Presence Of Micrometastases Research Articles

Micrometastasis in lymph nodes of colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Postoperative adjuvant chemotherapy is recommended for node‐positive stage III patients. A systematic meta‐analysis reported that the presence of micrometastases in regional lymph nodes (LNs) was associated with poor survival in patients with node‐negative CRC. Because most data employed in the meta‐analysis were based on retrospective studies, we conducted a prospective clinical trial and concluded that stage II is a transitional zone between stage I and stage III, where CRC tumors continuously increase the micrometastasis volume in LNs and proportionally raise the risk for tumor recurrence. The one‐step nucleic acid amplification (OSNA) assay is a simple and rapid technique to detect CK19 mRNA using the reverse‐transcription loop‐mediated isothermal amplification (RT‐LAMP) method. Using the OSNA assay, we and colleagues reported that the upstaging rates of pStages I, IIA, IIB, and IIC were 2.0%, 17.7%, 12.5%, and 25%, respectively, in 124 node‐negative patients. Survival analysis indicated that OSNA positive stage II CRC patients had a shorter 3‐y disease‐free survival rate than OSNA negative stage II CRC patients. In 2017, AJCC TNM staging (the 8th version) revised the definition of LN metastasis in colon cancer and it is stated that micrometastasis should be considered as a standard LN metastasis. To our surprise, this revision was based on a meta‐analysis to which our previous study on micrometastasis largely contributed. The remaining questions to be addressed are how to find micrometastases efficiently and whether postadjuvant chemotherapy is effective to prevent disease recurrence and to contribute to longer survival.

Abstract P1-06-01: Insights from rapid autopsy shed light on mechanisms of cancer dissemination in metastatic breast cancer

Abstract Previously we presented our initial findings from a 9-patient rapid autopsy pilot for metastatic breast cancer (MBC). At the time of procurement, one third of subjects exhibited clinically unidentified diseased sites in organs not commonly associated with breast cancer metastases, including ovary, kidney, and pancreas. In two other instances, “resolved” bone specimens (as measured by absence of FTG uptake in PET/CT imaging) were later determined to be >30% tumor positive when assessed by a pathologist. We now expand upon these findings in a more in-depth exploration of the presence of micro-metastases in presumed tumor-negative tissues. A subset of tumor-free tissues were selected from each patient (average of 10 specimens per patient). All selected specimens were negative by clinical imaging, appeared grossly normal at procurement, and were reported to be tumor negative by H&E assessment by a clinical pathologist. We included organs both commonly and uncommonly involved in MBC, including lung, bone, spleen, pancreas, kidney, and non-tumor draining lymph nodes. Tissues were stained for one or more of the markers, pan-cytokeratin, GATA-3, HMFG, MUC1, and ER (if patient was previously ER+), depending on tissue type. Of the 87 total specimens assessed, we identified micro-metastases in 13 specimens from 4 individual patients. Across these 4 patients, micro-metastases were found in lung, bone, pancreas, spleen, and several non-tumor draining lymph nodes. While lung and bone are commonly involved in MBC and these results are not entirely surprising, pancreas and spleen involvement is extraordinarily rare. Further surprising was the identification of micro-metastases in several lymph nodes that were not located anatomically downstream from a disease-involved organ. Image patterns demonstrate tumor cell infiltration into the lymph node within the subcapsular sinus. Presence of micro-metastases in tumor-negative tissue did not correlate with tumor hormone status or cancer type (e.g. lobular vs DCIS). Combined with our previous findings, we now report unexpected and clinically undiagnosed disease involvement in 6/9, or two-thirds, of our patients. Based on these findings, we hypothesize that cancer stem cells and/or micro-metastases are present throughout the body, in all tissue types, and that their ability to grow into tumors is regulated by the local immune microenvironment. Lastly, the differing roles and mechanics of lymphatic vs hematological spread in metastatic disease has long been discussed. Our findings provide strong evidence for cancer dissemination through the lymphatics system. Further study is necessary to better understand the timing of metastatic spread, whether systemic dissemination occurs early or later in disease, and if conducive metastatic or pre-metastatic niches are already present throughout the body at the time of primary diagnosis or if these permissive environments develop slowly overtime. Citation Format: Eliza R. Bacon, Kena Ihle, Colt Egelston, Weihua Guo, Diana Simons, Dan Schmolze, Christina Wei, Lusine Tumyan, Peter P Lee, James R. Waisman. Insights from rapid autopsy shed light on mechanisms of cancer dissemination in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-01.

