Prognostic value of disseminated tumor cells in unresectable pancreatic ductal adenocarcinoma: a prospective observational study.

BACKGROUND: Although pancreatic ductal adenocarcinoma (PDAC) rarely metastasizes to the skeleton, disseminated tumor cells have been detected in bone marrow samples from patients with this disease. The prognostic value of such findings is currently unclear. Thus, the current study aimed to clarify the prognostic information associated with disseminated tumor cell detection in samples from patients with PDAC. METHODS: Bone marrow aspirates were obtained from 48 patients with locally advanced (n = 11) or metastatic (n = 37) PDAC, before and after 2 months of chemotherapy. Disseminated tumor cells were detected with an mRNA panel and quantitative reverse transcription PCR. We used the highest levels measured in healthy bone marrow (n = 30) as a threshold to define the positive detection of disseminated tumor cells. Progression-free and overall survival were analyzed with Kaplan-Meier and Cox proportional hazards regression analyses. RESULTS: Disseminated tumor cells were detected in 15/48 (31%) bone marrow samples obtained before starting chemotherapy and in 8/25 (32%) samples obtained during chemotherapy. Patients with disseminated tumor cells detected before therapy had significantly shorter progression-free (p = 0.03; HR = 2.0) and overall survival (p = 0.03; HR = 2.0), compared to those without disseminated tumor cells in the bone marrow. When restricting disseminated tumor cell detection to keratins KRT7 and KRT8, the prognostic information was substantially stronger (p = 1 × 10-6; HR = 22, and p = 2 × 10-5; HR = 7.7, respectively). The multivariable Cox regression analysis demonstrated that disseminated tumor cell detection prior to treatment had independent prognostic value. In contrast, disseminated tumor cells detected during treatment did not have prognostic value. CONCLUSIONS: Disseminated tumor cells detected before commencing chemotherapy had prognostic value in patients with inoperable PDAC.

Fragment size and level of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer.

BACKGROUND: It was recently demonstrated that the size of cell-free DNA (cfDNA) fragments that originates from tumor cells are shorter than cfDNA fragments that originates from non-malignant cells. We investigated whether cfDNA fragment size and cfDNA levels might have prognostic value in patients with advanced pancreatic cancer. METHODS: Blood samples were obtained from patients with advanced pancreatic cancer, before (n = 61) initiation of chemotherapy and after the first cycle of chemotherapy (n = 39). Samples were separated with density centrifugation and plasma DNA was isolated. Mode cfDNA fragment size and cfDNA levels were then determined using a 2100 Bioanalyzer. A cohort of partially age-matched healthy volunteers (n = 28) constituted the control group. RESULTS: Both a pre-treatment cfDNA fragment size of ≤ 167 bp (mode) and high pre-treatment cfDNA levels were associated with shorter progression-free survival (PFS) (p = 0.002 and p < 0.001, respectively) and overall survival (OS) (p = 0.001 and p = 0.001, respectively). Furthermore, multivariable Cox regression analyses demonstrated that pre-treatment cfDNA levels could independently predict prognosis for both PFS (HR = 3.049, p = 0.005) and OS (HR = 2.236, p = 0.028). CONCLUSION: This study demonstrates that cfDNA fragment size and cfDNA levels can be used to predict disease outcome in patients with advanced pancreatic cancer. The described approach, using a rapid, economic and simple test to reveal prognostic information, has potential for future treatment stratification and monitoring.

Expression profiling and intracellular localization studies of the novel Proline-, Histidine-, and Glycine-rich protein 1 suggest an essential role in gastro-intestinal epithelium and a potential clinical application in colorectal cancer diagnostics.

