LGR5 and CD133 as prognostic and predictive markers for fluoropyrimidine-based adjuvant chemotherapy in colorectal cancer

<p><b>Background:</b> Expression of leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) gene is associated with a metastatic phenotype and poor prognosis in colorectal cancer (CRC). CD133 expression is a putative cancer stem cell marker and a proposed prognostic marker in CRC, whereas the predictive value of CD133 expression for effect of adjuvant chemotherapy in CRC is unclear.</p> <p><b>Material and methods:</b> For the study of LGR5 mRNA and CD133 expression, tissue microarrays from 409 primary CRC stage II and III tumors, where patients had been randomized to adjuvant chemotherapy or surgery only, were available. LGR5 mRNA and CD133 expression were assessed by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. LGR5 mRNA and CD133 expression as prognostic and predictive markers were evaluated by univariate and multivariate analyses.</p> <p><b>Results:</b> For all CRC patients, positive LGR5 mRNA and CD133 expression were associated with classic adenocarcinoma histology type (p = 0.001 and p = 0.014, respectively). Positive LGR5 mRNA expression was also associated with smaller tumor diameter for CRC stage II (p = 0.005), but not for CRC stage III (p = 0.054). For CRC stage II, lack of LGR5 mRNA expression was associated with longer time to recurrence (TTR) in Kaplan-Meier (p = 0.045) and in multivariate Cox analysis (HR 0.27, 95% CI 0.08–0.95, p = 0.041). For colon cancer stage III patients, lack of CD133 expression was associated with better effect of adjuvant chemotherapy (p = 0.016) in Kaplan-Meier univariate analysis, but the interaction between CD133 and adjuvant chemotherapy was not statistically significant in multivariate analysis (HR 0.59, 95% CI 0.18–1.89, p = 0.374).</p> <p><b>Conclusion:</b> LGR5 mRNA expression is a prognostic factor for CRC stage II patients, whereas the value of CD133 expression as prognostic and predictive biomarker is inconclusive.</p

Validity of Norwegian Rectal Cancer Registry data at a major university hospital 1997–2005

<div><p><b>Background.</b> The Norwegian Rectal Cancer Registry (NRCR) has been used extensively to monitor patient treatment and outcomes since its establishment in 1993. Control of data validity is crucial to ensure reliable information, but an audit of the NRCR data validity has not been performed so far. This study aims to validate NRCR data on patients diagnosed in the period 1997–2005, Department of Surgery, Haukeland University Hospital.</p><p><b>Material and methods.</b> The material comprises NRCR data on all 482 patients diagnosed with rectal cancer in the period 1997–2005 at a major Norwegian university hospital. We checked 50 variables for discrepancies by comparing NRCR data with the medical records. All erroneous registrations were recorded.</p><p><b>Results.</b> One hundred patients (21%) had one or more data discrepancies in the registry, and 131 errors (0.5%) were noted in total. Sixteen variables (32%) had no erroneous registrations. Pre-operative CT and type of surgical procedure had the highest proportion of erroneous registrations (2.1%). Recorded errors were grouped into five variable categories: Pre-operative evaluation and adjuvant treatment (40 errors), surgical treatment (44 errors), pathological evaluation (20 errors), complications (7 errors) and oncological outcomes (20 errors). The majority of erroneous registrations (45%) were considered minor in severity, 27% were moderate and 28% were major.</p><p><b>Conclusion.</b> Assessment of the NRCR data from a nine-year period showed a good data validity in this hospital cohort.</p></div

Lymphocyte subsets during follow-up in the KTS-1-2008 and KTS-2-2010 trials.

