Bone Marrow Micrometastases Research Articles

Stromal Co-Cultivation for Modeling Breast Cancer Dormancy in the Bone Marrow.

Simple SummaryCancer cells travel to far away parts of the body before the original cancer can be detected. Breast cancer cells travel mainly to the bone marrow, where they are kept in a quiet, non-dividing, dormant state for many years by cells and proteins in the bone marrow. These metastatic cancer cells wake up at a steady rate over the years and result in incurable disease. This paper outlines how to study the interactions of cells that live in the bone marrow with cancer cells in the laboratory. We describe how to grow mixtures of a variety of bone marrow-residing cells to generate cell layers upon which cancer cells can be grown. Using this system, we can study their effects on cancer cell growth or dormancy and the biology of the interactions. With this approach, we can study how to either eliminate the cancer cells or prevent them from waking up.Cancers metastasize to the bone marrow before primary tumors can be detected. Bone marrow micrometastases are resistant to therapy, and while they are able to remain dormant for decades, they recur steadily and result in incurable metastatic disease. The bone marrow microenvironment maintains the dormancy and chemoresistance of micrometastases through interactions with multiple cell types and through structural and soluble factors. Modeling dormancy in vitro can identify the mechanisms of these interactions. Modeling also identifies mechanisms able to disrupt these interactions or define novel interactions that promote the reawakening of dormant cells. The in vitro modeling of the interactions of cancer cells with various bone marrow elements can generate hypotheses on the mechanisms that control dormancy, treatment resistance and reawakening in vivo. These hypotheses can guide in vivo murine experiments that have high probabilities of succeeding in order to verify in vitro findings while minimizing the use of animals in experiments. This review outlines the existing data on predominant stromal cell types and their use in 2D co-cultures with cancer cells.

Increasing Immune Dysfunction is Associated with Increasing Matrix-Metalloproteinase-2 Expression and Predicts Biochemical Failure in Men with Bone Marrow Micro-Metastasis Positive Localized Prostate Cancer.

To determine if there was an association of the ALC (absolute lymphocyte count) and LCP (lymphocytopenia) with the expression of MMP-2 in bone marrow micro-metastasis, the changes occurring during follow-up and association with biochemical failure. One month after surgery blood and bone marrow samples were taken to determine the presence of micro-metastasis, the presence of circulating prostate cells (CPCs) and ALC. CPCs and micro-metastasis were detected using immunocytochemistry and MMP-2 expression determined in micro-metastasis. Only men positive for micro-metastasis participated in the study. At end follow blood was taken for serum PSA, ALC and CPCs, if the ALC decreased by more than 10% bone marrow sampling was repeated and MMP-2 expression determined, similarly for men with BF. Men who had stable ALCs had an end of study evaluation of the bone marrow. 402 men underwent radical prostatectomy, one month post surgery 79 men were positive for only bone marrow micro-metastasis and formed the study group; of whom 36/79 (45%) underwent BF. Clinical pathological findings were not significantly different between men with or without BF. In men with BF the ALC was significantly lower one-month post surgery. The 5 and 10 year Kaplan-Meier survival was 100% at 5-years and 65% at 10-years for the whole cohort. Men without BF had stable ALCs. A decrease of >10% in the ALC was associated with increasing MMP-2 expression in the micro-metastasis and surrounding stromal tissue, the appearance of CPCs 6-12 months later and BF. the immune host-tumour cell interaction in the microenvironment is dynamic and changes with time. A decreasing ALC may be a valuable marker in identifying men with high risk of BF and changes in immune mediated dormancy before the PSA rises.

Disseminated tumour cells in bladder cancer

Molecular analysis of disseminated tumour cells (DTC) may aid in predicting the course of the disease and response to therapies in individual patients. It has been shown in bladder cancer and many other cancer types that the presence of disseminated tumour cells or occult micrometastases in bone marrow or lymph nodes is associated with shorter survival. This type of analysis is particularly important for patients who have been declared disease-free after postsurgery histopathological and clinical imaging analysis. However, comprehensive molecular analysis of disseminated tumour cells is challenging due to the low amount of material and great heterogeneity of the disease. Therefore, currently the routine molecular analysis of these cells is hardly possible in daily clinical practice. Nevertheless, we see daily advances in clinical utility of analysis of cellular or cell-free liquid biopsy analytes taken before, during or after surgery. These advances will enable an integration of translational research workflows into clinical decision-making.