BACKGROUND: The primary function of the intestines is the absorption of water and nutrients. Although our knowledge about these processes on the cellular level is extensive, a number of important intracellular elements remain unknown. Here, we characterize the novel proline-, histidine-, glycine-rich 1 (PHGR1) mRNA and protein on the molecular level and propose a functional role of the PHGR1 protein in the intestinal and gastric epithelium. METHODS: PHGR1 mRNA and protein expression in human tissues and cell lines were characterized by quantitative RT-PCR, in situ hybridization, Northern blotting, Western blotting, and immunohistochemistry. Glycosylation was assessed by a chemical deglycosylation assay, whereas intracellular localization was studied by immunofluorescent staining of cell line cells. PHGR1 mRNA levels in HT29 cells was reduced by RNA interference and the resulting global changes in gene expression assessed by microarray hybridization. RESULTS: PHGR1 mRNA and protein were found to be expressed specifically in epithelial cells of intestinal mucosa, with the highest expression in the most mature and differentiated cells. PHGR1 protein was found to be glycosylated and to localize to both the cytoplasm and nucleus. Transcript profiling and gene ontology analysis of HT29 cells subjected to PHGR1 knockdown suggested a functional relationship with transport and metabolic processes. Examination of PHGR1 mRNA and protein levels in lymph nodes with known colorectal cancer metastases indicated that they may serve as biomarkers for detection of such metastases. CONCLUSIONS: Functional analyses of the novel PHGR1 mRNA and protein suggest an essential role in gastrointestinal epithelium and a clinical application in detection of colorectal cancer lymph node metastases.

The Prognostic Relevance of Sentinel Lymph Node Metastases Assessed by PHGR1 mRNA Quantification in Stage I to III Colon Cancer.

BACKGROUND: Regional lymph node (LN) metastasis is a strong and well-established prognostic factor in colon cancer, and recent data suggest a prognostic value of detecting micrometastases and isolated tumor cells in regional LNs. The aim of the study was to investigate the clinical relevance of detecting sentinel lymph node (SLN) metastases in colon cancer patients by measuring the novel metastasis marker PHGR1 mRNA. METHODS: Using quantitative reverse-transcription polymerase chain reaction, we measured PHGR1 mRNA levels in SLNs and primary tumors from 206 patients surgically treated for stage I to III colon cancer and 52 normal LNs from patients undergoing surgery for benign colon diseases. The prognostic impact of these findings was evaluated by Kaplan-Meier analysis and Cox proportional-hazards regression. RESULTS: Compared to normal LNs, elevated PHGR1 mRNA levels were detected in SLNs from 56 (89%) of the 63 patients with pN+ disease. Furthermore, 68 (48%) of the 143 node-negative (pN0) patients had elevated PHGR1 mRNA levels in SLNs, suggesting occult metastases. With a median follow-up of 7.2 years, a significantly shorter recurrence-free (P=.005) and disease-specific (P=.02) survival was observed in patients with elevated PHGR1 mRNA levels in SLNs. Multivariable modeling showed that the SLN PHGR1 mRNA level was an independent prognostic factor. However, when the survival analyses were restricted to pN0 patients, no significant prognostic information was found. CONCLUSION: Measuring PHGR1 mRNA in SLNs provided independent prognostic information on operable colon cancer patients but not in the pN0 subgroup.

Neoadjuvant chemotherapy versus surgery first for resectable pancreatic cancer (Norwegian Pancreatic Cancer Trial - 1 (NorPACT-1)) - study protocol for a national multicentre randomized controlled trial.

