, with scores with scores being lower in bigger cities  and higher in smaller cities . For clients who rated 3 and above on the Likert scale of 5 for all items in the questionnaire, the mean score for satisfaction with methadone service  was set as 100 (range: 58–131), while the score over 80 was designated as “much satisfaction”. While 88.6%  .Among the respondents, those with a higher quality of life score on physical, psychological, social and environmental categories, those who were satisfied with their current marital and leisure status, and those who consumed alcohol had higher MMT treatment satisfaction scores of over 80  were considered in the regression analysis. The backward stepwise binary logistic regression analysis was done among the significant parameters in the model to identify final predictors to the outcome group with MMT satisfaction , and those who had good physical health  were 12 times  more satisfied with their MMT program. When checking for the multi collinearity, mean variance inflation factor (vif) was 1.61 and none of the variables had more than 10. The regression model alpha ratio was set at 0.05.After adjusting the potential confounding variables, this analysis estimated the association between the independent variables and the outcome variable of the service satisfaction score category. Those who used alcohol were 6 times  of respondents were on high-dose methadone (more than 80 mg/ day) and the rest 63.5% (n = 132) on low-dose methadone (less than 80 mg/ day). Almost half , of the respondents reported hepatitis C virus infection (HCV), 37% (n = 74/200) reported Human Immunodeficiency Virus (HIV) infection, while 16% (n = 34) reported HIV and HCV co-infection. Forty-five respondents (21.53%) reported sexually transmitted infections, and 15 respondents (8.29%) reported Hepatitis B infection. Out of reported HIV respondents, 68 (92%) were on antiretroviral therapy (ART) with an average ART duration of 30 months . Taking high methadone dose (more than 80 mg) was found to have a significant association with having antiretroviral therapy (p = 0.039) [About one-third  .p < 0.05).We also ran a univariate cox proportional hazard model analysis to estimate treatment satisfaction based on the acquired data. In this survival analysis, which is similar to that of , “the mep = 0.000), treatment satisfaction was 2 times higher among HIV negative patients compared with HIV positive patients, and the satisfaction was 1.7 times higher among non-co-infected patients compared with co-infected patients . The higher rate of satisfaction incidence among HIV negative respondents was 1.49% and among HIV positive respondents was 0.72% after adjusting the methadone dose (p = 0.000). A higher dose was associated with increased satisfaction scores (p = 0.037) in HIV negative respondents [The results show that, after controlling the methadone dose variable . This Cox regression, in which univariate analysis was done for the interested variable “Treatment satisfaction”, and this analysis did not take into account other confounding variables. Despite the limitation of cross-sectional study type and potential bias, the estimation of the incidence rate of “treatment satisfaction” was analysed with a similar way of analysis [Higher satisfaction incidence was 1.2% among non-co-infected patients and 0.71% among co-infected patients after adjusting for doses. In other words, co-infected patients were less likely to be satisfied with the program than non-co-infected patients (n = 186) of clients were highly satisfied with the MMT program implemented in their respective localities in Myanmar.This study found that 88.6%  scored the highest (3.89 out of 5), while items in specific intervention category scored the lowest. A Vietnamese study  also repRegarding the program categories, the ratings for satisfaction in this study were higher than those reported by a study from Spain (3.83 vs 3.5 for basic intervention items and 3.42 vs 3.1 for specific intervention) . People Many factors influence MMT treatment satisfaction. These include recent heroin injections, use of benzodiazepines, HCV infection, high addiction severity index (ASI), alcohol consumption and family/ social status. Limitations or barriers in methadone treatment such as dosing spaces, waiting areas and staff shortage , methadoOur stepwise binary logistic regression result showed alcohol consumption and physical health were significantly associated with methadone treatment satisfaction. Although Myanmar is a majority Buddhist country, alcohol consumption is common among its people.There were a few limitations in this study. First, respondents for this study were randomly recruited from a few MMT clinics. Hence there could be bias by not enrolling clients unsatisfied with their MMT program. There is a limitation to make generalisations from the results bias the data were based on one-time self-reporting. The result’s further potential bias could be contributed by the selection criteria of a minimum 6-month treatment. The National indicator from the DDTRU (Drug Dependency Treatment and Research Unit) specified the client retention in the methadone treatment as “those clients who were taking methadone at least 6-month period”, and this survey excluded the satisfactory situation of the clients who were less than 6 months of the treatment. The 2018 annual data  showed 7