The prognostic significance of disseminated tumor cells in bone marrow in patients with operable breast cancer: An updated analysis.

e13013 Background: The main cause of death in breast cancer patients is the consequence of the spread and outgrowth of tumor cells at distant sites. The presence of disseminated tumor cells (DTCs) in the bone marrow at diagnosis is considered a predictor for metastasis. Methods: This is an updated analysis of a study in 102 patients with operable breast cancer on the significance of bone marrow micrometastases. The mean follow-up time is 127 months (range 10 - 142, median 129 months). Bone marrow aspirates were analysed for the presence of DTCs by a real time-polymerase chain reaction (RT-PCR) for cytokeratin 19 (CK) and mammaglobin (MAM).The aim of this study is to confirm the association between DTCs and disease-specific survival (DSS) as well as metastasis-free survival (MFS). Results: CK positivity was borderline significant for DSS (p = 0.054). MAM positivity was significantly prognostic for DSS with a hazard ratio (HR) of 3,110 (1,416-6,832) (p = 0,005). Patients positive for both CK and MAM did have a significant worse outcome with a HR for DSS of 7,329 (2,361 – 22,752) (p = 0,001). No significant influence on MFS was identified for CK (p = 0,658) or MAM positivity (p = 0,095). The combination of CK and MAM positivity did however confer a significantly increased risk for MFS (p = 0,043) at a HR of 4,126 (1,409-12,082). Conclusions: This study confirms the role of DTCs as a negative, prognostic factor in patients with operable breast cancer. The combination of CK and MAM is useful to identify this increased risk.

Improvement in the Neutrophil-Lymphocyte Ratio after Combined Fluorouracil, Leucovorina and Oxaliplatino based (FOLFOX) Chemotherapy for Stage III Colon Cancer is Associated with Improved Minimal Residual Disease and Outcome.

Introduction:Minimal residual disease (MRD) is the net result of the biological properties of disseminated tumour cells and the effect of the immune system and treatment to eliminate them. The aim of this study was to analyse the effect of combined chemotherapy on the immune function as determined by the neutrophil-lymphocyte ratio (NLR) and if it was associated with changes in the subtype of minimal residual disease and outcome in stage III colon cancer. Methods and Patients:A prospective, single centre observational study; the NLR was determined immediately prior to and one, two and three months after completing chemotherapy. Circulating tumour cells (CTCs) and bone marrow micro-metastasis (mM) using immunocytochemistry with anti-CEA were determined prior to and one month after chemotherapy. The association of changes in the NLR with MRD subtypes classified as Group I (negative for CTCs and mM), Group II (positive for mM) and Group III (positive for CTCs) as a result of chemotherapy and five-year disease free progression (DFS) analysed. Results:One hundred and eighty eight patients participated of whom 83 (44.9%) relapsed. In non-relapsing patients the NLR significantly increased and was higher after chemotherapy compared with relapsing patients. Significant increases in the NLR were associated with changes to a better MRD prognostic subtype and decreases with a worse MRD subtype. Neither baseline NLR nor MRD subtype predicted response to chemotherapy. DFS for MRD subgroups were 88%, 56% and 6% for Groups I to III respectively. Conclusions:Immune function as measured by the NLR is associated with MRD prognostic subtypes, improvements in the NLR are associated with improvements in MRD post chemotherapy but neither baseline NLR or MRD predicted outcome.

The Simultaneous Presence of Isolated Tumour Cells and Bone Marrow Micrometastases in Stage I and II Colon Cancer\u2014Challenging the Theory of a Chronological Pathway of Tumour Cell Dissemination

BackgroundAccording to the common tenet, tumour progression is a chronological process starting with lymphatic invasion. In this respect, the meaning of bone marrow micrometastases (BMM) in patients with lymph node negative colon cancer (CC) is unclear. This study examines the relationship of isolated tumour cells (ITC) in sentinel lymph nodes (SLN) and BMM in patients in early CC.MethodsBM aspirates were taken from both pelvic crests and in vivo SLN mapping was done during open oncologic colon resection in patients with stage I and II CC. Stainings were performed with the pancytokeratin markers A45-B/B3 and AE1/AE3 as well as H&E. The correlation between the occurrence of ITC+ and BMM+ and their effects on survival was examined using Cox regression analysis.ResultsIn a total of 78 patients with stage I and II CC, 11 patients (14%) were ITC+, 29 patients (37%) BMM+. Of these patients, only two demonstrated simultaneous ITC+ /BMM+. The occurrence of BMM+ was neither associated with ITC+ in standard correlation (kappa = − 0.13 [95% confidence interval [CI] = − 0.4–0.14], p = 0.342) nor univariate (odds ratio [OR] = 0.39, 95%CI:0.07–1.50, p = 0.180) or multivariate (OR = 0.58, 95%CI: 0.09–2.95, p = 0.519) analyses. Combined detection of ITC+ /BMM+ demonstrated the poorest overall (HR = 61.60, 95%CI:17.69–214.52, p = 0.032) and recurrence free survival (HR = 61.60, 95%CI: 17.69–214.5, p = 0.032).ConclusionsThese results indicate that simultaneous and not interdependent presence of very early lymphatic and haematologic tumour spread may be considered as a relevant prognostic risk factor for patients with stage I and II CC, thereby suggesting the possible need to reconsider the common assumptions on tumour spread proposed by the prevalent theory of sequential tumour progression.