BACKGROUND: Pancreatic cancer is the fourth leading cause of cancer-related death. While surgical resection remains the foundation for potentially curative treatment, survival benefit is achieved with adjuvant oncological treatment. Thus, completion of multimodality treatment (surgical resection and (neo)adjuvant chemotherapy) to all patients and early treatment of micrometastatic disease is the ideal goal. NorPACT-1 aims to test the hypothesis that overall mortality at one year after allocation of treatment can be reduced with neoadjuvant chemotherapy in surgically treated patients with resectable pancreatic cancer. METHODS/DESIGN: The NorPACT- 1 is a multicentre, randomized controlled phase III trial organized by the Norwegian Gastrointestinal Cancer Group for Hepato-Pancreato-Biliary cancer. Patients with resectable adenocarcinoma of the pancreatic head are randomized to receive either surgery first (Group 1: SF/control) or neoadjuvant chemotherapy (Group 2: NT/intervention) with four cycles FOLFIRINOX followed by resection. Both groups receive adjuvant chemotherapy with gemicitabine and capecitabine (six cycles in Group 1, four cycles in Group 2). In total 90 patients will be randomized in all the five Norwegian university hospitals performing pancreatic surgery. Primary endpoint is overall mortality at one year following commencement of treatment for those who ultimately undergo resection. Secondary endpoints are overall survival after date of randomization (intention to treat), overall survival after resection, disease-free survival, histopathological response, complication rates after surgery, feasibility of neoadjuvant and adjuvant chemotherapy, completion rates of all parts of multimodal treatment, and quality-of-life. Bolt-on to the study is a translational research program that aims at identifying factors that are predictive of response to NT, the risk of distant cancer spread, and patient outcome. DISCUSSION: NorPACT- 1 is designed to investigate the additional benefit of NT compared to standard treatment only (surgery + adjuvant chemotherapy) for resectable cancer of the pancreatic head to decrease early mortality (within one year) in resected patients. TRIAL REGISTRATION: Trial open for accrual 01.02.2017. ClinicalTrials.gov Identifier: NCT02919787 . Date of registration: September 14, 2016.

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

BACKGROUND: Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. METHODS: Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. RESULTS: Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. CONCLUSION: The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

MINDEC-An Enhanced Negative Depletion Strategy for Circulating Tumour Cell Enrichment.

Most current methods of circulating tumour cell (CTC) enrichment target the epithelial protein EpCAM, which is commonly expressed in adenocarcinoma cells. However, such methods will not recover the fraction of CTCs that have a non-epithelial phenotype due to epithelial-mesenchymal transition. For phenotype-independent CTC enrichment, we developed a new enhanced negative depletion strategy-termed MINDEC-that is based on multi-marker (CD45, CD16, CD19, CD163, and CD235a/GYPA) depletion of blood cells rather than targeted enrichment of CTCs. Here we validated the performance of MINDEC using epithelial and mesenchymal cancer cell lines, demonstrating a mean recovery of 82 ± 10%, high depletion (437 ± 350 residual white blood cells (WBCs)/mL peripheral blood), linearity between spiked and recovered cells (correlation coefficient: r = 0.995), and a low detection limit (≥1 cell recovered in all four replicates spiked with 3 cells). For clinical validation of this method, we enumerated CTCs in peripheral blood samples from patients with metastatic pancreatic cancer, detecting CTCs in 15 of 21 blood samples (71%) from 9 patients. The promising performance of the MINDEC enrichment strategy in our study encourages validation in larger clinical trials.

Clinical relevance of circulating KRAS mutated DNA in plasma from patients with advanced pancreatic cancer.

We used KRAS mutations to investigate the clinical relevance of circulating tumor DNA (ctDNA) measurements in patients with advanced pancreatic cancer. Fifty-three blood samples were collected from 14 prospectively recruited patients prior to chemotherapy (gemcitabine or FOLFIRINOX) and subsequently every month during treatment. Samples were processed by density centrifugation and plasma DNA isolation. A Peptide-nucleic acid-clamp PCR was then used to detect KRAS mutations (present in >90% of pancreatic cancers) as a surrogate marker for ctDNA. Plasma samples from 29 healthy individuals were analyzed as a reference group. Results were compared to conventional monitoring measures and survival data. Median follow-up time was 3.7 months (range 0.6-12.9 months). Ten (71%) patients had a positive KRAS status in the plasma samples obtained prior to chemotherapy, indicating the presence of ctDNA. Among the patients who were ctDNA-positive before chemotherapy, nine (90%) experienced disease progression during follow-up, compared to one (25%) of four ctDNA-negative patients (P = 0.01). The pre-therapy ctDNA level was a statistically significant predictor of both progression-free and overall survival (P = 0.014 and 0.010, respectively). Of the 14 patients, ten had ≥2 follow-up samples; in several of these patients, the ctDNA level changed substantially during the course of chemotherapy. Changes in ctDNA levels corresponded both with radiological follow-up data and CA19-9 levels for several patients. This pilot study supports the hypothesis that ctDNA may be used as a marker for monitoring treatment efficacy and disease progression in pancreatic cancer patients. Recruitment of more patients is ongoing to corroborate these findings.