Abstract PS14-27: Oncogenetic caracterization and imunohistochemistry patterns of male cancer breast diagnosticated in Haroldo JuaÇaba hospital, Northeast of Brazil

Abstract INTRODUCTION: The male breast cancer (MBC) is a rare disease, responsible for about 0.2% of all cancers and 0.1% of deaths for male cancer. Despite the rarity of the disease, statistics indicate that the incidence of MBC has been increasing significantly. The main risk factors for the development of MBC include old age, hormonal imbalance, radiation exposure, and family history of breast cancer. Hereditary breast and ovarian cancer (HBOC) is a syndrome associated with mutations in the BRCA1 and BRCA2 genes that have been related to MBC that as a rare disease, still poorly understood. METHODOLOGY: Family history analyzes, type and tumor staging, molecular markers expression, hormone receptors (RE, RP, and HER2), and cell proliferation marker (KI67) analysis were performed by immunohistochemistry. The presence of genetic mutations and the frequency of these mutations were evaluated in 31 genes of HBOC suspected patients treated at the Haroldo Juaçaba Hospital between the years 2009 to 2020. RESULTS: A total of 236 patients were diagnosed with breast cancer and submitted to a genetic test. Six of them were male patients aged between 54 and 73 years. His family history indicated that 83.3% had first-degree relatives affected by breast cancer. Different pathological staging was founded after tumors evaluation with the presence of micrometastases (P3) and higher levels of invasion in the lymph nodes. The expression of the cell proliferation marker (KI67) indicated low levels of expression except for patient P5. All patients presented expression of two hormone receptors (ER and RP) and no expression for HER2, except for one patient (P1), who had bilateral breast cancer, with HER2 expression (+2) on the right breast (first tumor) and no RP and HER2 expression in the left breast (second tumor), besides a moderate expression of KI67 (40%) (Table 1). The oncogenetic evaluation indicated that 83.33% of the patients had mutations described in the clinical database (ClinVar) 60% of them were pathogenic. The variants founded were in the PALB2 (16.66%), BRCA1 (33.33%), and BRCA2 (33.33%) genes (Table 1). The pathogenic mutations founded were located in the BRCA 1 and BRCA2 genes. One patient (P5) did not present mutations or VUS. The patient without the mutation (P5) presented a tumor with T3N1 pathological staging, low expression of estrogen (10%) and progesterone (10%) receptors, absence of HER2 expression, and high capacity of proliferation, indicated by the expression of KI67 (80%). The results indicate heterogeneity in the histological and molecular patterns of the patients evaluated, besides the oncogenetic patterns that may be associated with HBOC, especially those whose variants founded were pathogenic.CONCLUSION: Due to the low frequency of male breast cancer, the oncological data of these patients are relevant for epidemiological, pathological, and oncogenetic characterization, improving the characterization and identification of patterns for this pathology.Research Sponsor: Ministerio da Saude, Brasil (PRONON) Table 1. Histopathologic molecular markers and variation gene characterization of male breast cancer evaluated in Hospital Haroldo Juaçaba, Northest of Brazil, between 2009 and 2020. *VUS #Pathogenic variation.AgeFamiliar historyMolecular markersVariationPatientDiagnosticLast clinical evaluationBCOtherSubtype tumorPathological stagingRERPHER2KI67GeneSpecificityP14857YesPancreas; CCPLUMINAL A(R) pT1cpN1a(L) pT1B(R) 80%(L) 80%(R) 20%(L) negative(R) +2(L) negative(R) 18%(L) 40%PALB2*c. 3257G>A:Arg1086GlnP27274YesNoLUMINAL B(L)pT2 pN3a90%100%Negative20%BRCA1*c. 754C>T:Arg252CysP36466NoNoLUMINAL ApT2pN1mic90%70%Negative10%BRCA1#c. 5266dupC: p.Gln1756Pro fs74P45457YesNoLUMINAL A(L)T4bN290%90%Negative10%BRCA2#c. 4808delA: pAsn1603Thr fs14P55858YesProstaticLUMINAL A(R) T3N110%10%Negative80%Not detectedNot detectedP65858YesProstaticLUMINAL B(R) T4dN060%60%Negative30%BRCA2#c. 4808delA: pAsn1603Thr fs14 Citation Format: Maria Claudia dos Santos Luciano, Maria Júlia Barbosa Bezerra, Isabelle JoyceLima Silva-Fernandes, Paulo Goberlanio de Barros Silva, Clarissa Gondim Picanço-Albuquerque, Rosane Oliveira Sant´anna, Francisca Fernanda Barbosa Oliveira, Flavio Silveira Bitencourt, Marcos Venicio Alves Lima. Oncogenetic caracterization and imunohistochemistry patterns of male cancer breast diagnosticated in Haroldo JuaÇaba hospital, Northeast of Brazil [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS14-27.

