Time dependent effect of cold ischemia on the phosphoproteome and protein kinase activity in fresh-frozen colorectal cancer tissue obtained from patients.

BACKGROUND: Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. METHODS: Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. RESULTS: Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p < 0.01 and fold change > 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p < 0.01). Only two phosphopeptides were significantly changed in all time points, including one peptide (PKP3) with a fold change > 2. CONCLUSIONS: The vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.

The influence of micrometastases on prognosis and survival in stage I-II colon cancer patients: the Enroute⊕ Study.

BACKGROUND: The presence of lymph node metastases remains the most reliable prognostic predictor and the gold indicator for adjuvant treatment in colon cancer (CC). In spite of a potentially curative resection, 20 to 30% of CC patients testing negative for lymph node metastases (i.e. pN0) will subsequently develop locoregional and/or systemic metastases within 5 years. The presence of occult nodal isolated tumor cells (ITCs) and/or micrometastases (MMs) at the time of resection predisposes CC patients to high risk for disease recurrence. These pN0(micro+) patients harbouring occult micrometastases may benefit from adjuvant treatment. The purpose of the present study is to delineate the subset of pN0 patients with micrometastases (pN0(micro+)) and evaluate the benefits from adjuvant chemotherapy in pN0(micro+) CC patients. METHODS/DESIGN: EnRoute+ is an open label, multicenter, randomized controlled clinical trial. All CC patients (age above 18 years) without synchronous locoregional lymph node and/or systemic metastases (clinical stage I-II disease) and operated upon with curative intent are eligible for inclusion. All resected specimens of patients are subject to an ex vivo sentinel lymph node mapping procedure (SLNM) following curative resection. The investigation for micrometastases in pN0 patients is done by extended serial sectioning and immunohistochemistry for pan-cytokeratin in sentinel lymph nodes which are tumour negative upon standard pathological examination. Patients with ITC/MM-positive sentinel lymph nodes (pN0(micro+)) are randomized for adjuvant chemotherapy following the CAPOX treatment scheme or observation. The primary endpoint is 3-year disease free survival (DFS). DISCUSSION: The EnRoute+ study is designed to improve prognosis in high-risk stage I/II pN0(micro+) CC patients by reducing disease recurrence by adjuvant chemotherapy. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01097265.

Poor compliance with perioperative chemotherapy for resectable gastric cancer and its impact on survival.

BACKGROUND: In several Western European countries it is recommended to treat gastric cancer patients with perioperative chemotherapy if they are eligible for surgery. However, little is known about its use in daily clinical practice. This study examines the use of perioperative treatment and its impact on survival in the Netherlands. METHODS: Patients diagnosed with potentially resectable gastric cancer (cT1N+/cT2-T3,X any cN, cM0,X) between 2006 and 2014 were selected from the Netherlands Cancer Registry (N = 5824). Treatment trends were examined. Propensity score matching was used to create a subsample to reduce selection bias. Cox regression analysis was used to assess differences in overall survival. RESULTS: The percentage of patients treated with perioperative treatment increased from 3% in 2006 to 26% in 2014 and the use of only surgery decreased from 60% to 26%. 35% of all patients did not undergo surgery. Of the patients who underwent preoperative chemotherapy and surgery, 43% did not commence postoperative treatment. Cox regression analysis showed a better overall survival for patients who underwent perioperative treatment compared to patients who underwent preoperative treatment only (HR = 0.80 95%CI 0.70-0.93; propensity matched sample: HR = 0.84 95%CI 0.71-0.99), whereas survival was comparable for patients who underwent preoperative chemotherapy versus surgery alone (HR = 0.89 95%CI 0.77-1.02, propensity matched sample: HR = 0.85 95%CI 0.72-1.01). CONCLUSION: This population-based study highlights that a significant proportion of the patients did not receive perioperative treatment. More research is necessary to elucidate the importance of the individual components of perioperative treatment.

The impact of age on first-line systemic therapy in patients with metachronous metastases from colorectal cancer.