Molecular subtypes in stage II-III colon cancer defined by genomic instability: early recurrence-risk associated with a high copy-number variation and loss of RUNX3 and CDKN2A.

OBJECTIVE: We sought to investigate various molecular subtypes defined by genomic instability that may be related to early death and recurrence in colon cancer. METHODS: We sought to investigate various molecular subtypes defined by instability at microsatellites (MSI), changes in methylation patterns (CpG island methylator phenotype, CIMP) or copy number variation (CNV) in 8 genes. Stage II-III colon cancers (n = 64) were investigated by methylation-specific multiplex ligated probe amplification (MS-MLPA). Correlation of CNV, CIMP and MSI, with mutations in KRAS and BRAFV600E were assessed for overlap in molecular subtypes and early recurrence risk by uni- and multivariate regression. RESULTS: The CIMP phenotype occurred in 34% (22/64) and MSI in 27% (16/60) of the tumors, with noted CIMP/MSI overlap. Among the molecular subtypes, a high CNV phenotype had an associated odds ratio (OR) for recurrence of 3.2 (95% CI 1.1-9.3; P = 0.026). Losses of CACNA1G (OR of 2.9, 95% CI 1.4-6.0; P = 0.001), IGF2 (OR of 4.3, 95% CI 1.1-15.8; P = 0.007), CDKN2A (p16) (OR of 2.0, 95% CI 1.1-3.6; P = 0.024), and RUNX3 (OR of 3.4, 95% CI 1.3-8.7; P = 0.002) were associated with early recurrence, while MSI, CIMP, KRAS or BRAF V600E mutations were not. The CNV was significantly higher in deceased patients (CNV in 6 of 8) compared to survivors (CNV in 3 of 8). Only stage and loss of RUNX3 and CDKN2A were significant in the multivariable risk-model for early recurrence. CONCLUSIONS: A high copy number variation phenotype is a strong predictor of early recurrence and death, and may indicate a dose-dependent relationship between genetic instability and outcome. Loss of tumor suppressors RUNX3 and CDKN2A were related to recurrence-risk and warrants further investigation.

Comparison of molecular and immunocytochemical methods for detection of disseminated tumor cells in bone marrow from early breast cancer patients.

BACKGROUND: Disseminated tumor cells (DTCs) have potential to predict the effect of adjuvant treatment. The purpose of this study was to compare two methods, reverse transcription quantitative PCR (RT-qPCR) and immunocytochemisty (ICC), for detecting breast cancer DTCs in bone marrow (BM) from early breast cancer patients. METHODS: We investigated a subset (n = 313) of BM samples obtained from 271 early breast cancer patients in the "Secondary Adjuvant Taxotere Treatment" (SATT)-trial. All patients in this study had node positive or intermediate/high-risk node negative non-metastatic disease. The DTCs were detected by ICC using AE1-AE3 anti-cytokeratin monoclonal antibodies. Patients with DTCs detected in their BM by ICC after standard adjuvant fluorouracil, cyclophosphamide, epirubicin (FEC) chemotherapy were offered docetaxel treatment. For comparison, 5 × 106 mononuclear cells from the aliquoted BM samples were also analyzed by RT-qPCR using a multimarker (MM) assay based on the tumor cell mRNA markers keratin 19 (KRT19), mammaglobin A (hMAM), and TWIST1. In the MM-assay, a sample was defined as positive for DTCs if at least one of the mRNA markers was positive. RESULTS: The MM RT-qPCR assay identified DTCs in 124 (40%) of the 313 BM samples compared with 23/313 (7%) of the samples analyzed by ICC. The concordance between the MM RT-qPCR and ICC was 61% (Kappa value = 0.04) and twelve of the BM samples were positive by both methods. By RT-qPCR, 46/313 (15%) samples were positive for KRT19, 97/313 (31%) for TWIST1, and 3/313 (1%) for hMAM mRNA. There were no statistically significant associations between the individual mRNA markers. CONCLUSION: The RT-qPCR based method demonstrated more DTC-positive samples than ICC. The relatively low concordance of positive DTC-status between the two different assessment methods suggests that they may be complementary. The clinical relevance of the methods will be evaluated based on future clinical outcome data. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00248703.

