Hepatocytes undergo punctuated expansion dynamics from a periportal stem cell niche in normal human liver.

BACKGROUND & AIMS: While normal human liver is thought to be generally quiescent, clonal hepatocyte expansions have been observed, though neither their cellular source nor their expansion dynamics have been determined. Knowing the hepatocyte cell of origin, and their subsequent dynamics and trajectory within the human liver will provide an important basis to understand disease-associated dysregulation. METHODS: Herein, we use in vivo lineage tracing and methylation sequence analysis to demonstrate normal human hepatocyte ancestry. We exploit next-generation mitochondrial sequencing to determine hepatocyte clonal expansion dynamics across spatially distinct areas of laser-captured, microdissected, clones, in tandem with computational modelling in morphologically normal human liver. RESULTS: Hepatocyte clones and rare SOX9+ hepatocyte progenitors commonly associate with portal tracts and we present evidence that clones can lineage-trace with cholangiocytes, indicating the presence of a bipotential common ancestor at this niche. Within clones, we demonstrate methylation CpG sequence diversity patterns indicative of periportal not pericentral ancestral origins, indicating a portal to central vein expansion trajectory. Using spatial analysis of mitochondrial DNA variants by next-generation sequencing coupled with mathematical modelling and Bayesian inference across the portal-central axis, we demonstrate that patterns of mitochondrial DNA variants reveal large numbers of spatially restricted mutations in conjunction with limited numbers of clonal mutations. CONCLUSIONS: These datasets support the existence of a periportal progenitor niche and indicate that clonal patches exhibit punctuated but slow growth, then quiesce, likely due to acute environmental stimuli. These findings crucially contribute to our understanding of hepatocyte dynamics in the normal human liver. IMPACT AND IMPLICATIONS: The liver is mainly composed of hepatocytes, but we know little regarding the source of these cells or how they multiply over time within the disease-free human liver. In this study, we determine a source of new hepatocytes by combining many different lab-based methods and computational predictions to show that hepatocytes share a common cell of origin with bile ducts. Both our experimental and computational data also demonstrate hepatocyte clones are likely to expand in slow waves across the liver in a specific trajectory, but often lie dormant for many years. These data show for the first time the expansion dynamics of hepatocytes in normal liver and their cell of origin enabling the accurate measurment of changes to their dynamics that may lead to liver disease. These findings are important for researchers determining cancer risk in human liver.

Clonal Transitions and Phenotypic Evolution in Barrett's Esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) is a risk factor for esophageal adenocarcinoma but our understanding of how it evolves is poorly understood. We investigated BE gland phenotype distribution, the clonal nature of phenotypic change, and how phenotypic diversity plays a role in progression. METHODS: Using immunohistochemistry and histology, we analyzed the distribution and the diversity of gland phenotype between and within biopsy specimens from patients with nondysplastic BE and those who had progressed to dysplasia or had developed postesophagectomy BE. Clonal relationships were determined by the presence of shared mutations between distinct gland types using laser capture microdissection sequencing of the mitochondrial genome. RESULTS: We identified 5 different gland phenotypes in a cohort of 51 nondysplastic patients where biopsy specimens were taken at the same anatomic site (1.0-2.0 cm superior to the gastroesophageal junction. Here, we observed the same number of glands with 1 and 2 phenotypes, but 3 phenotypes were rare. We showed a common ancestor between parietal cell-containing, mature gastric (oxyntocardiac) and goblet cell-containing, intestinal (specialized) gland phenotypes. Similarly, we have shown a clonal relationship between cardiac-type glands and specialized and mature intestinal glands. Using the Shannon diversity index as a marker of gland diversity, we observed significantly increased phenotypic diversity in patients with BE adjacent to dysplasia and predysplasia compared to nondysplastic BE and postesophagectomy BE, suggesting that diversity develops over time. CONCLUSIONS: We showed that the range of BE phenotypes represents an evolutionary process and that changes in gland diversity may play a role in progression. Furthermore, we showed a common ancestry between gastric and intestinal-type glands in BE.

IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution.

Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells' dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

High-throughput sequencing of IgH gene in minor salivary glands from Sjögren's syndrome patients reveals dynamic B cell recirculation between ectopic lymphoid structures.