Does the presence of nodal micrometastases or isolated tumor cells increase the risk of recurrent endometrioid adenocarcinoma? A case control study

Objective: The purpose of this case control study was to explore whether the presence of micrometastases (MMs) or isolated tumor cells (ITCs) is associated with an increased the risk for recurrence in women with fully staged, stage I–II endometrioid endometrial cancer.

Micrometastases in Sentinel Lymph Nodes Represent a Significant Negative Prognostic Factor in Early-Stage Cervical Cancer: A Single-Institutional Retrospective Cohort Study.

The data on the prognostic significance of low volume metastases in lymph nodes (LN) are inconsistent. The aim of this study was to retrospectively analyze the outcome of a large group of patients treated with sentinel lymph node (SLN) biopsy at a single referral center. Patients with cervical cancer, stage T1a-T2b, common tumor types, negative LN on preoperative staging, treated by primary surgery between 01/2007 and 12/2016, with at least unilateral SLN detection were included. Patients with abandoned radical surgery due to intraoperative SLN positivity detected by frozen section were excluded. All SLNs were postoperatively processed by an intensive protocol for pathological ultrastaging. Altogether, 226 patients were analyzed. Positive LN were detected in 38 (17%) cases; macrometastases (MAC), micrometastases (MIC), isolated tumor cells (ITC) in 14, 16, and 8 patients. With the median follow-up of 65 months, 22 recurrences occurred. Disease-free survival (DFS) reached 90% in the whole group, 93% in LN-negative cases, 89% in cases with MAC, 69% with MIC, and 87% with ITC. The presence of MIC in SLN was associated with significantly decreased DFS and OS. Patients with MIC and MAC should be managed similarly, and SLN ultrastaging should become an integral part of the management of patients with early-stage cervical cancer.

Abstract OT3-01-03: Neonod Study: Conservation of axillary lymph nodes in the presence of micrometastases in the sentinel lymph node if cN- After neoadjuvant chemotherapy

Abstract Background: In the last 20 years it was observed a clear trend to develop and perform less and less demolitive axillary surgery in breast cancer patients. The development of identification, dissection and intraoperative analysis of sentinel lymph node (SLNB, Sentinel Lymph Node Biopsy) contributed to give all informations on the need to proceed or not to axillary lymph node dissection (ALND), while neoadjuvant chemotherapy (NAC) permitted to enable surgical treatment in initially non-operable cancers or to reduce tumor size in order to perform a less demolitive surgery. In Patients with residual axillary disease after NAC, it is not completely clear if the prognosis worsens according to the entity of residual disease (isolated tumor cells or ITC, micrometastases, macrometastases). In particular, IBCSG 23-01 study demonstrated that, in patients with ypN1mi early breast cancer, axillary lymph node dissection (ALND) could be avoided without worsening the prognosis. Assuming that patients cN+ before, but ypN1mi after NAC are clinically equivalent to patients in IBSCG study, complete ALND would be useless even if in patients subjected to NAC for that subgroup. Still it is to demonstrate if axillary micrometastases after NAC could have a prognostic value similar to micrometastasis in patients subjected to adjuvant treatment. To overcome these limitations, we propose this non-controlled clinical study to verify the effect of avoiding ALND on relapse and survival rate in cN+ patients, turned ycN0 (clinically) and ypN1mi (pathologically) after NAC. Materials and methods: This is a non-controlled clinical study designed as a non-inferiority study to verify if the avoidance of ALND in ypN1mi patients after NAC does not involve a significant worsening of survival or locoregional or distant relapse risk compared to ypN0 patients, in which the avoidance of ALND actually represents the standard treatment. Patients with macrometastatic SLN (ypN≥1) after NAC subjected to ALND as a standard procedure represents an internal control group for the demonstrated worse prognosis compared to ypN0 and ypN1mi patients. This study includes patients with cN+ lymph nodes assessed in the initial diagnosis, resulted negative (with clinical and instrumental studies) after NAC. On the base of histopathologic definitive evaluation on harvested sentinel lymph nodes, patients will be allocated in one of the two groups (standard or experimental). Group 1 (experimental) will include patients with micrometastatic SLN or micrometastatic parasentinel lymph node (ypN1mi), in which ALND will not be performed. Group 2 (standard) will include patients with negative SLN or SLN with ITC in it (ypN0/ypN0(i+)). ALND will not be performed in these patients. Patients with macrometastatic SLN or another macrometastatic lymph node (ypN≥1) will be included in a third internal control group (group 3) not utilized in statistical comparison with the other two groups, but finalized to the evaluation of the case study’s appropriateness. In these patients, standard ALND will be performed. Patients enrolling in the study protocol will last 3 years. Patients should be studied for at least 5 years with clinical and instrumental follow-up periodic controls, prevised from actual standard guidelines. Endpoints: The primary endpoint of the study is the disease free survival rate (DFS) and will be evaluated from the entry date in the study to the date of the last contact, the date of the distant or locoregional relapse, or the date of death for any cause. Secondary endpoints are overall survival (OS), locoregional disease-free survival (LRDFS) and distant disease-free survival (DDFS) Citation Format: Corrado Tinterri, Emilia Marrazzo, Wolfgang Gatzemeier, Erika Barbieri, Andrea Sagona, Alberto Bottini, Alberto Testori, Valentina Errico, Giuseppe Canavese. Neonod Study: Conservation of axillary lymph nodes in the presence of micrometastases in the sentinel lymph node if cN- After neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT3-01-03.