Improvement in immune dysfunction after FOLFOX chemotherapy for Stage III colon cancer is associated with improved minimal residual disease prognostic subtype and outcome

Minimal residual disease (MRD) is the net result of the biological properties of disseminated tumour cells and the effect of the immune system and treatment to eliminate them. The aim of this work is to report the changes in MRD status and immune function (lymphocyte count) after FOLFOX chemotherapy, and the outcome in Stage III colon cancer patients. This study is a prospective, single-centre observational study. Lymphocyte counts were determined prior to and 1, 2 and 3months after the completion of chemotherapy. Circulating tumour cells (CTCs) and bone marrow micrometastases were determined using immunocytochemistry with anticarcinoembryonic antigen prior to and 1month after chemotherapy. MRD was classified as negative (Group I), micrometastasis positive only (Group II) and CTC positive (Group III). Changes in lymphocyte counts and MRD subtype following chemotherapy and relapse-free progression were analysed. Of the total of 185 patients, 83 (44.9%) relapsed. The risk of relapse significantly increased from Groups I to III (p<0.001) and with decreasing lymphocyte count (p<0.01). The lymphocyte count significantly decreased from Groups I to III (p<0.001). Multivariance Cox regression analysis showed hazard ratios of 3.58 (Group II), 17.43 (Group III) and 0.39 (lymphocyte count) in predicting relapse. Following chemotherapy, improved lymphocyte count was associated with improved MRD subtype (p<0.0001). Neither baseline lymphocyte count nor MRD subtype predicted response to chemotherapy. Five-year relapse-free survival for combined lymphocyte-MRD subtypes was 95%, 57% and 5% for Groups I to III, respectively (p<0.001). Following chemotherapy, improvements in immune function were associated with improved MRD subtype and a better relapse-free survival.

Subtypes of minimal residual disease and outcome for stage II colon cancer treated by surgery alone.

IntroductionTwenty-five percent of stage II colon cancer (CC) patients relapse within 5 years due to minimal residual disease (MRD) not eliminated by surgery. We hypothesise that subtypes of MRD, defined by circulating tumour cells (CTCs) and bone marrow micrometastasis (mM), have different types and kinetics of relapse.Methods and patientsOne month after surgery, blood and bone marrow samples were taken to detect CTCs and mM using immunocytochemistry with anti-carcinoembryonic antigen (CEA). Follow-up was for up to 5 years or relapse. Disease-free survival curves using Kaplan–Meier (DFS) and restricted mean disease-free survival times (RMST) were calculated for three prognostic groups: A: MRD (−), B: mM (+) CTC (−) MRD and C: CTC (+) MRD.ResultsOne hundred and eighty-one patients (82 men) have participated, mean age was 68 years and median follow-up was 4.04 years (A (N = 105), B (N = 36) and C (N = 40)). For the whole cohort of 5 years, DFS was 70%, median DFS has not reached (Groups A: 98%, B: 63% and C: 7%) and median DFS for Groups A and B have not reached. RMST for the whole cohort of 4.1 years, Group A was 4.9 years, B was 4.1 years and C was 1.7 years. Serum CEA was significantly higher in Group C. No significant differences for sex, age or high-risk adverse prognostic factors between groups were detected.ConclusionsMRD subtypes define relapse patterns and may be useful to define the risk of relapse in stage II CC patients, in which patients may benefit or not from additional therapy and warrants further studies with a larger number of patients.

The Epstein criteria predict for organ-confined prostate cancer but not for minimal residual disease and outcome after radical prostatectomy.

The Epstein criteria (EC) used to select men for active surveillance do not predict biologically insignificant diseases. Minimal residual disease (MRD) is an undetected microscopic disease that remains after radical prostectomy (RP) and is a biological classification associated with the risk of treatment failure. Subtypes of MRD, the 10-year biochemical failure free survival (BFFS), and restricted mean biochemical failure free survival time (RMST) were determined and compared in EC patients treated with RP. Consecutive patients with a Gleason 6 biopsy treated at a single institution were divided into those who did or did not fulfill the EC and underwent RP. One month after surgery, samples were taken for the detection of circulating prostate cells (CPCs) and bone marrow micrometastasis. MRD was defined as negative for both CPCs and micrometastasis; patients were positive for micrometastasis and CPCs separately. BFFS for up to 10 years and RMST were determined for each MRD subgroup for EC positive and negative patients. EC positive men (137/426) were significantly older (p<0.05) and had negative MRD, pT2 (pathologically organ confined) disease (<0.02), and lower frequency of upgrading (p<0.02). Of the EC positive men, 71% were MRD negative, 13% were positive for micrometastasis, and 16% were positive for CPCs with respective 10-year BFFS of 99%, 89%, and 21% (<0.001) (hazard ratio: 1.00, 1.76, 4.03, respectively) with no signficant differences between the 10-year BFFS or RMST for MRD subgroups for EC positive and negative patients. EC predict pT2, MRD negative disease; however, 29% are MRD positive with a high risk of treatment failure.

