Fludarabine, cyclophosphamide, and rituximab as first-line treatment in patients with chronic lymphocytic leukemia: A long-term analysis of the German CLL Study Group (GCLLSG) registry.

Long-term data of chronic lymphocytic leukemia (CLL) patients with favorable risk who were treated with fludarabine, cyclophosphamide, and rituximab (FCR) within clinical trials show good efficacy. We here report long-term data collected within the GCLLSG registry. Altogether, 417 CLL patients who received first-line treatment with FCR were analyzed, of which 293 (70.3%) were treated outside of clinical trials. The median observation time from first-line was 95.8 (interquartile range 58.7-126.8) months. Focusing on data of 194 (46.5%) patients who received FCR first-line treatment after 2013 (start of data collection within GCLLSG registry), responses were documented in 85% of the patients, non-responses in 15%, and for 3.6% the assessment was missing. Median event-free survival (EFS, time until disease progression, subsequent treatment, or death) was 60.2 months with a 5-year EFS-rate of 50.6%. Patients with higher-risk disease, characterized by unmutated IGHV (N = 78), had a median EFS of 45.4 months with a 5-year EFS rate of 36.3%, while the median EFS was 77.5 months with a 5-year EFS rate of 60.3% in patients with mutated IGHV (N = 40). Median overall survival was not reached with a 5-year survival rate of 92.7%. In summary, first-line FCR was associated with long EFS, especially in patients exhibiting a mutated IGHV status.

Clinical outcome after total neoadjuvant treatment (CAO/ARO/AIO-12) versus intensified neoadjuvant and adjuvant treatment (CAO/ARO/AIO-04) a comparison between two multicenter randomized phase II/III trials.

BACKGROUND: Total neoadjuvant therapy (TNT) can enhance local tumor regression, but its survival benefits compared to intensified chemoradiotherapy (CRT) followed by adjuvant chemotherapy (CT) remain unclear. METHODS: This is a secondary comparison between 607 patients treated with intensified 5-FU/Oxaliplatin neoadjuvant CRT and adjuvant CT within the experimental arm of the CAO/ARO/AIO-04 phase III trial, and 306 patients treated with TNT within the CAO/ARO/AIO-12 phase II trial. Comparison between clinical-pathological characteristics, surgical quality, and post-surgical complications were analyzed using the Pearson's Chi-squared or Mann-Whitney U test. Oncological outcome was examined with log-rank, Gray's test, and multivariate cox regression. In addition, further subgroup analyses and propensity score matching were performed to optimize the balance of baseline covariates. FINDINGS: Patients treated with CRT followed by consolidation CT had a significantly higher rate of pathological complete remission (pCR) compared to patients treated within the experimental arm of the CAO/ARO/AIO-04 trial (25.3 % vs 17.3 %, P = 0.04). Post-surgical complications were less common in the CAO/ARO/AIO-12 trial. After a median follow-up of 46 months, clinical outcome did not differ significantly in the overall cohort, in any subgroup or after propensity score matching. In multivariate analysis, disease-free survival (DFS) was similar between the experimental arm of the CAO/ARO/AIO-04 trial and treatments arms of the CAO/ARO/AIO-12 trial (vs arm A: HR 0.92 [95 % CI 0.62-1.37], P = 0.69; vs arm B: HR 1.06 [95 % CI 0.72-1.58], P = 0.76). INTERPRETATION: Notwithstanding the limitations of intertrial comparison, TNT did not improve long term oncological outcome in our study compared to the intensified neoadjuvant CRT and adjuvant CT treatment in the CAO/ARO/AIO-04 trial. Improved response rates after TNT offers an attractive option to explore organ preservation in selective patients with locally advanced rectal cancer.

Total Neoadjuvant Therapy for Rectal Cancer in the CAO/ARO/AIO-12 Randomized Phase 2 Trial: Early Surrogate Endpoints Revisited.