Circulating tumor cells in pancreatic cancer patients: methods of detection and clinical implications.

The poor prognosis of pancreatic cancer patients is associated with the frequent and early dissemination of the disease, as well as late detection due to unspecific and late symptoms from the primary tumor. Pancreatic cancers frequently spread to the liver, lung and skeletal system, suggesting that pancreatic tumor cells must be able to intravasate and travel through the circulation to distant organs. Circulating tumor cells (CTCs) are tumor cells that have acquired the ability to enter the circulatory system; this cell population is ultimately responsible for the development of metastases in distant organs. Clinical studies have revealed that the presence of CTCs in blood is correlated with disease progression for other cancers, such as breast, colorectal and prostate cancer. However, as CTCs are extremely rare, both enrichment and sensitive methods of detection are required for their enumeration. This review highlights various enrichment procedures and methods for the detection of CTCs. Furthermore, we systematically review previously reported studies of the clinical relevance of CTC detection in pancreatic cancer patients. There is evidence that the presence of CTCs also correlates with an unfavorable outcome in pancreatic cancer patients. However, technical/methodological issues may explain why some studies only show a trend toward an association between CTC detection and disease progression. Larger studies, as well as characterization of the CTC population, are required to achieve further insight into the clinical implications of CTC detection in pancreatic cancer patients.

Influence of microsatellite instability and KRAS and BRAF mutations on lymph node harvest in stage I-III colon cancers.

Lymph node (LN) harvest is influenced by several factors, including tumor genetics. Microsatellite instability (MSI) is associated with improved node harvest, but the association to other genetic factors is largely unknown. Research methods included a prospective series of stage I-III colon cancer patients undergoing ex vivo sentinel-node sampling. The presence of MSI, KRAS mutations in codons 12 and 13, and BRAF V600E mutations was analyzed. Uni- and multivariate regression models for node sampling were adjusted for clinical, pathological and molecular features. Of 204 patients, 67% had an adequate harvest (≥ 12 nodes). Adequate harvest was highest in patients whose tumors exhibited MSI (79%; odds ratio [OR] 2.5, 95% confidence interval [CI] 1.2-4.9; P = 0.007) or were located in the proximal colon (73%; 2.8, 1.5-5.3; P = 0.002). In multiple linear regression, MSI was a significant predictor of the total LN count (P = 0.02). Total node count was highest for cancers with MSI and no KRAS/BRAF mutations. The independent association between MSI and a high LN count persisted for stage I and II cancers (P = 0.04). Tumor location in the proximal colon was the only significant predictor of an adequate LN harvest (adjusted OR 2.4, 95% CI 1.2-4.9; P = 0.01). An increase in the total number of nodes harvested was not associated with an increase in nodal metastasis. In conclusion, number of nodes harvested is highest for cancers of the proximal colon and with MSI. The nodal harvest associated with MSI is influenced by BRAF and KRAS genotypes, even for cancers of proximal location. Mechanisms behind the molecular diversity and node yield should be further explored.

Fatigue and weight loss predict survival on circadian chemotherapy for metastatic colorectal cancer.