Bone marrow mammaglobin-1 (SCGB2A2) immunohistochemistry expression as a breast cancer specific marker for early detection of bone marrow micrometastases

Despite all the advances in the management of breast cancer (BC), patients with distance metastasis are still considered incurable with poor prognosis. For that reason, early detection of the metastatic lesions is crucial to improve patients’ life span as well as quality of life. Many markers were proposed to be used as biomarkers for metastatic BC lesions, however many of them lack organ specificity. This highlights the need for novel markers that are more specific in detecting disseminated BC lesions. Here, we investigated mammaglobin-1 expression as a potential and specific marker for metastatic BC lesions using our patient cohort consisting of 30 newly diagnosed BC patients. For all patients, bone marrow (BM) aspiration, BM biopsy stained by H&E and BM immunohistochemically stained for mammaglobin-1 were performed. In addition, the CA15-3 in both serum and bone marrow plasma was also evaluated for each patient. Indeed, mammaglobin-1 immuno-staining was able to detect BM micrometastases in 16/30 patients (53.3%) compared to only 5/30 patients (16.7%) in BM biopsy stained by H&E and no cases detected by BM aspirate (0%). In addition, our results showed a trend of association between mammaglobin-1 immunoreactivity and the serum and BM plasma CA15-3. Further validation was done using large publicly available databases. Our results showed that mammaglobin-1 gene expression to be specifically upregulated in BC patients’ samples compared to normal tissue as well as samples from other cancers. Moreover, our findings also showed mammaglobin-1 expression to be a marker of tumour progression presented as lymph nodes involvement and distant metastasis. These results provide an initial evidence for the use of mammaglobin-1 (SCGB2A2) immunostaining in bone marrow as a tool to investigate early BM micrometastases in breast cancer.

The CAPRA score versus sub-types of minimal residual disease to predict biochemical failure after external beam radiotherapy.

IntroductionExternal beam radiotherapy is a treatment option for clinically localised prostate cancer; however, some 15% of patients will undergo treatment failure within 5 years. The objective was to compare the Cancer of the Prostate Risk Assessment (CAPRA) score (based on the clinical-pathological findings) and the sub-types of minimal residual disease (MRD) (based on the biological properties of the cancer cells) risk classifications to predict biochemical failure (BF) after external beam radiotherapy.Methods and PatientsClinical-pathological findings were obtained from the prostate biopsy to determine the CAPRA score and used to define low-, intermediate- and high-risk patients. Blood and bone marrow were obtained 3 months after radiotherapy; circulating prostate cells (CPCs) and micro-metastasis were detected using immunocytochemistry with anti-prostate specific antigen. CPCs and micro-metastasis were classified as positive if at least one cell was detected in the sample. Three subgroups were formed Group A (MRD negative), Group B (micro-metastasis positive, CPC negative) and Group C (CPC positive)Patients were followed up for 10 years or until biochemical failure. Biochemical failure free survival (BFFS) curves were constructed using Kaplan–Meier (observed), a flexible parameter model (predicted survival) and the restricted mean survival time (RMST) was calculated for each sub-group.Results309 men participated with a median follow-up of 8 years. The risk of biochemical failure increased proportionally with increasing CAPRA score, hazard ratio 1.18 for intermediate and 1.69 for high risk patients. After 10 years, the percentage BFFS and RMST to failure were 74%, 49%, 16% and 9, 7 and 6 years, respectively. The agreement between observed and predicted BFFS was acceptable (Harrell´s C 0.62). The BFFS curves for MRD were different and not proportional, survival curves for men MRD negative and only micro-metastasis were similar up to 5 years, and then there was increasing failure in the micro-metastasis only group. After 10 years, the percentage BFFS and RMST to failure were 95%, 59%, 28% and 10, 9 and 6 years, respectively. The CAPRA score failed to distinguish between Groups A and B, one third of high risk Group C had low risk CAPRA scores. The agreement between observed and predicted BFFS was very good (Harrell´s C 0.92). Minimal residual disease hazard ratios were Group B 1.84 and Group C 4.51.ConclusionsThe MRD prognostic classification is based on the biological characteristics of the tumour cell-microenvironment interaction, to give three groups, MRD negative, only bone marrow micro-metastasis and CPC positive prostate cancer. Differing from the CAPRA score classification the risk of treatment failure changes with time, differentiating between early and late treatment failures and incorporates the concept of dormancy. It proved to be superior to the CAPRA score in predicting biochemical failure and the results need to be confirmed in larger studies.