OBJECTIVES: B cells play a central role in Sjögren's syndrome (SS) whereby autoreactive B-cells populate ectopic germinal centres (GC) in SS salivary glands (SG) and undergo somatic hypermutation (SHM) and class-switch recombination of the immunoglobulin genes. However, the capacity of specific B cell clones to seed ectopic GC in different SG and undergo clonal diversification is unclear. To unravel the dynamics of B cell recirculation among minor SG biopsies, we investigated the immunoglobulin heavy chain (IgH) gene usage and the pattern of SHM using a high-throughput sequencing approach. METHODS: We generated ~166,000 reads longer than 350bp and detected 1631 clonotypes across eight samples from four different SS patients, all characterised by the presence of functional ectopic GC as demonstrated by the expression of activation-induced cytidine deaminase. RESULTS: A large number of shared clonotypes were observed among paired mSG biopsies from each patient but not across different patients. Lineage tree analysis revealed significant clonal expansion within the mSG with the identification of shared dominant B cell clones suggestive of extensive recirculation across different SG. Several shared clonotypes with high proliferating capacity displayed IgH-VH gene usage common in autoreactive B cells, including VH1-69, which is typical of rheumatoid factor+ B cells representing potential lymphoma precursors. CONCLUSIONS: The complex dynamic recirculation of B cells that we observed within ectopic GC responses linked with their ability to independently proliferate, undergo ongoing SHM and Ig class-switching within individual glands may explain the difficulty in achieving consistent eradication of ectopic GCs following B cell depleting agents reported in different studies.

H and L Chain Affinity Maturation and/or Fab N-Glycosylation Influence Immunoreactivity toward Neutrophil Extracellular Trap Antigens in Rheumatoid Arthritis Synovial B Cell Clones.

We previously showed that anti-neutrophil extracellular trap (NET) rheumatoid arthritis (RA)-rmAbs derived from CD19+ B cells within RA human synovial tissues frequently react against NETs. In this study, we aimed to characterize the importance of affinity maturation via somatic hypermutation (SHM) within the Ig variable H (VH) and variable L (VL) chains and Fab-N-linked glycosylation in RA synovial B cell clones reactive to NETs and NET-derived Ags such as citrullinated histones. Selected anti-NET RA-rmAbs derived from synovial RA CD19+ B cells were subjected to overlap-PCR to generate germline (GL; VH and VL reverted into GL), hybrid clones (VH/VL region reverted into GL), and N-glycosylation mutants (N→Q) and analyzed for anti-NETs and citrullinated histones (cit-H2B) immunoreactivity. Anti-NET/cit-H2B immunoreactivity of selected RA-rmAbs was abrogated in the VH and VL GL counterpart. In RA B cell hybrid clone RA015/11.88 and RA056/11.23.2, NET and/or cit-H2B immunoreactivity was solely dependent on SHM in the IgVH region whereas RA B cell hybrid clone RA015/11.91 required affinity maturation of both VH and VL for efficient binding to cit-H2B. In 7/80 RA-rmAb, SHM resulted in ex novo N-glycosylation sites in VH and/or VL regions. Removal of Fab-linked glycans in RA056/11.23.2 in the N-mutant counterpart resulted in 90% reduction in immunoreactivity to cit-H2B. Thus, SHM in the IgVH and/or VL regions of RA synovial B cells is necessary for the immunoreactivity to NET-Ags. Fab-N-linked-glycosylation introduction sites are observed in a minority of anti-NET B cell clones but can strongly influence NET-Ag binding.

Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs.

OBJECTIVES: Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium. METHODS: Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA. RESULTS: RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a 'citrullinome' multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS-) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-ß, two master regulators of ELS. CONCLUSION: We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.

Molecular characteristics of early-onset pancreatic ductal adenocarcinoma.

The median age of patients with pancreatic ductal adenocarcinoma (PDAC) at diagnosis is 71 years; however, around 10% present with early-onset pancreatic cancer (EOPC), i.e., before age 50. The molecular mechanisms underlying such an early onset are unknown. We assessed the role of common PDAC drivers (KRAS, TP53, CDKN2A and SMAD4) and determined their mutational status and protein expression in 90 formalin-fixed, paraffin-embedded tissues, including multiple primary and matched metastases, from 37 EOPC patients. KRAS was mutated in 88% of patients; p53 was altered in 94%, and p16 and SMAD4 were lost in 86% and 71% of patients, respectively. Meta-synthesis showed a higher rate of p53 alterations in EOPC than in late-onset PDAC (94% vs. 69%, P = 0.0009) and significantly higher loss of SMAD4 (71% vs. 44%, P = 0.0025). The majority of EOPC patients accumulated aberrations in all four drivers; in addition, high tumour heterogeneity was observed across all tissues. The cumulative effect of an exceptionally high rate of alterations in all common PDAC driver genes combined with high tumour heterogeneity suggests an important mechanism underlying the early onset of PDAC.