OBJECTIVES: The paucity of evidence for the optimal use of systemic therapy in elderly patients with metastatic colorectal cancer (mCRC) poses significant challenges to cancer specialists. The present population-based study provides insight into the impact of age on palliative systemic therapy in patients with metachronous metastases from CRC, in order to optimize the decision-making process. METHODS: Data on the development and treatment of metachronous metastases were collected for patients with primary resected CRC diagnosed between 2003 and 2008 in the Eindhoven area of the Netherlands Cancer Registry. Patients undergoing surgery for metastases were excluded, resulting in a study population treated with palliative intent, with or without systemic therapy (n=746). RESULTS: 385 patients received palliative systemic therapy (52%). Patients aged ≥75years were less likely to receive systemic therapy (31% ≥75years vs 73% <60years) and more likely to receive single-agent chemotherapy than combination-chemotherapy. Elderly patients (≥75years) treated with capecitabine-oxaliplatin (CAPOX) received fewer cycles (51% ≤3 oxaliplatin cycles, 43% ≤3 capecitabine cycles) and lower cumulative dosages compared to patients aged <75years, although initial dosages were similar. If capecitabine monotherapy (CapMono) was administered, starting dosages were 2414mg/m2/d<75years and 1992mg/m2/d≥75years (p<0.05), but no differences in number of received cycles or cumulative dosages were observed. CONCLUSION: Age beginning at 75years significantly influenced palliative systemic therapy. Even in selected elderly patients, first-line treatment with CAPOX was associated with less cycles and lower cumulative dosages compared to younger patients. With single-agent fluoropyrimidine therapy, however, no such results were observed.

Successful Transfer of Umbilical Cord Blood CD34+ Hematopoietic Stem and Progenitor-derived NK Cells in Older Acute Myeloid Leukemia Patients.

Purpose: Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units.Experimental Design: Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 106/kg body weight) after lymphodepleting chemotherapy without cytokine boosting.Results: HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1 × 104 T cells/kg and <3 × 105 B cells/kg body weight. HSPC-NK cells were well tolerated, and neither graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting being given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, which was accompanied by augmented IL15 plasma levels. Moreover, donor chimerism up to 3.5% was found in bone marrow. Interestingly, in vivo HSPC-NK cell maturation was observed, indicated by the rapid acquisition of CD16 and KIR expression, while expression of most activating receptors was sustained. Notably, 2 of 4 patients with minimal residual disease (MRD) in bone marrow before infusion became MRD negative (<0.1%), which lasted for 6 months.Conclusions: These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential "off-the-shelf" translational immunotherapy approach in AML. Clin Cancer Res; 23(15); 4107-18. ©2017 AACR.

Deciding on adjuvant chemotherapy for elderly patients with stage III colon cancer: a qualitative insight into the perspectives of surgeons and medical oncologists.

OBJECTIVE: The aim of this study is to identify doctor-related factors determining the decision-making for adjuvant chemotherapy for patients with stage III colon cancer aged ≥75years. MATERIALS AND METHODS: 21 surgeons and 15 medical oncologists from 10 community hospitals were asked to complete a short questionnaire including tick-box questions regarding motives for non-referral/non-treatment, consultation of geriatricians, chemotherapy schemes prescribed and an open question regarding tolerability of chemotherapy. RESULTS: 29 medical specialists returned a completed questionnaire (response 81%). The motives for non-referral/non-treatment reported most often were comorbidity/bad general health condition of the patient; surgical complications; and treatment offered but refused by patient/family. 39% of the surgeons and 55% of the medical oncologists reported consultation of a geriatrician in 2-30% of their decisions. CAPOX and capecitabine were reported by medical oncologists as the most frequently prescribed regimens. Factors that influenced the decision for monotherapy or combination therapy were comorbidity; general health condition of the patient; and toxicity profile of the chemotherapeutics. In general, medical oncologists defined grade ≤2 toxicities as tolerable, with the exception of neuropathy, for which grade ≤1 toxicity was accepted. CONCLUSIONS: In case medical oncologists prescribe adjuvant chemotherapy to elderly patients with stage III colon cancer, the chemotherapy schemes used are in line with clinical guidelines and they agree on acceptable levels of toxicity. However, the variation among surgeons and medical oncologists in motives for non-referral, non-treatment and consultation of geriatricians when deciding on adjuvant chemotherapy for elderly patients with stage III colon cancer, shows the complexity and need for specific knowledge.