The expression of matrix-metalloproteinase-2 in bone marrow micro-metastasis is associated with the presence of circulating prostate cells and a worse prognosis in men treated with radical prostatectomy for prostate cancer

The expression of matrix-metalloproteinase-2 (MMP-2) in the primary tumor is associated with a worse prognosis but little is known at this time regarding the expression in micro-metastasis, the association with circulating prostate cells (CPCs), and outcome. This was a prospective study of men undergoing radical prostatectomy. Bone marrow and blood samples were taken at one month after surgery. Micro-metastasis and CPCs were identified using immunocytochemistry with anti-prostate specific-antigen and MMP-2 expression determined with anti-MMP-2. Pathological stage, Gleason score, and time to biochemical failure were recorded; meanwhile, Kaplan-Meier biochemical failure-free survival and restricted mean biochemical failure-free survival times for 10 years were determined. A total of 282 men participated, 54 (19%) of whom had micro-metastasis but not CPCs (group B) and 88 (31%) of whom had micro-metastasis and CPCs (group C). Men in group C had a higher frequency of MMP-2 expressing micro-metastasis at 63% versus 12% (p<0.001), and MMP-2 expression in bone marrow micro-metastasis was associated with a higher Gleason score (p<0.05) as well as a higher frequency of and shorter time to treatment failure. Also, a 10-year Kaplan-Meier biochemical failure-free survival rate of 0% versus 7.7% (MMP-2 positive versus negative) and a mean time to biochemical failure of 2.6 versus 4.0 years were recorded. The expression of MMP-2 in bone marrow micro-metastasis is associated with a higher Gleason score, the presence of CPCs, and a higher frequency of and shorter time to failure and could be clinically useful for identifying men at high risk of treatment failure.

Lymph Node and Bone Marrow Micrometastases Define the Prognosis of Patients with pN0 Esophageal Cancer.

Background: Pathological routine lymph node staging is postulated to be the main oncological prognosticator in esophageal cancer (EC). However, micrometastases in lymph nodes (LNMM) and bone marrow (BNMM) are discussed as the key events in tumor recurrence. We assessed the prognostic significance of the LNMM/BNMM status in initially pN0 staged patients with curative esophagectomy. Methods: From 110 patients bone marrow aspirates and lymph node tissues were analyzed. For LNMM detection immunohistochemistry was performed using the anticytokeratin antibody AE1/AE3. To detect micrometastases in the bone marrow a staining with the pan-keratin antibody A45-B/B3 was done. Results were correlated with clinicopathologic parameters as well as recurrence and death during follow-up time. Results: Thirty-eight (34.5%) patients showed LNMM, whereas in 54 (49.1%) patients BNMM could be detected. LNMM and BNMM positive patients showed a correlation to an increased pT category (p = 0.017). Univariate and multivariate analyses revealed that the LNMM/BNMM status and especially LNMM skipping the anatomical lymph node chain were significant independent predictors of overall survival and recurrence-free survival. Conclusions: This study indicates that routine pathological staging of EC is insufficient. Micrometastases in lymph nodes and the bone marrow seem to be the main reason for tumor recurrence and they are a strong prognosticator following curative treatment of pN0 EC.

Subtypes of Minimal Residual Disease and Outcome for Stage II Colon Cancer Treated by Surgery Alone

Introduction: 25% of stage II colon cancer (CC) patients relapse within 5 years due to minimal residual disease (MRD) not eliminated by surgery. We hypothesize that subtypes of MRD, defined by circulating tumor cells (CTCs) and bone marrow micro-metastasis (mM) have different types and kinetics of relapse. Methods and Patients: One month after curative surgery for stage II CC, blood and bone marrow samples were taken to detect CTCs and mM using immunocytochemistry with anti-CEA. Follow-up was for up to 5 years or relapse, defined as new images detected on CT scan. Survival curves using Kaplan-Meier (KM) and restricted mean survival times (RMST) were calculated for 3 prognostic groups; A: MRD (-); B: mM (+) CTC (-) MRD and C: CTC (+) MRD. Results: 181 patients (82 men) participated, mean age 68 years, median follow up 4.04 years; A: N=105, B: N= 36, C: N=40. For the whole cohort 5yr KM was 70%, median survival not reached, Group A: 98%, B 63%, C 7%, median survivals for Group A and B were not reached. RMST for the whole cohort 4.1 years, Group A 4.9 years, B 4.1 years, C 1.7 years. Serum CEA was significantly higher in Group C. No significant differences for sex or age between groups were detected. Conclusions: MRD subtypes define relapse patterns, Group A MRD (-) had an excellent prognosis, 98% disease free survival at 5 years, Group B had a similar outcome for up to 2 years post-surgery, then increasing relapse occurred (late failure), while Group C had a high risk of early failure. MRD sub-classification may be useful to define risk of relapse in stage II CC patients, which patients may benefit or not from additional therapy and warrants further studies with a larger number of patients. Funding Statement: The study was funded by a Western Metropolitan Health Authority Research Grant. Declaration of Interests: Dr Murray has received consultancy fees from ViatarCTC Solutions, Boston, USA. All other authors reported no conflicts of interest. Ethics Approval Statement: The study was approved by the local ethics committee and fully complied with the Declaration of Helsinki and Chilean law on patient’s rights. All patients provided written informed consent.

