Evaluation of the inflammation-based modified Glasgow Prognostic Score (mGPS) as a prognostic and predictive biomarker in patients with metastatic colorectal cancer receiving first-line chemotherapy: a post hoc analysis of the randomized phase III XELAVIRI trial (AIO KRK0110).

BACKGROUND: The inflammation-based modified Glasgow Prognostic Score (mGPS) combines serum levels of C-reactive protein and albumin and was shown to predict survival in advanced cancer. We aimed to elucidate the prognostic impact of mGPS on survival as well as its predictive value when combined with gender in unselected metastatic colorectal cancer (mCRC) patients receiving first-line chemotherapy in the randomized phase III XELAVIRI trial. PATIENTS AND METHODS: In XELAVIRI, mCRC patients were treated with either fluoropyrimidine/bevacizumab followed by additional irinotecan at first progression (sequential treatment arm; Arm A) or upfront combination of fluoropyrimidine/bevacizumab/irinotecan (intensive treatment arm; Arm B). In the present post hoc analysis, survival was evaluated with respect to the assorted mGPS categories 0, 1 or 2. Interaction between mGPS and gender was analyzed. RESULTS: Out of 421 mCRC patients treated in XELAVIRI, 362 [119 women (32.9%) and 243 men (67.1%)] were assessable. For the entire study population a significant association between mGPS and overall survival (OS) was observed [mGPS = 0: median 28.9 months, 95% confidence interval (CI) 25.9-33.6 months; mGPS = 1: median 21.4 months, 95% CI 17.6-26.1 months; mGPS = 2: median 16.8 months, 95% CI 14.3-21.2 months; P < 0.00001]. Similar results were found when comparing progression-free survival between groups. The effect of mGPS on survival did not depend on the applied treatment regimen (P = 0.21). In female patients, a trend towards longer OS was observed in Arm A versus Arm B, with this effect being clearly more pronounced in the mGPS cohort 0 (41.6 versus 25.5 months; P = 0.056). By contrast, median OS was longer in male patients with an mGPS of 1-2 treated in Arm B versus Arm A (20.8 versus 17.4 months; P = 0.022). CONCLUSION: We demonstrate the role of mGPS as an independent predictor of OS regardless of the treatment regimen in mCRC patients receiving first-line treatment. mGPS may help identify gender-specific subgroups that benefit more or less from upfront intensive therapy.

Relevance of baseline carcinoembryonic antigen for first-line treatment against metastatic colorectal cancer with FOLFIRI plus cetuximab or bevacizumab (FIRE-3 trial).

PURPOSE: Increased baseline carcinoembryonic antigen (CEA) serum level is associated with inferior overall survival (OS) in metastatic colorectal cancer (mCRC). However, limited data exist on its predictive relevance for targeted therapies. Therefore, we analysed its relevance in FIRE-3, a randomised phase III study. EXPERIMENTAL DESIGN: FIRE-3 evaluated first-line FOLFIRI plus cetuximab (FOLFIRI/Cet) versus FOLFIRI plus bevacizumab (FOLFIRI/Bev) in mCRC patients with RAS-WT tumour (i.e. wild-type in KRAS and NRAS exons 2-4). Herein, the impact of CEA on patient outcome was investigated. RESULTS: Of 400 patients, 356 (89.0%) were evaluable for CEA. High CEA (>10 ng/ml; N = 237) compared to low CEA (≤10 ng/ml; N = 119) was associated with shorter OS in the FOLFIRI/Bev arm (hazard ratio [HR] = 1.50; P = 0.036), while no significant OS difference was observed in the FOLFIRI/Cet arm (HR = 1.07; P = 0.74). In patients with high CEA, FOLFIRI/Cet compared to FOLFIRI/Bev showed a greater OS benefit (HR = 0.56; P < 0.001) than in patients with low CEA (HR = 0.78; P = 0.30). Furthermore, FOLFIRI/Cet exhibited significantly superior objective response rate in patients with high CEA (odds ratio = 2.21; P = 0.006) in contrast to patients with low CEA (odds ratio = 0.90; P = 0.85). CONCLUSION: In patients with RAS-WT mCRC receiving first-line chemotherapy with FOLFIRI/Cet versus FOLFIRI/Bev, elevated CEA was associated with inferior survival in the bevacizumab arm, while this was not the case when cetuximab was applied. Comparison of OS and objective response rate according to treatment arms indicated that cetuximab was greatly superior to bevacizumab in patients with elevated CEA, while this effect was markedly lower and lost statistical significance in patients with low CEA.

Surgical treatment options following chemotherapy plus cetuximab or bevacizumab in metastatic colorectal cancer-central evaluation of FIRE-3.