High serum levels of periostin are associated with a poor survival in breast cancer.

Periostin is a secreted extracellular matrix protein, which was originally described in osteoblasts. It supports osteoblastic differentiation and bone formation and has been implicated in the pathogenesis of several human malignancies, including breast cancer. However, little is known about the prognostic value of serum periostin levels in breast cancer. In this study, we analyzed serum levels of periostin in a cohort of 509 primary, non-metastatic breast cancer patients. Disseminated tumor cell (DTC) status was determined using bone marrow aspirates obtained from the anterior iliac crests. Periostin levels were stratified according to several clinical parameters and Pearson correlation analyses were performed. Kaplan-Meier survival curves were assessed by using the log-rank (Mantel-Cox) test. To identify prognostic factors, multivariate Cox regression analyses were used. Mean serum levels of periostin were 505 ± 179pmol/l. In older patients (> 60years), periostin serum levels were significantly increased compared to younger patients (540 ± 184pmol/l vs. 469 ± 167pmol/l; p < 0.0001) and age was positively correlated with periostin expression (p < 0.0001). When stratifying the cohort according to periostin serum concentrations, the overall and breast cancer-specific mortality were significantly higher in those patients with high serum periostin (above median) compared to those with low periostin during a mean follow-up of 8.5years (17.7% vs. 11.4% breast cancer-specific death; p = 0.03; hazard ratio 1.65). Periostin was confirmed to be an independent prognostic marker for breast cancer-specific survival (p = 0.017; hazard ratio 1.79). No significant differences in serum periostin were observed when stratifying the patients according to their DTC status. Our findings emphasize the relevance of periostin in breast cancer and reveal serum periostin as a potential marker for disease prediction, independent on the presence of micrometastases.

KRAS Mutation Predicted More Mirometastases and Closer Resection Margins in Patients with Colorectal Cancer Liver Metastases.

The presence of micrometastases is a source of recurrence after surgical resection of colorectal liver metastases (CRLM). The KRAS mutation is common in colorectal cancer, however the correlation between KRAS status and micrometastases has not been thoroughly clarified. We enrolled a cohort of 251 consecutive CRLM patients who received complete liver surgery with known KRAS mutation status, and collected clinicopathological information, including micrometastases, margin status, preoperative chemotherapy, and liver recurrence-free survival (LRFS) and overall survival (OS) rates. KRAS-mutant (mutKRAS) patients had a higher incidence (60.3 vs. 40.8%; p = 0.002) and higher number of micrometastases [2.0 (range 0-38.0) vs. 0 (range 0-15.0); p < 0.001] than KRAS wild-type (wtKRAS) patients. The micrometastases in the mutKRAS group were more distant than those in the wtKRAS group [0.7 (range 0.1-9.0) vs. 0.6 (range 0.2-5.0) mm; p = 0.018). The mutKRAS group had more involved margin resections (21.5 vs. 9.2%; p = 0.07) and narrower margin widths [2.0 (range 0-40.0) vs. 4.3 (0-50.0) mm; p = 0.002] than the wtKRAS group. In addition, preoperative chemotherapy was associated with a lower rate of micrometastases in mutKRAS CRLM tumors (p < 0.05). mutKRAS status, positive margins, and micrometastases were all related to worse LRFS and OS (p < 0.05); however, micrometastases were not significantly correlated with OS in the multivariate analysis (p = 0.106). mutKRAS patients had more micrometastases, increased R1 resections, and narrower margins. The presence of micrometastases may have led to the narrow margin width observed in these cases.