BACKGROUND: Early efficacy outcome measures in rectal cancer after total neoadjuvant treatment are increasingly investigated. We examined the prognostic role of pathological complete response (pCR), tumor regression grading (TRG) and neoadjuvant rectal (NAR) score for disease-free survival (DFS) in patients with rectal carcinoma treated within the CAO/ARO/AIO-12 randomized phase 2 trial. METHODS: Distribution of pCR, TRG and NAR score was analyzed using the Pearson's chi-squared test. Univariable analyses were performed using the log-rank test, stratified by treatment arm. Discrimination ability of non-pCR for DFS was assessed by analyzing the ROC curve as a function of time. RESULTS: Of the 311 patients enrolled, 306 patients were evaluable (Arm A:156, Arm B:150). After a median follow-up of 43 months, the 3-year DFS was 73% in both groups (HR, 0.95, 95% CI, 0.63-1.45, p = 0.82). pCR tended to be higher in Arm B (17% vs. 25%, p = 0.086). In both treatment arms, pCR, TRG and NAR were significant prognostic factors for DFS, whereas survival in subgroups defined by pCR, TRG or NAR did not significantly differ between the treatment arms. The discrimination ability of non-pCR for DFS remained constant over time (C-Index 0.58) but was slightly better in Arm B (0.61 vs. 0.56). CONCLUSION: Although pCR, TRG and NAR were strong prognostic factors for DFS in the CAO/ARO/AIO-12 trial, their value in selecting one TNT approach over another could not be confirmed. Hence, the conclusion of a long-term survival benefit of one treatment arm based on early surrogate endpoints should be stated with caution.

Chemoradiotherapy Plus Induction or Consolidation Chemotherapy as Total Neoadjuvant Therapy for Patients With Locally Advanced Rectal Cancer: Long-term Results of the CAO/ARO/AIO-12 Randomized Clinical Trial.

IMPORTANCE: Total neoadjuvant therapy has been increasingly adopted for multimodal rectal cancer treatment. The optimal sequence of chemoradiotherapy (CRT) and chemotherapy needs to be established. OBJECTIVE: To report the long-term results of the secondary end points prespecified in the Randomized Phase 2 Trial of Chemoradiotherapy Plus Induction or Consolidation Chemotherapy as Total Neoadjuvant Therapy (CAO/ARO/AIO-12 trial) for Locally Advanced Rectal Cancer. DESIGN, SETTING, AND PARTICIPANTS: This secondary analysis of a randomized clinical trial included 311 patients who were recruited from the accrued CAO/ARO/AIO-12 trial population from June 15, 2015, to January 31, 2018, from 18 centers in Germany. Patients with cT3-4 and/or node-positive rectal adenocarcinoma were included in the analysis. Data were analyzed from June 15, 2015, to January 31, 2018. The follow-up analysis was conducted between January 31, 2018, and November 30, 2020. INTERVENTIONS: Patients were randomly assigned to group A for 3 cycles of fluorouracil, leucovorin, and oxaliplatin before fluorouracil/oxaliplatin CRT (50.4 Gy), or to group B for CRT before chemotherapy. Total mesorectal excision was scheduled on day 123 after the start of total neoadjuvant therapy in both groups. MAIN OUTCOMES AND MEASURES: The end points assessed in this secondary analysis included long-term oncologic outcomes, chronic toxicity, patient-reported outcome measures for global health status (GHS) and quality of life (QoL), and the Wexner stool incontinence score. RESULTS: Of the 311 patients enrolled, 306 were evaluable, including 156 in group A (mean [SD] age, 60 [11] years; 106 men [68%]) and 150 in group B (mean [SD] age, 62 [10] years; 100 men [67%]). After a median follow-up of 43 months (range, 35-60 months), the 3-year disease-free survival was 73% in both groups (hazard ratio, 0.95; 95% CI, 0.63-1.45, P = .82); the 3-year cumulative incidence of locoregional recurrence (6% vs 5%, P = .67) and distant metastases (18% vs 16%, P = .52) were not significantly different. Chronic toxicity grade 3 to 4 occurred in 10 of 85 patients (11.8%) in group A and 8 of 66 patients (9.9%) in group B at 3 years. The GHS/QoL score decreased after total mesorectal excision but returned to pretreatment levels 1 year after randomization with no difference between the groups. Stool incontinence deteriorated 1 year after randomization in both groups and only improved slightly at 3 years, but never reached baseline levels. CONCLUSIONS AND RELEVANCE: This secondary analysis of a randomized clinical trial showed that CRT followed by chemotherapy resulted in higher pathological complete response without compromising disease-free survival, toxicity, QoL, or stool incontinence and is thus proposed as the preferred total neoadjuvant therapy sequence if organ preservation is a priority. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02363374.