BACKGROUND: The FIRE-3 trial investigated combination chemotherapy plus either cetuximab or bevacizumab in patients with untreated metastatic colorectal cancer (mCRC) not scheduled for upfront surgery. We aimed to determine the number of patients who present with potentially resectable disease during systemic first-line therapy and to compare the findings with study reports concerning resections and outcome. PATIENTS AND METHODS: This evaluation of 448 patients was performed as central review blinded for treatment, other reviewers' evaluations and conducted interventions. Resectability was defined if at least 50% of the reviewers recommended surgical-based intervention. Overall survival was assessed by Kaplan-Meier method. RESULTS: Resectability increased from 22% (97/448) at baseline before treatment to 53% (238/448) at best response (P < 0.001), compared with an actual secondary resection rate for metastases of 16% (72/448). At baseline (23% versus 20%) and best response (53% versus 53%), potential resectability of metastases in this molecular unselected population was similar in cetuximab-treated patients versus bevacizumab-treated patients and not limited to patients with one-organ disease. The actual resection rate of metastases was significantly associated with treatment setting (P = 0.02; university hospital versus hospital/practice). Overall survival was 51.3 months (95% confidence interval [CI] 35.9-66.7) in patients with resectable disease who received surgery, 30.8 months (95% CI 26.6-34.9) in patients with resectable disease without surgery and 18.6 months (95% CI 15.8-21.3) in patients with unresectable disease (P < 0.001). CONCLUSIONS: Our findings illustrate the potential for conversion to resectability in mCRC, certain reluctance towards metastatic resections in clinical practice and the need for pre-planned and continuous evaluation for metastatic resection in high-volume centres. CLINICALTRIALS. GOV-IDENTIFIER: NCT00433927.

Evaluation of survival across several treatment lines in metastatic colorectal cancer: Analysis of the FIRE-3 trial (AIO KRK0306).

BACKGROUND: We explored the impacts of sequential application of various treatment lines on survival kinetics. Therefore, differences in overall survival (OS) observed in FIRE-3 were investigated in the context of time and exposure to applied treatment. PATIENTS AND METHODS: OS analyses (stratified by treatment with FOLFIRI plus either cetuximab or bevacizumab) were performed according to time intervals as well as using a Cox model to define changes of hazard ratio (HR) over time. RESULTS: The fraction of patients with systemic treatment and time on treatment markedly decreases over treatment lines and time. OS evaluation by a Cox model indicated a trend towards a non-proportional hazard between treatment arms (P = 0.12/P = 0.09 for KRAS-intention-to-treat (ITT)/all-RAS wild-type populations, respectively). To improve the fit of the model, a change-point (point of curve separation) was estimated at 22.6 months (day 687) after randomisation. The HR between the two arms before 22.6 months was not significantly different from one. However, markedly different survival kinetics in favour of the cetuximab arm were apparent after the change-point (KRAS-ITT: P = 0.0018; HR, 0.60 [95% confidence interval [CI], 0.44-0.83] and RAS: P = 0.0006; HR, 0.51 [95% CI, 0.35-0.75]). CONCLUSION: The differences in OS favouring the cetuximab arm become apparent about 22.6 months after randomisation, indicating that only those patients who survive 22.6 months after randomisation benefit from the superiority of the cetuximab arm. When OS curves separate, only few patients receive active systemic treatment in short courses, suggesting that earlier treatment effects are responsible for later kinetics of survival curves.

Outcome according to KRAS-, NRAS- and BRAF-mutation as well as KRAS mutation variants: pooled analysis of five randomized trials in metastatic colorectal cancer by the AIO colorectal cancer study group.