Effect of FOLFOX on minimal residual disease in Stage III colon cancer and risk of relapse.

Introduction25% of Stage III colon cancer patients relapse within 5 years due to minimal residual disease (MRD) not eliminated by surgery and chemotherapy. We hypothesise that sub-types of MRD, defined by circulating tumour cells (CTCs) and bone marrow micro-metastasis (mM) have different types and kinetics of relapse.Patients and MethodsOne month of curative surgery and 1 month after completing six cycles of FOLFOX chemotherapy blood and bone marrow samples were taken to detect CTCs and mM using immunocytochemistry with anti-carcino-embryonic antigen (CEA). Follow up was up to 5 years or disease progression defined as new images on CT scanning. Survival curves using Kaplan–Meier (KM) and Restricted Mean Survival Time (RMST) were calculated for three prognostic groups: CTC and mM negative, CTC negative mM positive, and CTC positive.Results76 patients (39 men) participated, mean age 67 years, median follow-up 3.6 years. The response to chemotherapy was heterogeneous and MRD pre-treatment did not predict response to therapy. Of 21 patients MRD (−), 20 remained MRD negative and one patient became mM (+); of 21 patients mM (+), 10 became MRD (−), 8 remained the same and 3 became CTC (+); of the 34 CTC positive, 8 became MRD (−), 8 with only mM, and 18 remained positive.After chemotherapy, 38 patients were negative for CTC and mM, 17 were positive for only mM, and 21 for CTCs. For the whole cohort, the 5 year KM was 58%, the median survival was not reached. For the three prognostic groups, the KM 5-year survivals were 87%, 58%, and 4%, respectively, the median survival for patients MRD negative and mM only was not reached. RMST for the whole cohort was 3.6 years, for the three prognostic groups the RMST was 4.6 years, 4.0 years, and 1.5 years, respectively. Serum CEA was significantly higher pre-surgery in the CTC positive group. There were no significant differences with respect to age or sex between the three groups.ConclusionsMRD subtypes pre-chemotherapy did not predict treatment response. Post-chemotherapy MRD subtypes were associated with the pattern of failure and time to failure. MRD negative patients had an excellent prognosis with 87% disease-free survival at 5 years. Those with only mM had a similar outcome up to 2 years and then were at increasing risk of late failure. Patients who were CTC positive had a high risk of early failure. MRD subclassification may be useful to define the risk of relapse in Stage III colon cancer patients and warrants further studies with a larger number of patients.

Abstract 29: RANK-RANKL signaling inhibition delays early breast cancer bone metastasis formation