Single-cell microRNA sequencing method comparison and application to cell lines and circulating lung tumor cells.

Molecular single cell analyses provide insights into physiological and pathological processes. Here, in a stepwise approach, we first evaluate 19 protocols for single cell small RNA sequencing on MCF7 cells spiked with 1 pg of 1,006 miRNAs. Second, we analyze MCF7 single cell equivalents of the eight best protocols. Third, we sequence single cells from eight different cell lines and 67 circulating tumor cells (CTCs) from seven SCLC patients. Altogether, we analyze 244 different samples. We observe high reproducibility within protocols and reads covered a broad spectrum of RNAs. For the 67 CTCs, we detect a median of 68 miRNAs, with 10 miRNAs being expressed in 90% of tested cells. Enrichment analysis suggested the lung as the most likely organ of origin and enrichment of cancer-related categories. Even the identification of non-annotated candidate miRNAs was feasible, underlining the potential of single cell small RNA sequencing.

Durvalumab after definitive chemoradiotherapy in locally advanced NSCLC: Data of the German EAP.

Following the PACIFIC trial, durvalumab has been approved by the European Medicines Agency (EMA) for consolidation of locally advanced PD-L1-positive NSCLC after chemoradiotherapy (CRT). Patients were treated with durvalumab in the EAP from 22.11.2017 to 15.10.2018 allowing analysis of its efficacy and safety. 211 patients were registered by 90 German centres. Data were collected retrospectively by questionnaire and queries. 56 centres reported data on 126 patients who actually received at least one cycle of durvalumab. In contrast to the PACIFIC-trial population, some patients with oligometastatic disease and a history of autoimmune disease are included in the EAP population. Information on PD-L1 status was obtained for 111 patients. Baseline data include age, gender, ECOG, stage (IASLC 8th ed.), and smoking history. Treatment data include mode of chemoradiotherapy, used chemotherapy agent, and duration of durvalumab therapy. Adverse evants were documented according to CTAEC 5.0. Data were analysed for progression-free survival (PFS), overall survival (OS), and adverse events (AE). The results were published in Lung Cancer [1].

Durvalumab after definitive chemoradiotherapy in locally advanced unresectable non-small cell lung cancer (NSCLC): Real-world data on survival and safety from the German expanded-access program (EAP).

BACKGROUND: Following the PACIFIC trial, durvalumab has been approved by the European Medicines Agency (EMA) for consolidation of locally advanced PD-L1-positive NSCLC after chemoradiotherapy (CRT). Patients were treated with durvalumab in the EAP from 22.11.2017 to 15.10.2018 allowing analysis of its efficacy and safety. METHODS: Data from 56 centres were analysed for adverse events (AE), progression-free survival (PFS), overall survival (OS). RESULTS: 126 patients actually received at least 1 cycle durvalumab. Compared to the PACIFIC trial, the EAP population had more advanced stage and included "oligometastatic" stage IV patients and patients with autoimmune disease. PFS (20.1 months) and OS (not reached) were similar in the EAP and the PACIFIC trial. 42.9 % completed 12 months of durvalumab without deaths during FU. Stage IV patients (n = 7) had encouraging OS (not reached at 27 months). Autoimmune disease did not affect survival. PFS and OS were similar in PD-L1-negative patients (n = 32) and PD-L1-positive patients (n = 79). CONCLUSIONS: Survival in the EAP was comparable to the PACIFIC trial. Selected stage IV patients and patients with autoimmune disease may benefit from durvalumab consolidation and should be included in future immuno-oncological trials. PD-L1 did not predict survival challenging the exclusion of PD-L1-negative patients from durvalumab consolidation. In summary, durvalumab consolidation is safe and effective in a European real-world setting.