BACKGROUND: Chemotherapy-induced neutropenia has been associated with prolonged survival selectively in patients on a conventional schedule (combined 5-fluorouracil, leucovorin, and oxaliplatin [FOLFOX2]) but not on a chronomodulated schedule of the same drugs administered at specific circadian times (chronoFLO4). The authors hypothesized that the early occurrence of chemotherapy-induced symptoms correlated with circadian disruption would selectively hinder the efficacy of chronotherapy. METHODS: Fatigue and weight loss (FWL) were considered to be associated with circadian disruption based on previous data. Patients with metastatic colorectal cancer (n = 543) from an international phase 3 trial comparing FOLFOX2 with chronoFLO4 were categorized into 4 subgroups according to the occurrence of FWL or other clinically relevant toxicities during the initial 2 courses of chemotherapy. Multivariate Cox models were used to assess the role of toxicity on the time to progression (TTP) and overall survival (OS). RESULTS: The proportions of patients in the 4 subgroups were comparable in both treatment arms (P = .77). No toxicity was associated with TTP or OS on FOLFOX2. The median OS on FOLFOX2 ranged from 16.4 (95% confidence limits [CL], 7.2-25.6 months) to 19.8 months (95% CL, 17.7-22.0 months) according to toxicity subgroup (P = .45). Conversely, FWL, but no other toxicity, independently predicted for significantly shorter TTP (P < .0001) and OS (P = .001) on chronoFLO4. The median OS on chronoFLO4 was 13.8 months (95% CL, 10.4-17.2 months) or 21.1 months (95% CL, 19.0-23.1 months) according to presence or absence of chemotherapy-induced FWL, respectively. CONCLUSIONS: Early onset chemotherapy-induced FWL was an independent predictor of poor TTP and OS only on chronotherapy. Dynamic monitoring to detect early chemotherapy-induced circadian disruption could allow the optimization of rapid chronotherapy and concomitant improvements in safety and efficacy.

Novel molecular tumor cell markers in regional lymph nodes and blood samples from patients undergoing surgery for non-small cell lung cancer.

INTRODUCTION: Recent evidence suggests that microscopic lymph node metastases and circulating tumor cells may have clinical importance in lung cancer. The purpose of this study was to identify new molecular markers for tumor cells in regional lymph nodes (LNs) and peripheral blood (PB) from patients with non-small cell lung cancer (NSCLC). METHODS: Candidate markers were selected based on digital transcript profiling and previous literature. KRT19, CEACAM5, EPCAM, DSG3, SFTPA, SFTPC and SFTPB mRNA levels were initially validated by real-time reverse transcription PCR-based quantification in 16 NSCLC tumors and 22 LNs and 12 PB samples from individuals without known cancer. Five of the candidate markers were selected for secondary validation by quantification in parallel tumor biopsies, regional LNs and PB samples from 55 patients undergoing surgery for NSCLC. LN and PB marker status were compared to clinicopathological patient data. RESULTS: All selected markers except DSG3 were present at high levels in the primary tumors and at very low or non-detectable levels in normal LNs and PB in the first round of validation, indicating a potential for detecting tumor cells in NSCLC patients. The expression profiles of KRT19, CEACAM5, DSG3, SFTPA and SFTPC mRNA were confirmed in the larger group during the secondary validation. Using the highest normal LN level of each marker as threshold, 39 (71%) of the 55 patients had elevated levels of at least one marker in regional LNs. Similarly, 26 (47%) patients had elevated levels of at least one marker in PB. A significantly higher number of patients with adenocarcinomas had positive LN status for these markers, compared with other histological types (P = 0.004). CONCLUSIONS: Several promising molecular tumor cell markers in regional LNs and PB were identified, including the new SFTPA and SFTPC mRNAs. Clinical follow-up in a larger cohort is needed to elucidate their prognostic value.

Prognostic comparison of the proliferation markers (mitotic activity index, phosphohistone H3, Ki67), steroid receptors, HER2, high molecular weight cytokeratins and classical prognostic factors in T1₋2N0M0 breast cancer.

The proliferation factors: mitotic activity index (MAI), phosphohistone H3 (PPH3) and Ki67 have strong prognostic value in early breast cancer but their independent value to each other and other prognostic factors has not been evaluated. In 237 T1₋2N0M0 breast cancers without systemic adjuvant treatment, formalized MAI assessment and strictly standardized, fully automated quantitative immunohistochemistry (IHC) for Ki67, PPH3, estrogen (ER) and progesterone receptor (PR), HER2, cytokeratins-5/6 and -14, and automated digital image analysis (DIA) for measuring PPH3 and Ki67 were performed. Section thickness was measured to further control IHC measurements. All features were measured in the periphery of tumors. The different proliferation assessments and other well-established clinicopathological and biomarker prognostic factors were compared. DIA-Ki67 added prognostically to PPH3. None of the other biomarkers or clinicopathological variables added prognostically to this PPH3/Ki67 combination. However, when PPH3 is replaced by MAI the prognostic value is nearly the same. In early operable node negative breast cancer without adjuvant systemic treatment, Ki67 with a threshold of 6.5% assessed by digital image analysis in the periphery of the tumor is prognostically strong. The combination of either PPH3/Ki67 or MAI/Ki67 overshadowed the prognostic value of all other features including Ki67 alone.

Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer.

AIMS: The proliferation factor Ki67 is prognostic in breast cancer and included in international therapy guidelines, but measurement procedures differ between laboratories. We compared the reproducibility and prognostic value of different Ki67 sampling and measurement methods. METHODS AND RESULTS: In 237 T(1,2) N(0) M(0) breast cancers without adjuvant systemic treatment, strictly standardized section thickness, automated antigen retrieval and immunohistochemistry were used. The percentages of Ki67-positive nuclei were assessed using (i) a 'quick-scan rapid estimate', (ii) ocular-square-guided counts by independent pathologists, (iii) computerized point-grid-sampling interactive morphometry (CIM) and (iv) automated digital image analysis (DIA). Quick-scan rapid estimates were poorly reproducible. The optimal prognostic thresholds of Ki67 counts by two pathologists differed greatly (4%, 14%; kappa: 0.36), with many therapeutic differences. CIM-Ki67 and DIA-Ki67 were strongly prognostic (P<0.0001) and reproducible. DIA-Ki67 (threshold: 6.5%) was the strongest and most robust prognosticator (the threshold could vary from 4 to 15% without significant prognostic loss). Ki67 was prognostically strongest in the periphery of the tumour. CONCLUSION: In node-negative breast cancer without adjuvant systemic treatment, Ki67% by DIA, but not subjective counts, is reproducible and prognostically strong. This casts serious doubt on therapeutic guidelines using subjective counts of Ki67.

Prognostic relevance of occult metastases detected by cytokeratin 20 and mucin 2 mRNA levels in sentinel lymph nodes from colon cancer patients.

PURPOSE: To investigate the prognostic value of occult metastases detected by quantitative measurements of candidate biomarkers in sentinel lymph nodes (SLNs) from patients curatively resected for colon cancer. METHODS: Resection specimens from consecutive patients undergoing surgery for localized colon cancer were subjected to ex vivo SLN mapping. SLNs were examined for the presence of metastases by routine hematoxylin-erythrosin-safranin staining and by cytokeratin 20 (CK20) and mucin 2 (MUC2) mRNA quantification. The patients were stratified according to KRAS and BRAF mutation status and microsatellite instability status in their primary tumors. Survival end points were analyzed by Kaplan-Meier survival estimates and log-rank tests. RESULTS: A total of 817 SLNs were identified in 206 (97 %) of the 213 included patients. Routine histological examination of SLNs and other regional lymph nodes identified 63 patients with positive nodes (pN+), of which 42 (67 %) were positive in one or more SLNs (sensitivity 67 %, false-negative rate 33 %). On the basis of the CK20 and MUC2 mRNA levels in SLNs, occult metastases were suggested in 86 (60 %) and 52 (36 %) of the 143 otherwise LN-negative (pN0) patients, respectively. Survival analysis with a median 3.6-year follow-up revealed that MUC2 mRNA quantification had significant prognostic value in SLNs from all patients; however, occult SLN metastasis detection did not. CONCLUSIONS: Occult SLN metastases detected by CK20 and MUC2 mRNA quantification had limited prognostic value.

The prognostic value of MARCKS-like 1 in lymph node-negative breast cancer.