SNP rs6457327 in the HLA region on chromosome 6p is predictive of the transformation of follicular lymphoma.

Inherited risk determinants for follicular lymphoma (FL) have recently been described in the immune gene-rich human leukocyte antigen region on chromosome 6p. The known importance of host immune response to FL survival led us to evaluate these germline factors in FL outcome. We confirm the association of single nucleotide polymorphisms rs10484561 (P = 3.5 × 10⁻9) and rs6457327 (P = .008) with risk of FL and demonstrate that rs6457327 predicts both time to (P = .02) and risk of (P < .01) FL transformation independently of clinical variables, including the Follicular Lymphoma International Prognostic Index.

Mutational patterns along different evolution paths of follicular lymphoma.

Follicular lymphoma (FL) is an indolent disease, characterized by a median life expectancy of 18-20 years and by intermittent periods of relapse and remission. FL frequently transforms into the more aggressive diffuse large B cell lymphoma (t-FL). In previous studies, the analysis of immunoglobulin heavy chain variable region (IgHV) genes in sequential biopsies from the same patient revealed two different patterns of tumor clonal evolution: direct evolution, through acquisition of additional IgHV mutations over time, or divergent evolution, in which lymphoma clones from serial biopsies independently develop from a less-mutated common progenitor cell (CPC). Our goal in this study was to characterize the somatic hypermutation (SHM) patterns of IgHV genes in sequential FL samples from the same patients, and address the question of whether the mutation mechanisms (SHM targeting, DNA repair or both), or selection forces acting on the tumor clones, were different in FL samples compared to healthy control samples, or in late relapsed/transformed FL samples compared to earlier ones. Our analysis revealed differences in the distribution of mutations from each of the nucleotides when tumor and non-tumor clones were compared, while FL and transformed FL (t-FL) tumor clones displayed similar mutation distributions. Lineage tree measurements suggested that either initial clone affinity or selection thresholds were lower in FL samples compared to controls, but similar between FL and t-FL samples. Finally, we observed that both FL and t-FL tumor clones tend to accumulate larger numbers of potential N-glycosylation sites due to the introduction of new SHM. Taken together, these results suggest that transformation into t-FL, in contrast to initial FL development, is not associated with any major changes in DNA targeting or repair, or the selection threshold of the tumor clone.

Stem cell mimicry: key to transformation?

In this issue of Blood, Gentles and colleagues used a computational model to investigate FL and transformed DLBCL, and identified the acquisition of an ESC-like signature as a key feature of transformation.

Transformation of follicular lymphoma to diffuse large B-cell lymphoma may occur by divergent evolution from a common progenitor cell or by direct evolution from the follicular lymphoma clone.

To investigate the cell of origin linking follicular (FL) and transformed (t-FL) lymphomas, we analyzed the somatic hypermutation (SHM) pattern of the variable region of the immunoglobulin heavy gene (IgH-VH) in 18 sequential FL/t-FL samples and a father (donor) and son (recipient), who developed FL and t-FL, after transplantation. Genealogic trees showed a pattern compatible with a common progenitor cell (CPC) origin in 13 cases. The identification of the t-FL clonotype in the previous FL sample and of the putative CPC sequence in both the FL/t-FL biopsies showed that the intraclonal diversity of FL and t-FL germinal centers (GCs) is more intricate than previously described, and all 3 clonotypes (CPC, FL, t-FL) may occur simultaneously within the same lymph node. On the basis of the father/son model, this CPC must be long-lived, providing a possible explanation for the incurable nature of this disease.

The presence of TP53 mutation at diagnosis of follicular lymphoma identifies a high-risk group of patients with shortened time to disease progression and poorer overall survival.