Randomized Phase II Trial of Chemoradiotherapy Plus Induction or Consolidation Chemotherapy as Total Neoadjuvant Therapy for Locally Advanced Rectal Cancer: CAO/ARO/AIO-12.

PURPOSE: Total neoadjuvant therapy is a new paradigm for rectal cancer treatment. Optimal scheduling of preoperative chemoradiotherapy (CRT) and chemotherapy remains to be established. PATIENTS AND METHODS: We conducted a multicenter, randomized, phase II trial using a pick-the-winner design on the basis of the hypothesis of an increased pathologic complete response (pCR) of 25% after total neoadjuvant therapy compared with standard 15% after preoperative CRT. Patients with stage II or III rectal cancer were assigned to group A for induction chemotherapy using three cycles of fluorouracil, leucovorin, and oxaliplatin before fluorouracil/oxaliplatin CRT (50.4 Gy) or to group B for consolidation chemotherapy after CRT. Secondary end points included toxicity, compliance, and surgical morbidity. RESULTS: Of the 311 patients enrolled, 306 patients were evaluable (156 in group A and 150 in group B). CRT-related grade 3 or 4 toxicity was lower (37% v 27%) and compliance with CRT higher in group B (91%, 78%, and 76% v 97%, 87%, and 93% received full-dose radiotherapy, concomitant fluorouracil, and concomitant oxaliplatin in groups A and B, respectively); 92% versus 85% completed all induction/consolidation chemotherapy cycles, respectively. The longer interval between completion of CRT and surgery in group B (median 90 v 45 days in group A) did not increase surgical morbidity. A pCR in the intention-to-treat population was achieved in 17% in group A and in 25% in group B. Thus, only group B (P < .001), but not group A (P = .210), fulfilled the predefined statistical hypothesis. CONCLUSION: Up-front CRT followed by chemotherapy resulted in better compliance with CRT but worse compliance with chemotherapy compared with group A. Long-term follow-up will assess whether improved pCR in group B translates to better oncologic outcome.

Oxaliplatin added to fluorouracil-based preoperative chemoradiotherapy and postoperative chemotherapy of locally advanced rectal cancer (the German CAO/ARO/AIO-04 study): final results of the multicentre, open-label, randomised, phase 3 trial.