<p>Panels A-D: Lymphocyte subsets among patients included in the randomized KTS-1-2008 study, in the rituximab group (red) and the placebo group (black), determined by flowcytometry and shown as million cells per liter at baseline (0 months), and at 1, 2, 3, 4, 6, 8, 10, and 12 months follow-up (n = 30). Panel A: CD3+ T-cells, panel B: CD4+ T helper cells, panel C: CD8+ cytotoxic T-cells, panel D: CD56+/16+ NK-cells. For 29 patients, data on lymphocyte populations were missing for 16 out of 261 (6.1%) time points. For General linear model (GLM) for repeated measures these were replaced by interpolation between preceding and succeeding values. The missing values were not replaced in the plots. Panels E-J: T-cell activation markers among patients included in the KTS-1-2008 trial, at baseline and through 12-months follow-up, and shown as percentage of the CD4+ T helper cell population, as determined by flowcytometry. Panel E: Regulatory T-cells, panel F: CD69+ cells, panel G: CD154+ cells, panel H: CD278+ (ICOS) cells, panel I: HLA-DR+ cells. Panel J shows the percentage of HLA-DR+ cells among the CD8+ cytotoxic T-cells. For 29 patients, data on T-regulatory cells and T-cell activation parameters were missing for 30 out of 261 (11.5%) time points. For GLM, missing values were replaced by interpolation between the preceding and succeeding analyses (not replaced in the plots). Panels K-O: Lymphocyte subsets among patients in the KTS-2-2010 study receiving rituximab maintenance therapy, determined by flowcytometry and shown as million cells per liter at baseline (0 months), and 3, 6, 12, 15, 20, and 24-months follow-up. Responders to B-cell depletion therapy are shown in red and non-responders in blue. Panel K: Ratio of CD4+/CD8+ T-cells, panel L: CD3+T- cells, panel M: CD4+ T helper cells, panel N: CD8+ cytotoxic T-cells, panel O: CD56/16+ NK-cells. In three patients, data on lymphocyte subpopulations for at least three time points were missing, these three were omitted leaving 23 patients for GLM analyses to assess the interaction between time and response group. In these 23 patients, 15/161 (9.3%) data were missing; these were replaced by interpolation between preceding and succeeding values (not replaced in the plots). Analyses for interaction between time and intervention group, i.e. assessing for difference in course of the variables over time, between the rituximab and placebo groups (panels A-J), or between responders and nonresponders to rituximab maintenance treatment (panels K-O), were performed using GLM for repeated measures. Error bars denote 95% confidence intervals (CI) for the mean values. The dotted lines indicate lower and upper normal reference values as established at Haukeland University Hospital.</p

Serum BAFF levels during follow-up in the KTS-1-2008 and KTS-2-2010 trials.

<p>Panel A: Serum BAFF levels at baseline and at 3, 6, and 8-months follow-up in the placebo arm of the KTS-1-2008 trial, relative to baseline levels (n = 13). Panel B: Serum BAFF levels at baseline, and at 3, 6, and 8-months follow-up in the rituximab arm of the randomized KTS-1-2008 trial, relative to baseline levels (n = 14). Panel C: Serum BAFF levels at baseline and at 3, 6, and 10-months follow-up in the open-label KTS-2-2010 rituximab maintenance trial, relative to baseline levels (n = 22). Panel D: Serum APRIL levels at baseline, and at 3 and 8-months follow-up in the placebo arm of the KTS-1-2008 trial, relative to baseline levels (n = 12). Panel E: Serum APRIL levels at baseline, and at 3 and 8-months follow-up in the rituximab arm of the KTS-1-2008 trial, relative to baseline levels (n = 13). P-values from Repeated measures One-way ANOVA with Dunnett’s test for adjusted p-values for individual comparisons of the different time points to baseline. Error bars denote mean and 95% CI.</p

Serum BAFF levels at baseline.

<p>Panel A: Baseline serum BAFF levels (pg/mL) in 70 ME/CFS patients and in 56 healthy controls. Serum samples before treatment in the KTS-1-2008 and KTS-2-2010 trials (n = 38), and in addition 32 samples from ME/CFS patients fulfilling Canadian diagnostic criteria, were included. Panel B: Baseline APRIL serum concentrations (ng/mL), in ME/CFS patients included in KTS-1-2008 study (n = 28) and in healthy controls (n = 22). P-values from unpaired t-test (equal variances not assumed). Error bars denote mean and 95% confidence intervals (CI).</p

Serum immunoglobulin levels during 24-months follow-up, in KTS-2-2010 trial.

<p>Immunoglobulin levels in serum during 24 months follow-up, for patients included in the KTS-2-2010 trial with rituximab maintenance therapy, and shown as grams per liter (g/L). In panels A, B and C are shown serum levels for IgG, IgA, and IgM, respectively, with corresponding values at baseline and at 24-months, for each of 23 patients included in KTS-2-2010. P-values from paired t-tests. Panel D: Baseline levels of serum IgG (g/L), shown for patients with subsequent clinical response to B-cell depletion therapy during follow-up, or patients with no response. P-value from unpaired Mann-Whitney U test. The dotted lines indicate normal reference values as established by Haukeland University Hospital.</p

B-Lymphocyte Depletion in Myalgic Encephalopathy/ Chronic Fatigue Syndrome. An Open-Label Phase II Study with Rituximab Maintenance Treatment

