follicular lymphoma ( fl ) is the most common indolent non - hodgkin lymphoma ( nhl ) , accounting for 2030% of all nhl in western countries.1 it is characterized by a chronic course , with a projected survival of more than 18 years in the modern chemo - immunotherapy era.2 while some patients with limited stage disease may be cured , those presenting with advance stage or relapsing after local radiotherapy are generally considered not curable with standard treatments.1 early studies have shown that deferring treatment in asymptomatic patients with low tumor burden is not associated to a worse survival and , in many cases , the disease can remain stable for several years.3,4 the usefulness of watchful waiting has been later on confirmed in the rituximab era.5 thus only patients bearing a high tumor burden disease and/or symptomatic are currently treated with chemo - immunotherapy . standard , first line treatment includes the use of rituximab plus chemotherapy with an expected overall response rates of more than 90% and complete remissions in the range of 2570% with median progression - free survival ( pfs ) exceeding 4 years.6 a two years maintenance with rituximab in responders results in significant prolongation of pfs , but not overall survival ( os).7,8 therefore , despite the excellent improvement gained by chemo - immunotherapy , the majority of the patients eventually progress or relapse . several factors have been identified as of key importance in predicting pfs and os , among those the quality of first line response have been shown to be remarkably associated to survival outcomes.9 traditionally , response evaluation in fl has been made with the use of contrast enhanced computed tomography ( ct)-scan and bone marrow biopsy ( bmb ) along with standard laboratory tests and clinical parameters.10,11 immunohistochemical staining of bmb is the standard technique to assess lymphoma infiltration , but more sensitive assays have been developed to detect subclinical involvement . the presence of a hybrid bcl2/igh gene in 8090% of fl has spurred the interest in applying polymerase chain reaction ( pcr ) techniques to test the bone marrow ( bm ) and peripheral blood ( pb ) of patients before and after treatment.12,13 in the current review we will discuss the methodological aspects of molecular monitoring and its clinical significance in the modern chemo - immunotherapy era . the genetic hallmark of fl is the t(14;18)(q32;q21 ) translocation that leads to deregulated expression of the anti - apoptotic gene bcl2 in tumor cells , thus allowing for the acquisition of secondary chromosomal alterations in the germinal center environment , where the most non - neoplastic b cells undergo apoptosis.14 the resulting hybrid gene bcl2/igh is highly attractive for pcr based assays as it is a disease specific clonal sequence directly linked to fl pathogenesis and thus represents a highly stable marker . five different clusters of bcl2/igh rearrangements occur : the major breakpoint region ( mbr ) , the minor clustering region ( mcr ) , the intermediate cluster region ( icr ) , the 3-bcl-2 region and the 5-mcr region ( figure 1).15 to date , the molecular detection of minimal residual disease ( mrd ) has been almost entirely based only on the study of mbr and mcr which account for about 50 and 10% of all bcl2 rearrangements , respectively.15 qualitative ( nested ) pcr ( npcr ) has been widely used in molecular testing fl patients and proved as a highly reproducible method with an excellent sensitivity level able to detect 1 neoplastic cell in about a hundred thousand normal cells ( 110).16 the advent of taqman - based approaches allowed the introduction of quantitative pcr methods i.e. real - time quantitative pcr ( rq - pcr ) , a significant step forward from the mere presence or absence of a bcl2/igh rearrangement.17,18 this latter technique made possible the quantification of bcl2/igh tumor burden at diagnosis and the dynamic of its reduction with treatment with lower risk of contamination and higher inter - laboratory reproducibility . conversely , rq - pcr has lower sensitivity than npcr , probably as less amount of dna is tested , and it is more expensive and technically complex needing the construction of standard reference curves.19 the occurrence of non - neoplastic bcl2/igh rearrangements in the peripheral blood of healthy donors or patients without lymphoma was regarded as a possible confounder factor for mrd studies.20,21 the low chimeric gene levels found in non - fl patients and its clearance after chemotherapy , however , confirmed the feasibility of mrd testing in this setting.22 another key point for the diffusion of mrd assessment is standardization of methodologies and definitions of common mrd terms . standardization of rq - pcr , including data interpretation and reporting , has been made by the efforts of the european network project euro - mrd and has been applied in clinical trials.19,2326 new technical approaches could in the near future improve the frequency and the feasibility of bcl2/igh rearrangements identification , as next generation sequencing ( ngs ) or droplet digital pcr.27,28 twenty - five years have passed since the first observations that mrd negativity plays a role in predicting the outcome of patients with fl.13 early studies showed that standard chemotherapy programs can achieve a molecular remission ( mr ) in a minority of patients . first line anthracycline containing protocols could attain a mr in about 3050% of the patients,29,30 while intensification with autologous stem - cell transplant ( sct ) can lead up to 6070% of mrd negativity.31 conversely , the proportion was negligible in those with relapsed disease.32 in all of these studies , patients achieving a mr were characterized by a significantly prolonged disease control . notably , long term results of two trials aiming at reducing neoplastic cell contamination before asct with ex vivo purging , confirmed that