Abstract Denosumab is a fully human monoclonal antibody to human RANKL, currently approved in the management of established bone metastases, mainly due to its anti-resorptive activity which blocks tumor-induced osteoclastogenesis and osteolysis. Our aims are to determine the role of RANK expression in breast cancer (BC) bone metastasis and the efficacy of RANKL blocking by AMG161 (IgG1 equivalent to Denosumab). AMG161 does not recognize murine RANKL, so we use a BalB/c nude knock-in (KI) mouse model, which expresses a chimeric murine/human RANKL. Moreover, non-invasive in vivo imaging techniques such as bioluminescence and µCT were used to monitor metastatic tumor burden and bone degradation, respectively. To evaluate bone marrow early colonization by BC cells, DiD labelling and ex vivo laser scanning multiphoton confocal microscopy were used. Long-term studies showed that intra-arterial (i.a.) injection of MDA-MB231 RANK versus MDA-MB231 leads to a poorer overall survival (OS), with the most striking differences seen in the earlier time-points. In this respect, the extent of radiographic osteolytic lesions was higher in the RANK overexpressing group than in the control group. Similar findings were observed following intra-tibial inoculation of RANK-expressing MCF-7 cells, when compared to that observed with parental MCF-7 cells. However, at later time points, bone and lung metastatic burden was similar between the two groups, suggesting that the poor OS of the RANK overexpressing group was due to a faster tumor progression owing to enhanced bone homing and colonization. Indeed, using short-term studies (5 and 14 days), i.a. injection of MDA-MB231 RANK led to higher incidence and extension of bone marrow micro-metastases than MDA-MB231, whereas there was no difference for lung micro-metastases. Daily subcutaneous injections of 1,5mg/kg AMG161 antibody to MDA-MB231RANK tumor-bearing animals, decreased bone micro-metastases and early bone marrow colonization without affecting lung micro-metastasis. We therefore hypothesized that RANK overexpressing BC cells interact with RANKL produced by osteocytes, osteoblasts and other cells in the osteogenic niche, favoring survival and colonization of BC cells in bone. To test this hypothesis, we made use of heterotypic mammosphere assays in which BC cells were cultured with or without cells of the osteogenic niche (e.g. pre-osteoblasts, mesenchymal stem cells), recombinant RANKL and anti-RANK antibody or AMG161. Co-culture with recombinant RANKL or a cellular source of RANKL increased BC cell mammosphere formation, and blocking RANK/RANKL signaling impaired it. In conclusion, RANK/RANKL signaling promotes the early engraftment of BC cells in the bone marrow. Blocking this signaling with AMG161 decreases bone marrow micro-metastasis formation in vivo, suggesting that adjuvant treatment of early-stage breast cancer patients with denosumab may prevent bone relapse. Citation Format: Sofia Sousa, Evelyne Gineyts, Sandra Geraci, Martine Croset, Philippe Clézardin. RANK-RANKL signaling inhibition delays early breast cancer bone metastasis formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 29.

Role of Immunohistochemistry in Early Detection of Bone Marrow Micrometastases in Breast Cancer

BackgroundIn Egypt, breast cancer (BC) accounted for 37.7% as reported from WHO in 2008. Mammaglobin (MAM) is BC specific marker, which is almost expressed in breast epithelial cells. It is over expressed in subset of 70–80% of primary and metastatic BC tissues and can be used for identification of BC cells in bone marrow (BM).ObjectivesTo evaluate the significance of immunohistochemical (IHC) method applied on bone marrow trephine biopsy (BMB) in increasing the detection capacity for BC micrometastases in apparently non stage‐IV patients. To compare these results with those obtained by cytological examination of BM aspirate and the different biochemical marrow investigations.Materials and MethodsThirty prospective cases, presented with different stages (0‐III) of BC and who received no chemotherapy, were selected from Alexandria Armed Forces Hospital, Egypt, during the period from March 2014 to December 2015. All patient were subjected to the following work up: Reviewing patients' medical records for all clinical and demographic data. BM cytological examination (at least 6–8 Leishman's‐stained BMA smears) was meticulously scanned for any sheets of non‐haemopoietic malignant cells. Histological examination of BM core biopsy (at least 2.5 cm long); minimum of 2 H&amp;E stained sections were thoroughly examined by two histopathologists for any sheets of nonhaemopoietic malignant cells. Morphological interpretation was guided by the standardized morphological criteria. IHC staining was performed on the BMB sections using MAM monoclonal antibody. CA15‐3 serum and BMA plasma levels were assessed using the fully automated Abbott “Axsym” system. Results 22 patients were diagnosed with infiltrating duct carcinoma (IDC) (73.3%), while the rest (26.7%) were diagnosed with infiltrating lobular carcinoma (ILC). Three patients with IDC were classified as grade I (13.6%), 10 as grade II (45.5%), and 9 as grade III (40.9%). Regarding the stage, 2 patients (6.7%) presented with stage 0, 6 patients (20%) with stage I, 14 patients (46.7%) with stage II and 8 patients (26.7%) with stage III (Figure 1). Examination of the BM aspirates of the 30 patients were completely negative (100%), while examination of the H&amp;E stained sections from the BMB were as follows: 5 patients (16.7%) were positive for BM micrometastases showing hypercellularity, fibrosis, inflammatory infiltration (plasma cells, macrophage and eosinophils), angiogenesis and osteoclastic hyperplasia (Figure 2), while 25 patients (83.3%) were negative. 16/30 cases (53.3%) yielded positive results for the MAM IHC staining on BMB (Figures 3 and 4). There was statistical significant differences between using MAM IHC staining and BMB H&amp;E examination (P=0.0013). By measuring CA15.3 in both serum and BM plasma, there was no statistical significant difference regarding serum and BM plasma CA15.3. Conclusions Superiority of BMB over BMA in detection of BM micrometastatic disseminated tumor cells (DTCs) in patients diagnosed with primary BC. Combining BMB with IHC staining using MAM significantly increases the detection capacity for DTCs in the BM when diagnosing patients with primary BC. In spite of the correlation present between BM plasma and serum levels of CA15‐3, neither CA15‐3 serum levels nor BM plasma levels could be reliable markers for diagnosing or detecting early occult micrometastatic DTCs in the BM at initial diagnosis of primary BC This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Minimum Residual Disease in Patients Post Radical Prostatectomy for Prostate Cancer: Theoretical Considerations, Clinical Implications and Treatment Outcome