<div><p>Background</p><p>Myalgic Encephalopathy/Chronic Fatigue Syndrome (ME/CFS) is a disease of unknown etiology. We previously reported a pilot case series followed by a small, randomized, placebo-controlled phase II study, suggesting that B-cell depletion using the monoclonal anti-CD20 antibody rituximab can yield clinical benefit in ME/CFS.</p><p>Methods</p><p>In this single-center, open-label, one-armed phase II study (NCT01156909), 29 patients were included for treatment with rituximab (500 mg/m²) two infusions two weeks apart, followed by maintenance rituximab infusions after 3, 6, 10 and 15 months, and with follow-up for 36 months.</p><p>Findings</p><p>Major or moderate responses, predefined as lasting improvements in self-reported <i>Fatigue score</i>, were detected in 18 out of 29 patients (intention to treat). Clinically significant responses were seen in 18 out of 28 patients (64%) receiving rituximab maintenance treatment. For these 18 patients, the mean response durations within the 156 weeks study period were 105 weeks in 14 major responders, and 69 weeks in four moderate responders. At end of follow-up (36 months), 11 out of 18 responding patients were still in ongoing clinical remission. For major responders, the mean lag time from first rituximab infusion until start of clinical response was 23 weeks (range 8–66). Among the nine patients from the placebo group in the previous randomized study with no significant improvement during 12 months follow-up after saline infusions, six achieved a clinical response before 12 months after rituximab maintenance infusions in the present study. Two patients had an allergic reaction to rituximab and two had an episode of uncomplicated late-onset neutropenia. Eight patients experienced one or more transient symptom flares after rituximab infusions. There was no unexpected toxicity.</p><p>Conclusion</p><p>In a subgroup of ME/CFS patients, prolonged B-cell depletion with rituximab maintenance infusions was associated with sustained clinical responses. The observed patterns of delayed responses and relapse after B-cell depletion and regeneration, a three times higher disease prevalence in women than in men, and a previously demonstrated increase in B-cell lymphoma risk for elderly ME/CFS patients, suggest that ME/CFS may be a variant of an autoimmune disease.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT01156909" target="_blank">NCT01156909</a></p></div

Function level and “SF-36mean5”.

<p>Mean values of SF-36 raw scores for the five subdimensions Physical function (PF), Bodily pain (BP), Vitality (V), Social function (SF) and General health (GH) are shown (denoted “SF-36mean5”, scale 0–100), at baseline and at 3, 6, 10, 15, 20, 24, 30 and 36 months follow-up. SF-36mean5 scores for each time point during follow-up were compared to baseline scores. P-values were calculated using Repeated Measures One-way ANOVA, with Dunnett’s multiple comparison adjustments, and are indicated at the top of each panel. ns: not significant; **: p<0.01; ***: p<0.001; ****: p<0.0001. To be able to analyze for differences at each time point relative to baseline, a missing value at a time point for a patient was replaced with value interpolated between the previous and next values during follow-up for that patient (but not replaced in the plot). Panel A shows “SF-36mean5” (raw) scores for 27 patients in this study. One pilot patient did not fill in SF-36 forms. In addition 22/243 (9.1%) data were missing, including for patients out of study before end of follow-up, as explained in M&M. Panel C shows “SF-36mean5” scores for 17 patients with clinically significant response (data from pilot 2, and in addition 10/153 (6.5%) data were missing). Panel E shows “SF-36mean5” scores for 10 patients with no clinically significant response during follow-up (12/90 (13.3%) data were missing). Panels B, D and F show self-reported Function levels (according to a form with examples, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s005" target="_blank">S3 Fig</a>), at baseline and at 15, 24 and 36 months. In panel B for 28 patients included in the KTS-2-2010 study, in panel D for 18 patients with clinically significant responses, and in panel F for 10 patients without clinically significant responses. In panels B and D, two moderate responders registered their Function level at 32 and 33 months (instead of 36 months), respectively. Panel G shows a correlation plot between “SF-36mean5” raw scores and self-reported Function levels (both with scale 0–100), with pooled data from baseline and at 15, 24 and 36 months follow-up. Panel H shows a Bland-Altman plot for difference (“SF-36mean5”—Function level) versus average.</p

Consort 2010 Flow Diagram for the KTS-2-2010 study.

<p>Consort Flow Diagram for the KTS-2-2010 study, with enrollment, allocation to induction and maintenance rituximab treatment, and follow-up showing number of patients who withdrew from study before 36 months.</p

ME/CFS disease characteristics and selected response data, for 29 patients included in the study.