BACKGROUND: Preoperative chemoradiotherapy with infusional fluorouracil, total mesorectal excision surgery, and postoperative chemotherapy with fluorouracil was established by the German CAO/ARO/AIO-94 trial as a standard combined modality treatment for locally advanced rectal cancer. Here we compare the previously established regimen with an investigational regimen in which oxaliplatin was added to both preoperative chemoradiotherapy and postoperative chemotherapy. METHODS: In this multicentre, open-label, randomised, phase 3 study we randomly assigned patients with rectal adenocarcinoma, clinically staged as cT3-4 or any node-positive disease, to two groups: a control group receiving standard fluorouracil-based combined modality treatment, consisting of preoperative radiotherapy of 50·4 Gy in 28 fractions plus infusional fluorouracil (1000 mg/m(2) on days 1-5 and 29-33), followed by surgery and four cycles of bolus fluorouracil (500 mg/m(2) on days 1-5 and 29); or to an investigational group receiving preoperative radiotherapy of 50·4 Gy in 28 fractions plus infusional fluorouracil (250 mg/m(2) on days 1-14 and 22-35) and oxaliplatin (50 mg/m(2) on days 1, 8, 22, and 29), followed by surgery and eight cycles of oxaliplatin (100 mg/m(2) on days 1 and 15), leucovorin (400 mg/m(2) on days 1 and 15), and infusional fluorouracil (2400 mg/m(2) on days 1-2 and 15-16). Randomisation was done with computer-generated block-randomisation codes stratified by centre, clinical T category (cT1-3 vs cT4), and clinical N category (cN0 vs cN1-2) without masking. The primary endpoint was disease-free survival, defined as the time between randomisation and non-radical surgery of the primary tumour (R2 resection), locoregional recurrence after R0/1 resection, metastatic disease or progression, or death from any cause, whichever occurred first. Survival and cumulative incidence of recurrence analyses followed the intention-to-treat principle; toxicity analyses included all patients treated. Enrolment of patients in this trial is completed and follow-up is ongoing. This study is registered with ClinicalTrials.gov, number NCT00349076. FINDINGS: Of the 1265 patients initially enrolled, 1236 were assessable (613 in the investigational group and 623 in the control group). With a median follow-up of 50 months (IQR 38-61), disease-free survival at 3 years was 75·9% (95% CI 72·4-79·5) in the investigational group and 71·2% (95% CI 67·6-74·9) in the control group (hazard ratio [HR] 0·79, 95% CI 0·64-0·98; p=0·03). Preoperative grade 3-4 toxic effects occurred in 144 (24%) of 607 patients who actually received fluorouracil and oxaliplatin during chemoradiotherapy and in 128 (20%) of 625 patients who actually received fluorouracil chemoradiotherapy. Of 445 patients who actually received adjuvant fluorouracil and leucovorin and oxaliplatin, 158 (36%) had grade 3-4 toxic effects, as did 170 (36%) of 470 patients who actually received adjuvant fluorouracil. Late grade 3-4 adverse events in patients who received protocol-specified preoperative and postoperative treatment occurred in 112 (25%) of 445 patients in the investigational group, and in 100 (21%) of 470 patients in the control group. INTERPRETATION: Adding oxaliplatin to fluorouracil-based neoadjuvant chemoradiotherapy and adjuvant chemotherapy (at the doses and intensities used in this trial) significantly improved disease-free survival of patients with clinically staged cT3-4 or cN1-2 rectal cancer compared with our former fluorouracil-based combined modality regimen (based on CAO/ARO/AIO-94). The regimen established by CAO/ARO/AIO-04 can be deemed a new treatment option for patients with locally advanced rectal cancer. FUNDING: German Cancer Aid (Deutsche Krebshilfe).

Preoperative chemoradiotherapy and postoperative chemotherapy with fluorouracil and oxaliplatin versus fluorouracil alone in locally advanced rectal cancer: initial results of the German CAO/ARO/AIO-04 randomised phase 3 trial.