Introduction:Minimal residual disease (MRD) remaining after curative therapy for prostate cancer has the potential for growth and can result in metastasis. Circulating prostate cells (CPCs) and bone marrow micro-metastasis (mM) could represent different types of MRD. We here determined; biochemical failure free survival rates; time to BF after 10 years follow-up; and the presence of CPCs and mM in patients treated with radical prostatectomy (RP) for prostate cancer.Methods and Patients:One month after RP, blood and bone marrow were sampled for assessment of CPCs and mM. Cases were classified as: group A, CPC negative and mM negative; group B, CPC negative and mM positive; Group C, CPC positive and mM negative; and Group D, CPC positive and mM positive. Subjects were followed with serial determination of PSA levels, recording the time at which BF occurred defined as a serum PSA >0.2ng/ml. After ten years of follow-up Kaplan-Meier survival curves were generated and the restricted mean survival time (RMST) for each group calculated.Results:A total of 321 men participated, 140 in group A with survival of 92.7% (86.3 to 96.2), 39 in group B with 55.8% (37.2 to 70.9); 54 in group C with 6.41% (1.19 to 18.21) and 88 in group D with 4.96%(1.64 to 11.13%. The RMST (in years) were: group A, 9.47 (9.24 to 9.69); group B, 9.23 (8.87 to 9.58); group C, 4.62 (4.46 to 4.77); and group D, 3.57 (3.52 and 3.63) (p-value<0.001 between groups: A versus C and D, B versus C and D).Conclusions:CPC positive men have more aggressive disease, with increased early failure; men who are only positive for mM are at greater risk of late failure. These two forms of MRD represent different clinical entities with respect to biochemical failure and could be used to guide clinical treatment decisions.

The Results of Stricter Inclusion Criteria in an Immunomagnetic Detection Study of Micrometastatic Cells in Bone Marrow of Uveal Melanoma Patients - Relevance for Dormancy.

Approximately 50% of uveal melanoma patients develop metastases. We want to evaluate the effect of stricter criteria on our data from our previous study correlating survival and bone marrow (BM) micrometastasis results using our immunomagnetic separation (IMS) method. Mononuclear cell fractions (MNC) isolated from BM were examined for tumour cells and the patients were classified as BM positive (BM+) or BM negative (BM-). The study originally included 328 consecutive patients with uveal melanoma from 1997 to 2006. The cohort was limited to 217 patients when we introduced cyto- or histopathological verification of melanoma cells in the patient as a main new criterion for inclusion. Tumour cells were found in BM-samples in 38.7% (95% CI, 32-45) at enrolment. Until the latest work-up 43.8% (95% CI, 38-50) of patients had developed melanoma metastases. After a minimum follow-up time of 8.5years, 60.4% (95% CI, 54-66) of patients had died. The causes were: melanoma metastases 69.5%, another type of cancer 5.4% and non-cancerous causes 19.5%. Overall median survival was shorter for the BM- patients (11.3years) (95% CI, 10-12) compared to the BM+ (16.5years) (95% CI, 12-14), p=0.04, log rank test. All-cause mortality and specific melanoma mortality estimated after 12year follow-up showed a highly significant difference comparing BM- and BM+, p=0.010 and p=0,017, respectively. IMS yields a high fraction of BM+ samples due to micrometastasis at diagnosis and these cells appear to have a positive prognostic impact strengthening our previous report. The late recurrences support the concept of tumour dormancy.

Tracing the Seeds in the Soil.

Featured Article: Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC et al. A pooled analysis of BM micrometastasis in breast cancer. N Engl J Med. 2005;353:793–802.2 In our article featured here, we reported in a pooled analysis of >4700 patients with primary breast cancer (stage M0) that the presence of disseminated tumor cells (DTCs)3 in the bone marrow (BM) at the time of diagnosis was an independent predictor of poor prognosis. Metastasis formation in secondary organs, which is the major cause of death from solid tumors, originates from tumor seeds released from the primary lesion before it is removed by the surgeon. Our initial efforts to trace DTCs in BM were largely motivated by the fact that the bones are major sites of distant metastasis in breast cancer (and other solid tumors such as prostate and lung cancer). Moreover, the BM is accessible by needle aspiration, an invasive but safe diagnostic approach used for decades in patients with hematological malignancies. Finally, the BM is a mesenchymal organ, and breast cancers (as most other …