There is a need for new biomarkers to more correctly identify node-negative breast cancer patients with a good or bad prognosis. Myristoylated alanine-rich C kinase substrate like-1 (MARCKSL1) is a membrane-bound protein that is associated with cell spreading, integrin activation and exocytosis. Three hundred and five operable T(1,2)N(0)M(0) lymph node-negative breast cancer patients (median follow-up time 121 months, range 10-178 months) were evaluated for MARCKSL1 expression by immunohistochemistry and quantitative real-time PCR. The results were compared with classical prognosticators (age, tumor diameter, grade, estrogen receptor, and proliferation), using single (Kaplan-Meier) and multivariate survival analysis (Cox model). Forty-seven patients (15 %) developed distant metastases. With single and multivariate analysis of all features, MARCKSL1 protein expression was the strongest prognosticator (P < 0.001, HR = 5.1, 95 % CI = 2.7-9.8). Patients with high MARCKSL1 expression (n = 23) showed a 44 % survival versus 88 % in patients with low expression at 15-year follow-up. mRNA expression of MARCKSL1 in formalin fixed paraffin-embedded tissue was also prognostic (P = 0.002, HR = 3.6, 95 % CI = 1.5-8.3). However, the prognostic effect of high and low was opposite from the protein expression, i.e., low expression (relative expression ≤ 0.0264, n = 76) showed a 79 % survival versus 92 % in those with high expression of MARCKSL1 mRNA. Multivariate analysis of all features with distant metastases free survival as the end-point showed that the combination of MARCKSL1 protein and phosphohistone H3 (PPH3) has the strongest independent prognostic value. Patients with high expression (≥13) of PPH3 and high MARCKSL1 protein had 45 % survival versus 78 % survival for patients with low MARCKSL1 protein expression and high expression (≥13) of PPH3. In conclusion, MARCKSL1 has strong prognostic value in lymph node-negative breast cancer patients, especially in those with high proliferation.

miRNA expression profiling for identification of potential breast cancer biomarkers.

To identify micro RNA (miRNA) biomarker candidates for early detection of breast cancer and detection of minimal residual breast cancer, we performed miRNA expression profiling in pooled RNA samples from breast tumors, and from bone marrow mononuclear cells, peripheral blood mononuclear cells and plasma from healthy controls. We found substantially higher levels of five miRNAs in the breast tumors compared to the normal samples. However, validation of these miRNA levels, and seven other candidates selected from the literature, in individual samples from healthy controls and patients with non-metastatic breast cancer did not suggest further examination of their biomarker potential.

Persistent tumor cells in bone marrow of non-metastatic breast cancer patients after primary surgery are associated with inferior outcome.

BACKGROUND: To investigate the prognostic significance of disseminated tumor cells (DTCs) in bone marrow (BM) from non-metastatic breast cancer patients before and after surgery. METHODS: Patients with non-metastatic breast cancer were consecutively recruited to this project during the years 1998-2000. Real-time RT-PCR quantification of a DTC multimarker panel consisting of cytokeratin 19, mammaglobin A and TWIST1 mRNA was performed in BM samples obtained from 154 patients three weeks (BM2) and/or six months after surgery (BM3). The results were compared to previously published data from pre-operative BM analyses for the same patients. RESULTS: DTCs were identified in post-operative BM samples (BM2 and/or BM3) from 23 (15%) of the 154 patients investigated. During a median follow-up of 98 months, 10 (44%) of these patients experienced systemic relapse as compared to 16 (12%) of 131 DTC-negative patients. Kaplan-Meier estimates of systemic recurrence-free- and breast-cancer specific survival demonstrated significantly shorter survival for patients with persistent DTCs in BM after surgery (p≤0.001). By multivariate Cox regression analyses, persistent DTCs after surgery was an independent predictor of both systemic recurrence-free- (HR = 5.4, p < 0.001) and breast-cancer specific survival (HR = 5.3, p < 0.001). Furthermore, the prognostic value of DTCs in BM was similar for pre- and post surgery samples. However, patients with DTCs both before and after surgery (BM1 and BM2/3) had a particularly poor prognosis (systemic recurrence-free survival: HR = 7.2, p < 0.0001 and breast-cancer specific survival: HR = 8.0, p < 0.0001). CONCLUSIONS: Detection of persistent DTCs in BM samples obtained after surgery identified non-metastatic breast cancer patients at high risk for systemic relapse, and with reduced breast-cancer specific survival. Furthermore, patients with positive DTC status both before and after surgery had a particularly poor prognosis.