persistence of residual marrow involvement both at microscopic or molecular assessment were the only significant factors for long term remission , but not for survival.33 the association of rituximab with chemotherapy dramatically improved response rates , pfs and , most notably , os of advanced fl patients requiring treatment.34,35 the ability of rituximab to deplete fl neoplastic cells from peripheral blood and bone marrow , increased the rates of mr , accordingly . a clear demonstration of the rituximab activity on mrd was shown in a study in which only responsive patients after chop therapy not achieving a mr were treated with 4 weekly infusion of rituximab.30 overall , sequential administration of chop followed by rituximab resulted in complete bm and pb molecular response in more than 70% of patients . freedom from recurrence at 3 years was 5257% for those patients obtaining a durable mr after chop or chop plus rituximab , while it was significantly lower ( 20% ) for those failing to obtain or lost a mr . residual disease kinetic showed that most of the patients in mr after chop were negative at the first interim evaluation after 3 cycles . conversely , a delayed maximum effect was noted after rituximab , with 59% , 74% and 63% of the patients in mr at week + 12 , + 28 and + 44 after treatment . this late effect of rituximab was observed in other trials , as well as the more difficult clearance of mrd in the bm compared with the pb.23,24 rituximab have been used as consolidation therapy after autologous sct in small series of patients , and proved to be safe and effective both in increasing the quality of clinical response i.e. converting pr into cr , as well as in achieving mr.36,37 the efficacy of front line rituximab plus chemotherapy in inducing mr have been included as secondary end point in several large prospective trials ( table 1 ) . in two different controlled studies , r - chop resulted in mr of 3944%.23,25 similar results were obtained with fludarabine and antracycline - based induction regimens ( r - fm , r - fnd ) and with mitoxantrone , chlorambucil , and prednisolone ( r - mcp).23,24,38 indirect evidence suggests that the less intensive regimen r - cvp could lead to inferior results both in term of clinical response and mr rates , explaining the shorter pfs observed compared to r - chop / r - fm.23 conversely , intensive regimens including upfront autologous sct ( r - hds ) increase the molecular response rates up to 80% of patients.25 no data are currently available for the schema r - bendamustine in the front line setting , but bendamustine alone or combined with the novel anti - cd20 monoclonal antibody obinutuzumab in relapsed / refractory patients can induce mr.39 induction therapy with 90-ibritumomab - tiuxetan was associated with achievement of a mr in nearly all of the cr patients.40 most importantly , all these trials confirmed the significant improvement in disease control in terms of pfs or relapse / event free survival in those patients achieving and maintaining the mr . the result was independent from other prognostic factors , most importantly the quality of response ( cr vs pr ) , the chemotherapy induction regimen chosen and clinical risk factors as the fl prognostic index ( flipi ) . however , the good results achieved in terms of disease control , did not translate in survival improvement . the importance of mr was shown also for a non - chemotherapy based consolidation . in a phase iii trial 90-ibritumomab - tiuxetan was randomly given as consolidation therapy after standard first line therapy.41 interestingly , when compared to controls , consolidation with 90-ibritumomab - tiuxetan did not improve the pfs of patients who already were in mr while a significant prolongation of pfs was obtained in mrd positive patients ( 38.4 vs 8.2 months of the control group , p<0.01 ) . in the relapsed / refractory setting , a randomized phase iii study comparing chop vs r - chop therapy with subsequent rituximab maintenance vs observation further confirmed the predictive value of mr , as almost all of the few patients who were still bcl2/igh pcr positive at the end of the 2 years of maintenance treatment relapsed rapidly.42 quantitative pcr methods have been used to measure the bcl2/igh chimeric gene burden and early studies suggested that rq - pcr evaluation before and after autologous transplantation may predict the clinical course of these patients.43,44 in a clinical trial evaluating the sequential administration of chop and rituximab in mrd positive patients , a high lymphoma cell burden at diagnosis was associated with lower probability to achieve a clinical and molecular cr.45 the kinetic of bcl2/igh positive cells during treatment showed that chop and rituximab were both able to remove approximately 2 logs of tumor infiltration , thus explaining why patients with a limited lymphoma infiltration can achieve a molecular remission after chop chemotherapy alone , while the others necessitate the addition of rituximab . quantification of bcl2/igh chimeric gene burden in the bm , but not in pb , was associated to a better event free survival . the result of the mrd analysis of a large phase iii study confirmed the prognostic value of the molecular tumor burden both in term of likelihood to achieve a cr , and pfs.23 of note , high lymphoma cell burden at diagnosis was independent from flipi and clinical response in determining pfs . the importance of rq - pcr was additionally shown in a study in which significant reduction ( 2 logs ) of circulating lymphoma cells rather than the mere mrd negativity was associated with a favorable clinical response and prolonged event - free survival.38 however , not all the studies confirm these findings , probably due to the different induction regimen and rituximab schedule.24 compelling evidence indicates that mrd is a post treatment independent prognostic factor that can be consistently used to guide subsequent consolidation therapy in clinical