The International Prognostic Index and the Follicular Lymphoma International Prognostic Index are widely used for the risk assessment of follicular lymphoma (FL). Although molecular studies have provided insight into the biology of FL, no molecular marker has impacted on treatment stratification. Because TP53 mutations are associated with poor prognosis in hematologic malignancies, we investigated the prognostic value of TP53 mutation at diagnosis in FL. Heterozygous TP53 mutation was detected in 12 of 185 (6%) analyzed cases. Mutation was associated with older age (P = .02) and higher International Prognostic Index score (P = .04). On multivariate analysis, TP53 mutation correlated with shorter progression-free survival (P < .001) and overall survival (P = .009). TP53 mutation was associated with low expression of the immune-response 1 gene expression signature (P = .016) and with an unfavorable gene expression-based survival predictor score (P < .001), demonstrating for the first time that molecular features of the malignant cell may correlate with the nature of the immune response in FL.

FcgammaRIIIA and FcgammaRIIA polymorphisms do not predict clinical outcome of follicular non-Hodgkin's lymphoma patients treated with sequential CHOP and rituximab.

We analyzed FcgammaRIIIA-158V/F and FcgammaRIIA-131H/R polymorphisms in a cohort of 94 newly diagnosed follicular lymphoma (FL) patients sequentially treated with CHOP and Rituximab. With a median follow-up of 5.8 years, the overall survival at 8 years is 83%. Univariate and multivariate analysis showed no correlation between FcgammaRIIIA-158VV/VF and FcgammaRIIA-131HH/HR polymorphisms and the overall response rate, the molecular response and the event-free survival obtained after CHOP and Rituximab. By contrast, the achievement of a durable molecular clearance of BCL2/IgH+ cells detectable in the bone marrow is confirmed to be a reliable predictive factor of a better long-term clinical outcome.

High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution.

Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10-2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.

Integrated genomic analysis identifies recurrent mutations and evolution patterns driving the initiation and progression of follicular lymphoma.

Follicular lymphoma is an incurable malignancy, with transformation to an aggressive subtype representing a critical event during disease progression. Here we performed whole-genome or whole-exome sequencing on 10 follicular lymphoma-transformed follicular lymphoma pairs followed by deep sequencing of 28 genes in an extension cohort, and we report the key events and evolutionary processes governing tumor initiation and transformation. Tumor evolution occurred through either a 'rich' or 'sparse' ancestral common progenitor clone (CPC). We identified recurrent mutations in linker histone, JAK-STAT signaling, NF-κB signaling and B cell developmental genes. Longitudinal analyses identified early driver mutations in chromatin regulator genes (CREBBP, EZH2 and KMT2D (MLL2)), whereas mutations in EBF1 and regulators of NF-κB signaling (MYD88 and TNFAIP3) were gained at transformation. Collectively, this study provides new insights into the genetic basis of follicular lymphoma and the clonal dynamics of transformation and suggests that personalizing therapies to target key genetic alterations in the CPC represents an attractive therapeutic strategy.

Stage I/II follicular lymphoma: spread of bcl-2/IgH+ cells in blood and bone marrow from primary site of disease and possibility of clearance after involved field radiotherapy.

Stage I/IIA follicular lymphoma (FL) is considered a localised disease that can be adequately treated with radiotherapy alone. Bone marrow (BM) and peripheral blood (PB) involvement in FL was investigated by polymerase chain reaction (PCR) in a series of 24 consecutive patients with histologically revised diagnosis and treated with involved field radiotherapy. Despite the limited stage, Bcl-2/IgH+ cells were found at diagnosis in PB and/or BM of 16 patients (66.6%). After treatment, in 9/15 Bcl-2/IgH positive evaluable patients, a disappearance of Bcl-2/IgH+ cells was observed, which persisted after a median follow-up of 43.5 months (range 11-70) in all but one patient. Quantitative PCR demonstrated the feasibility of clearing PB and BM Bcl-2+ cells after local irradiation of the primary site of the disease only when the basal number of lymphoma cells was <1:100 000. Patients with Bcl-2/IgH+ cells at diagnosis or after treatment had a higher likelihood of relapse. Thus, despite a negative BM biopsy, the majority of localised FL Bcl-2/IgH+ cells were found in the PB and BM. Lymphoma cells can reversibly spread from the affected lymph node to PB and BM and, in a proportion of cases, durably disappear after irradiation. The possibility of a persistent lymphoma cell clearance is proportional to the amount of cells detected at presentation by quantitative PCR.

Quantitative PCR of bone marrow BCL2/IgH+ cells at diagnosis predicts treatment response and long-term outcome in follicular non-Hodgkin lymphoma.