BACKGROUND: To explore the impact of KRAS, NRAS and BRAF mutations as well as KRAS mutation variants in patients with metastatic colorectal cancer (mCRC) receiving first-line therapy. PATIENTS AND METHODS: A total of 1239 patients from five randomized trials (FIRE-1, FIRE-3, AIOKRK0207, AIOKRK0604, RO91) were included into the analysis. Outcome was evaluated by the Kaplan-Meier method, log-rank tests and Cox models. RESULTS: In 664 tumors, no mutation was detected, 462 tumors were diagnosed with KRAS-, 39 patients with NRAS- and 74 patients with BRAF-mutation. Mutations in KRAS were associated with inferior progression-free survival (PFS) and overall survival (OS) [multivariate hazard ratio (HR) for PFS: 1.20 (1.02-1.42), P = 0.03; multivariate HR for OS: 1.41 (1.17-1.70), P < 0.001]. BRAF mutation was also associated with inferior PFS [multivariate HR: 2.19 (1.59-3.02), P < 0.001] and OS [multivariate HR: 2.99 (2.10-4.25), P < 0.001]. Among specific KRAS mutation variants, the KRAS G12C-variant (n = 28) correlated with inferior OS compared with unmutated tumors [multivariate HR 2.26 (1.25-4.1), P = 0.001]. A similar trend for OS was seen in the KRAS G13D-variant [n = 71, multivariate HR 1.46 (0.96-2.22), P = 0.10]. More frequent KRAS exon 2 variants like G12D [n = 152, multivariate HR 1.17 (0.86-1.6), P = 0.81] and G12V [n = 92, multivariate HR 1.27 (0.87-1.86), P = 0.57] did not have significant impact on OS. CONCLUSION: Mutations in KRAS and BRAF were associated with inferior PFS and OS of mCRC patients compared with patients with non-mutated tumors. KRAS exon 2 mutation variants were associated with heterogeneous outcome compared with unmutated tumors with KRAS G12C and G13D (trend) being associated with rather poor survival.

Ultrastructural and molecular characterization of Pneumocystis carinii isolated from a rhesus monkey (Macaca mulatta).

High levels of heterogeneity have been observed among isolates of Pneumocystis carinii derived from different mammalian host species. We report the characterization of P. carinii isolated from a rhesus monkey (Macaca mulatta), which was immunosuppressed as a result of infection with a chimeric simian-human immunodeficiency virus (SHIVsbg). Histopathological examination showed evidence of severe P. carinii pneumonia with a large predominance of trophozoite forms. Alveolitis consisted of typical foamy, honeycomb exudate, with only a few alveolar macrophages. The lung inflammatory response was rather moderate without type-2 pneumocyte hyperplasia or collagenosis. P. carinii organisms were sometimes observed in the bronchiolar lumen. Ultrastructurally, macaque-derived P. carinii was more similar to human- or rabbit-derived parasites than to mouse-derived P. carinii. Molecular studies were carried out on the macaque-derived P. carinii DNA at two genetic loci: the genes encoding the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) and the mitochondrial small subunit ribosomal RNA (mt SSU rRNA). Comparison of the DNA sequences with those from P. carinii isolated from eight other host species demonstrated that the macaque-derived P. carinii was genetically distinct at both loci, and was more closely related to human-derived P. carinii than to P. carinii derived from non-primate sources. We propose that macaque-derived P. carinii be named Pneumocystis carinii f.sp. macacae.

VCAM-1 is internalized by a clathrin-related pathway in human endothelial cells but its alpha 4 beta 1 integrin counter-receptor remains associated with the plasma membrane in human T lymphocytes.

Lymphocyte extravasation involves a step(s) of de-adhesion to allow trans- and subendothelial migration in response to inflammatory signals. We show here that ligated VCAM-1 was rapidly internalized (t1/2 14.5 min) in ECV 304 endothelial cells and in TNF-alpha-primed human umbilical vein-derived endothelial cells (t1/2 11.2 min). The process required energy (ATP), intracellular Ca2+, an intact cytoskeletal network and active protein kinases. The internalization of VCAM-1 involved a clathrin-dependent pathway based on the observations that 1) it was inhibited in cells treated with lysosomotropic agents or with a hypertonic concentration of sucrose, and 2) internalized VCAM-1 colocalized with clathrin. In contrast, the cross-linked alpha 4 beta 1 integrin counter-receptor of VCAM-1 remained associated with the plasma membrane of purified peripheral T and Jurkat cells. Our results suggest a model where VCAM-1 would initially participate in the retention of T cells to the endothelium by binding alpha 4 beta 1 integrin. Lymphocyte de-adhesion would be facilitated as a result of the internalization of VCAM-1. The persistent cell surface expression of alpha 4 beta 1 integrin would allow the migrating T cells to interact with and receive signal(s) from its fibronectin ligand of the extracellular matrix.

Clustering the adhesion molecules VLA-4 (CD49d/CD29) in Jurkat T cells or VCAM-1 (CD106) in endothelial (ECV 304) cells activates the phosphoinositide pathway and triggers Ca2+ mobilization.