trials . firstly , in large randomized prospective trials where molecular evaluation has been performed routinely , a molecular marker could be detected in only 50 to 60% of the patients.2325 in a large study including samples from 415 patients , a molecular marker was present in 53% of the cases ; in particular , 67.5% of patients without bm infiltration were mrd positive , conversely 17.6% of patients with microscopic marrow involvement at bmb were lacking the molecular marker.23 several reasons could explain this finding , mainly the presence of uncommon rearrangements and the lack of significant marrow involvement in patients with nodal disease.15 despite the availability of primers and probes for detecting rare bcl2/igh breakpoints will increase the cases with molecular marker , a significant proportion of patients are eventually excluded from this strategy . persistence over time of mr is a major indication of sustained remission . as mrd detection is more informative on bm , especially in the rituximab era , the need of multiple invasive procedures additionally limits the feasibility of mrd monitoring over time . moreover , all the clinical trials reporting a prognostic implication of mr included the evaluation of response according to the 1999 or 2007 international working group ( iwg ) criteria with the use of the sole contrast enhanced ct - scan.10,11 the introduction of fdg - pet scan improved the accuracy of staging and response assessment in fdg - avid lymphomas and is currently recommended for the definition of response in fl.46 several trials showed that concordance in ct - based and pet - based response designation is critical especially for patients in pr or cr unconfirmed , as pet scan is able to identify those patients with metabolically active disease and thus can improve the predictive value of response assessment.4749 to date , no data is available regarding the integration of pet based response and mrd evaluation . the only report in this setting is a retrospective evaluation of a very limited proportion of patient ( 8% ) enrolled in a prospective trial.50 this study suggests that pet and mrd are not strongly correlated with each other , and thus could be used as complementary techniques at the end of therapy to optimally explore the nodal and bone marrow compartments , but further studies are necessary to confirm the independent role of the two techniques . the current clinical significance of mrd evaluation should also be evaluated when considering the evolving scenario of fl treatment . to date , given the satisfactory median results of chemo - immunotherapy and the lack of a survival impact of the chemotherapies available , the routine selection of induction treatment is guided more from the avoidance of unnecessary toxicity rather than the mere activity of the regimen.1 however , while most patients achieve a prolonged disease control , a sizeable subset of cases remains substantially refractory to front line treatment with a poor prognosis.9 clinical scores currently available as flipi or flipi2 fail in identifying such cases , and a growing numbers of prognostic factors before or after treatment have been developed with this aim.9,5153 thus , definition of high risk patient and , accordingly , end points for clinical trials are changing . treatment results are satisfactory in low risk patients and integration of new molecules should be made with great caution in this group.54 in this regard , achievement of sustained mr could allow the de - escalation of standard therapy in very low risk patients i.e. maintenance with rituximab . conversely , high risk patients are a group for which standard treatment need to be implemented and pfs should not represent per se the primary end point . efforts to consistently characterize this latter group are ongoing and surrogate end points for survival as 2-year pfs have been proposed.9,5153 although not yet integrated in clinical practice as compared to other setting such acute lymphoblastic leukemia,55 mrd evaluation is commonly integrated in clinical trials testing the efficacy of new treatment protocols in fl patients . in this setting mrd maintains its consistent and independent prognostic significance . achievement of mr is a marker of treatment sensibility that has been associated with good clinical outcome in term of pfs , but not os , independently from the specific therapy . some technical limitations such as the limited coverage of the different breakpoints present in the bcl2/igh rearrangements will be likely overcome in the near future by more appropriate molecular approaches.27,28 these laboratory improvements , most likely in combination with the new imaging technologies currently tested by an ongoing clinical trial ( nct02063685 ) , will probably lead to a reappraisal of mrd evaluation in fl patients
after 25 years , evaluation of minimal residual disease ( mrd ) in follicular lymphoma ( fl ) has become a standardized technique frequently integrated into clinical trials for its consistent and independent prognostic significance . 
 achievement of a sustained mrd negativity is a marker of treatment sensibility that has been associated with excellent clinical outcome in terms of clinical response and progression - free survival , independently from the employed therapy . 
 however , no survival advantages has been reported for mrd negative patients and despite the compelling results of clinical trials , mrd evaluation has currently no role in clinical practice . 
 ongoing clinical trials will help in clarifying the potential setting in which mrd monitoring may have a routine clinical application i.e. allowing de - escalation of standard maintenance therapy in very low risk patients . in this review 
 the clinical implications of mrd monitoring in rituximab - era are discussed in light of the current treatment paradigms most aimed at reducing toxicities , and the response definition that now routinely integrates pet scan .