By real-time quantitative polymerase chain reaction (RQ-PCR), we evaluated BCL2/IgH(+) cells in the bone marrow (BM) and peripheral blood (PB) from 86 patients with follicular lymphoma treated with the sequential administration of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) and rituximab. At diagnosis, the amount of BCL2/IgH(+) cells in the BM was low (1 BCL2/IgH(+) cell in 1000-100 000 normal cells) in 43% of patients, intermediate (1 in 100-1000) in 34%, and high (> 1 in 100) in 23%. A 2 log decrease of BCL2/IgH(+) cells was achieved after CHOP and an additional 2 log reduction following rituximab. By multivariate analysis, a low level of BCL2/IgH(+) cells in the BM at diagnosis was the best predictor for the achievement of a complete clinical and molecular response. At 5 years, the event-free survival rates of patients with a low/intermediate or high tumor infiltration in the BM were 59% and 32%, respectively. The freedom from recurrence of patients who achieved a molecular response in the BM, no matter whether after CHOP alone or CHOP and rituximab, was 64% as compared to 32% for patients who did not (P < .006). RQ-PCR performed at presentation on BM samples predicts treatment response and long-term clinical outcome in patients with follicular lymphoma.

Molecular characterization of a new recombination of the SIL/TAL-1 locus in a child with T-cell acute lymphoblastic leukaemia.

Deletions involving the SIL-TAL-1 locus are seen in 15% of T-acute lymphoblastic leukaemias (T-ALL). To date, seven deletions have been described, spreading over 90 kb of chromosome 1, fusing SIL to the TAL-1 gene and resulting in over expression of TAL-1. During the diagnostic screening of the TAL-1 deletion in 176 T-ALL patients, we identified one case showing a new SIL rearrangement. A novel fusion transcript was identified between the SIL exon 1a and an unknown sequence (633-cDNA). Polymerase chain reaction (PCR) screening of a human cDNA library confirmed the existence of this transcript. Using long-distance PCR on patient DNA, we obtained a genomic fragment containing SIL exon 1b, a portion of intron 1b, an unknown sequence and the 633 sequence. Using DNA from healthy donors, a partial genomic map of 633-DNA was found to be identical to the restriction map of the PCR fragment amplified from patient DNA. To define the chromosomal origin of 633-DNA, a YAC human genomic library was screened. Two clones containing 633-DNA were found, mapping to chromosomal region 1p32 and both contained SIL and TAL-1 sequences. By searching GenBank, we identified PAC RP1-18D14 which contains SIL, TAL-1 and 633-DNA, confirming this novel rearrangement as a new deletion of the SIL/TAL-1 locus.

Monitoring of minimal residual disease after CHOP and rituximab in previously untreated patients with follicular lymphoma.

Minimal residual disease (MRD) following sequential administration of CHOP and rituximab was studied in previously untreated patients with follicular lymphoma. At diagnosis, the presence of Bcl-2/IgH-positive cells in the peripheral blood (PB) and/or bone marrow (BM) was demonstrated in all patients (n = 128) by polymerase chain reaction (PCR) analysis. Patients who achieved a clinical response following CHOP but remained PCR-positive were eligible for rituximab (375 mg/m(2) intravenously, weekly for 4 weeks). After CHOP, 57% achieved a complete response (CR), 37% a partial response (PR), and 6% were nonresponders (NR). At this stage, patients proving PCR-negative (n = 41) or failing to achieve a clinical response (n = 8) were excluded from rituximab treatment. Seventy-seven patients received rituximab and entered a scheduled MRD follow-up program. At the first molecular follow-up (+12 weeks), 59% had converted to PCR negativity in the BM and PB, with a further increase documented at the second control (+28 weeks) with 74% PCR negative. At the last molecular follow-up (+44 weeks), 63% of the patients remained PCR negative. At 3 years, the estimated overall survival of all patients is 95% (95% confidence interval [CI], 86-98). For patients achieving PCR-negative status following CHOP and therefore excluded from rituximab treatment, freedom from recurrence (FFR) was 52% (95% CI, 28-71). For patients treated with rituximab, a durable PCR-negative status was associated with a better clinical outcome since FFR was 57% (95% CI, 23-81) compared with 20% (95% CI, 4-46) in patients who never achieved or lost the molecular negativity (P <.001).