Ligation of very late antigen (VLA)-4 (alpha 4 beta 1 integrin) with a cross-linked anti-alpha 4 subunit monoclonal antibody (mAb) triggered a biphasic Ca2+ response in Jurkat cell populations and in peripheral human lymphocytes. Cross-linking vascular cell adhesion molecule (VCAM)-1 (the counter-receptor of VLA-4) in ECV 304 endothelial cells generated a biphasic Ca2+ response. Tumor necrosis factor-alpha-primed human umbilical cord vascular endothelial cells also responded to the cross-linked mAb with a biphasic Ca2+ profile. Ligated VLA-4 (Jurkat cells) or VCAM-1 (ECV 304) stimulated the production of myo-inositol 1,4,5-trisphosphate. ECV 304 cells induced a biphasic Ca2+ response in Fura2-loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2-loaded ECV 304 cells. The Ca2+ responses in these experiments involved VLA-4/VCAM-1 interactions since they were significantly reduced (approximately 80%) by prior treatment of the target cells with the relevant noncross-linked mAb. Close contact between the cells triggered mutual Ca2+ signaling as shown by spectrofluorimetric and confocal microscopy time-dependent recordings. Fibronectin and its CS-1 fragment (V25) triggered a sustained Ca2+ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA-4 and VCAM-1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca2+.

A caffeine/ryanodine-sensitive Ca2+ pool is involved in triggering spontaneous variations of Ca2+ in Jurkat T lymphocytes by a Ca(2+)-induced Ca2+ release (CICR) mechanism.

Caffeine and ryanodine triggered an increase in [Ca2+]i (73 +/- 22 and 61 +/- 18 nM, respectively) in Jurkat cell populations that was independent of external Ca2+. In individual cells, caffeine and ryanodine induced Ca2+ spikes. Jurkat cell populations initially exposed to caffeine did not respond further to ryanodine and vice versa, suggesting an overlap of the Ca2+ pool that was contained within the thapsigargin-sensitive Ca2+ reserve. [3H]ryanodine bound to a single class of sites of Jurkat microsomes (KD, 18.4 +/- 5.7 nM; Bmax, 24.3 +/- 7.7 fmol/mg protein). Photolytic release (Nitr5) of caged Ca2+ induced a time-dependent increase of Ca2+ in individual Jurkat cells. The profile of the release of Ca2+ was characterized, 1) by a kinetic (0.55 +/- 0.07 nM s-1) slower than the Ca2+ response to caffeine (3.93 +/- 0.66 nM s-1) or to ryanodine (3.96 +/- 0.94 nM s-1), 2) by a release of Ca2+ (131 +/- 43 nM) that slowly returned to baseline and during which low amplitude oscillations were present (room temperature) or Ca2+ spikes (37 degrees C) and, 3) by a lack of dependency on an influx of Ca2+. Inhibitors of CICR (ruthenium red and 1-octanol) prevented the photolysis-dependent increase in [Ca2+]i but not the InsP3-dependent Ca2+ response. Our data suggest that Jurkat T cells possess at least two Ca2+ pools, one that is sensitive to InsP3 and one that is insensitive. These two Ca2+ pools may be involved in a CICR that generates spontaneous Ca2+ spikes and oscillations in these cells.

Effects of modulators of cytosolic Ca2+ on phytohemagglutin-dependent Ca2+ response and interleukin-2 production in Jurkat cells.

We have previously reported the presence, in Jurkat T cells, of outward K+ currents and inward currents that have been attributed to Ca2+ channels. Here, we have studied the effects of dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine-dicarboxylate (nifedipine) and 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxy-carbonylpyridine-3-carboxylate (PN200-110), two dihydropyridines (DHPs) known to inhibit voltage-dependent Ca2+ channel activity in different types of cells, and two inhibitors of internal Ca2+ release (muscle cells), ryanodine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), on the Phaseolus vulgaris phytohemagglutinin (PHA)-dependent responses in Jurkat T lymphocytes. Our results show that nifedipine and PN200-110 inhibit the PHA-dependent production of interleukin-2 except when 12-O-tetradecanoyl-13-O-acetyl phorbol is added to the cultures. Ryanodine and TMB-8 are not inhibitors. The PHA-dependent Ca2+ response is significantly reduced when the cells are preincubated in the presence of the DHPs. Under these conditions, ryanodine has only a small inhibitory effect and TMB-8 has no effect. In contrast, only ryanodine (50 microM) decreases the PHA-dependent cytosolic release of Ca2+i when the cells are bathed in a medium containing a low concentration of Ca2+ (60 nM). The inhibitory effects of nifedipine and PN200-110 may result from the binding of these DHPs to specific receptor sites as revealed by studies using [3H]PN200-110 (KD = 8.5 +/- 3.1 nM; 2300 +/- 500 apparent binding sites/cell). Photoaffinity labeling studies using [3H]azidopine as a probe showed specific incorporation of label into three glycoproteins of molecular mass (+/- SD) 170 +/- 13, 110 +/- 25, and 60 +/- 17 kd as analyzed by electrophoresis under reducing conditions.