<p>¹: The numbers refer to corresponding plots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s006" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s007" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s008" target="_blank">S6</a> Figs (i.e. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s006" target="_blank">S4A</a> means <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s006" target="_blank">S4 Fig</a> panel A) for each of 28 individual patients receiving rituximab maintenance treatment. Among 18 patients fulfilling the predefined response criterion, clinical significance (CS) was determined post-hoc as Major (MajR, n = 14) or Moderate (ModR, n = 4). One patient had a marginal response (MargR) and nine patients were non-responders (NonR).</p><p>*: one patient (last row in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.t001" target="_blank">Table 1</a>) had an allergic reaction to the first rituximab infusion and did not receive induction treatment or complete follow-up.</p><p>²: F: female, M: male. Age in years.</p><p>³: Indicating infection preceding ME/CFS onset. Mono: mononucleosis; Viral: viral infection not otherwise specified; GI: gastrointestinal infection; Airway: upper airways infection; Borrelia: Borrelia infection; Pneum.: pneumonia.</p><p>⁴: ME/CFS disease duration given in years (y). ME/CFS severity categorized as mild: mild grade; mild/mod: mild to moderate grade; mod: moderate grade, mod/sev: moderate to severe grade; sev: severe grade (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#sec007" target="_blank">Materials and Methods</a>).</p><p>⁵: Previous participation in clinical studies on ME/CFS. Pilot: pilot patient. KTS-1: participated in the KTS-1-2008 study; Pl,NoR: previously given placebo in KTS-1-2008 with no clinical response; Rx,R-Re: previously given rituximab in KTS-1-2008 (or as pilot) with clinical response and later relapse; Rx,NoR: previously given rituximab in KTS-1-2008 with no clinical response; No: the patient had not participated in previous studies. *: this patient (corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s006" target="_blank">S4 Fig</a> panel L) was a participant in KTS-1-2008 and also a pilot patient for this study.</p><p>⁶: Autoimmune diseases (AD) among first-degree relatives were present for 12 out of 29 included patients (41%). These AD included rheumatoid arthritis (in relatives of seven patients), Sjøgren’s syndrome (in relatives of two patients), ulcerative colitis (in relatives of three patients), thyroiditis (in relatives of two patients). Also, glomerulonephritis, lupus, juvenile arthritis, psoriasis, multiple sclerosis (all present in relatives of single patients).</p><p>⁷: Rx infus: number of rituximab (Rx) infusions given to each patient (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#sec007" target="_blank">Materials and Methods</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#sec016" target="_blank">Results</a>).</p><p>⁸: Clinical response periods according to predefined criteria, i.e. periods of <i>Fatigue score</i> ≥ 4.5 for at least six consecutive weeks which must include at least one recording of <i>Fatigue score</i> >5.0 during the response period. The numbers show the intervals with clinical response given in weeks during follow-up.</p><p>⁹: Response durations defined as the sum of individual clinical response periods during follow-up, and given in weeks. *: This patient had a moderate improvement from 58–112 weeks, and with a mean <i>Fatigue score</i> exactly 5.0 in the interval 80–112 weeks. He did not fulfill strict response criteria, but was post-hoc judged as a moderate responder.</p><p>**: This patient fulfilled predefined response criteria, but had a short lasting and late response period, interpreted post-hoc as probably not related to intervention and not clinically significant.</p><p>¹⁰: Clinical response status at end of study. Also shown the time of last recording for each patient. Yes: still in clinical response at end of follow-up; No: not in clinical response at end of follow-up; OoSt: out of study, also indicating time point during follow-up; SR: slight symptom increase but still in response;</p><p>*: allergic reaction to first rituximab infusion, did not complete induction therapy or follow-up.</p><p>¹¹: LON: late onset neutropenia; Airw.inf.: several airways infections; Rx-Wor: indicating number of transient worsening of ME/CFS-symptoms after rituximab infusions; Allergy: allergic reaction to rituximab; BC: breast cancer; ITP: idiopathic thrombocytopenic purpura (also indicated in the corresponding panels in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s006" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s007" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s008" target="_blank">S6</a> Figs);</p><p>*See comment: this patient had improvement during follow-up assessed by SF-36 scores and selfreported Function level, but did not fulfill criteria for clinical response; MargR KTS-1: this patient had a marginal response duration in the previous KTS-1-2008 study; No sign. response: not interpreted as a clinically significant response.</p><p>¹²: SF-36mean5 is the mean of raw scores for the five SF-36 subdimentions Physical function, Bodily pain, Vitality, General health, and Social function (scales 0–100), shown at 0, 15, 20 or 24, and 36 months follow-up (in three patients last recording at 31, 33 and 35 months, respectively); nd: not done.</p><p>¹³: Self-reported Function level, given as per cent (0–100%) in which 100% denotes completely healthy, according to a form with examples (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129898#pone.0129898.s005" target="_blank">S3 Fig</a>), recorded at 0, 15, 24 and 36 months follow-up (in three patients last recording at 31, 33 and 35 months, respectively); nd: not done.</p><p>ME/CFS disease characteristics and selected response data, for 29 patients included in the study.</p