BACKGROUND: Preoperative chemoradiotherapy, total mesorectal excision surgery, and adjuvant chemotherapy with fluorouracil is the standard combined modality treatment for rectal cancer. With the aim of improving disease-free survival (DFS), this phase 3 study (CAO/ARO/AIO-04) integrated oxaliplatin into standard treatment. METHODS: This was a multicentre, open-label, randomised, phase 3 study in patients with histologically proven carcinoma of the rectum with clinically staged T3-4 or any node-positive disease. Between July 25, 2006, and Feb 26, 2010, patients were randomly assigned to two groups: a control group receiving standard fluorouracil-based combined modality treatment, consisting of preoperative radiotherapy of 50·4 Gy plus infusional fluorouracil (1000 mg/m(2) days 1-5 and 29-33), followed by surgery and four cycles of bolus fluorouracil (500 mg/m(2) days 1-5 and 29; fluorouracil group); and an experimental group receiving preoperative radiotherapy of 50·4 Gy plus infusional fluorouracil (250 mg/m(2) days 1-14 and 22-35) and oxaliplatin (50 mg/m(2) days 1, 8, 22, and 29), followed by surgery and eight cycles of adjuvant chemotherapy with oxaliplatin (100 mg/m(2) days 1 and 15), leucovorin (400 mg/m(2) days 1 and 15), and infusional fluorouracil (2400 mg/m(2) days 1-2 and 15-16; fluorouracil plus oxaliplatin group). Randomisation was done with computer-generated block-randomisation codes stratified by centre, clinical T category (cT1-4 vs cT4), and clinical N category (cN0 vs cN1-2) without masking. DFS is the primary endpoint. Secondary endpoints, including toxicity, compliance, and histopathological response are reported here. Safety and compliance analyses included patients as treated, efficacy endpoints were analysed according to the intention-to-treat principle. This study is registered with ClinicalTrials.gov, number NCT00349076. FINDINGS: Of the 1265 patients initially enrolled, 1236 were evaluable (613 in the fluorouracil plus oxaliplatin group and 623 in the fluorouracil group). Preoperative grade 3-4 toxic effects occurred in 140 (23%) of 606 patients who actually received fluorouracil and oxaliplatin during chemoradiotherapy and in 127 (20%) of 624 patients who actually received fluorouracil chemoradiotherapy. Grade 3-4 diarrhoea was more common in those who received fluorouracil and oxaliplatin during chemoradiotherapy than in those who received fluorouracil during chemoradiotherapy (73 patients [12%] vs 52 patients [8%]), as was grade 3-4 nausea or vomiting (23 [4%] vs nine [1%]). 516 (85%) of the 606 patients who received fluorouracil and oxaliplatin-based chemoradiotherapy had the full dose of chemotherapy, and 571 (94%) had the full dose of radiotherapy; as did 495 (79%) and 601 (96%) of 624 patients who received fluorouracil-based chemoradiotherapy, respectively. A pathological complete response was achieved in 103 (17%) of 591 patients who underwent surgery in the fluorouracil and oxaliplatin group and in 81 (13%) of 606 patients who underwent surgery in the fluorouracil group (odds ratio 1·40, 95% CI 1·02-1·92; p=0·038). In the fluorouracil and oxaliplatin group, 352 (81%) of 435 patients who began adjuvant chemotherapy completed all cycles (with or without dose reduction), as did 386 (83%) of 463 patients in the fluorouracil group. INTERPRETATION: Inclusion of oxaliplatin into modified fluorouracil-based combined modality treatment was feasible and led to more patients achieving a pathological complete response than did standard treatment. Longer follow-up is needed to assess DFS. FUNDING: German Cancer Aid (Deutsche Krebshilfe).

Cell proliferation and migration in glioblastoma multiforme cell lines are influenced by insulin-like growth factor I in vitro.

BACKGROUND: Malignant gliomas continue to be a therapeutic challenge. One of the major problems is the early and rapidly infiltrating tumor growth. The role of the insulinlike growth factor (IGF) system in the progression of malignant glioma growth is poorly understood. In this study we investigated the expression of different members of the IGF system in malignant glioma cells and the influence of IGF-I and -II on the proliferation and migration of human glioma cell lines in vitro. MATERIALS AND METHODS: Expression of IGF-I and -II, IGF-receptor I and II and IGF binding proteins (IGFBP) 1 to 6 was analysed by PCR in cell lines T98G, A172, 86HG39 (glioblastoma multiforme) and U87MG (anaplastic astrocytoma). To investigate effects on cell-proliferation, the cells were treated with IGF-I or -II (0.001-100 ng/ml). The cell viability was assessed by MTT-assay. For testing migratory effects, the Boyden-chamber-assay with different combinations of IGF-I or -II or fetal calf serum (FCS) as chemotactic agents was used. RESULTS: All cell lines expressed IGF-I- and IGF-II-receptor, whereas none of the cells expressed IGF-I or -II. IGFBP 2-6 were found in all cell lines. IGF-I treated cell lines T98G and 86HG39 showed a weak dose-independent enhanced proliferation compared to controls, whereas A172 did not respond. None of the investigated cell lines changed proliferation when treated with IGF-II. All IGF-I (100 ng/ml) treated cells showed increased migration compared to controls. This effect was markedly enhanced by supplementation with 0.5% FCS. Again, IGF-II had no effect. CONCLUSION: These data demonstrate that IGF-I modulates proliferation and strongly stimulates migration of glioma cell lines in vitro.