Incidence and clinical relevance of non-small cell lung cancer lymph node micro-metastasis detected by staging endobronchial ultrasound-guided transbronchial needle aspiration.

Approximately twenty percent of lymph node (LN) negative non-small cell lung cancer (NSCLC) patients who undergo curative intent surgery have pan-cytokeratin immunohistochemistry (IHC)-detectable occult micro-metastases (MMs) in resected LNs. The presence of the MMs in NSCLC is associated worsened outcomes. As a substantial proportion of NSCLC LN staging is conducted using endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), we sought to determine the frequency of detection of occult MMs in EBUS-TBNA specimens and to evaluate the impact of MMs on progression-free and overall survival. We performed retrospective IHC staining for pan-cytokeratin of EBUS-TBNA specimens previously deemed negative by a cytopathologist based on conventional hematoxylin and eosin staining. The results were correlated with clinical variables, including survival outcomes. Of 887 patients screened, 44 patients were identified meeting inclusion criteria with sufficient additional tissue for testing. With respect to the time of the EBUS-TBNA procedure, 52% of patients were clinical stage I, 34% clinical stage II, and clinical 14% stage IIIa NSCLC. Three patients (6.8%) were found to have cytokeratin positive MMs. All 3 MMs detected were at N2 LN stations. The presence of MMs was associated with significantly decreased progression-free (median 210 vs. 1,293 days, P=0.0093) and overall survival (median 239 vs. 1,120 days, P=0.0357). Occult LN MMs can be detected in EBUS-TBNA specimens obtained during staging examinations and are associated with poor clinical outcomes. If prospectively confirmed, these results have significant implications for EBUS-TBNA specimen analyses and possibly for the NSCLC staging paradigm.

Abstract P4-08-16: Validation of the AJCC eighth edition prognostic stage compared with the anatomic stage for breast cancer with a Japanese single-institutional cohort

Abstract Background: The American Joint Committee for Cancer (AJCC) 8th edition cancer staging system for breast cancer incorporated biologic factors in addition to the 7th edition anatomic stage. We analyzed how the new AJCC 8th edition prognostic stage refined its stratification compared with the anatomic stage. Methods: We reviewed the data of 4,134 patients with stage I to III breast cancer who underwent surgery at Tokyo Metropolitan Komagome Hospital between 2000 and 2016. The anatomic stage and prognostic stage were re-staged according to the AJCC 8th edition staging manual. Patients who received neoadjuvant chemotherapy or had bilateral breast cancer and those with unknown clinicopathologic factors were excluded. The 21-gene Oncotype DX breast recurrence score was not used for staging in this study. Results: A total of 2,469 patients with a median follow-up of 4.7 years (range 0.1-15.5 years) were identified. According to the anatomic stage, there were 1,259 patients of stage IA, 132 of IB, 591 of IIA, 206 of IIB, 130 of IIIA, 14 of IIIB and 73 of IIIC. According to the prognostic stage, there were 1,610 patients of stage IA, 331 of IB, 236 of IIA, 73 of IIB, 85 of IIIA, 43 of IIIB and 27 of IIIC. Sixty-four patients (2.6%) could not be assigned using the new staging system for the presence of micrometastases in lymph nodes with tumors larger than 2 cm. The 5-year disease-free survival (DFS) rates according to the anatomic stage were 97.4% for stage IA, 97.1% for IB, 95.8% for IIA, 86.5% for IIB, 77.9% for IIIA, 49.2% for IIIB and 54.9% for IIIC. According to the prognostic stage, the 5-year DFS rates were 97.9% for stage IA, 92.9% for IB, 91.2% for IIA, 79.8% for IIB, 67.4% for IIIA, 53.3% for IIIB and 38.7% for IIIC. Compared with the AJCC anatomic stage, the prognostic stage was increased in 148 patients (6.2%) and decreased in 808 patients (32.8%). For those in whom the stage changed, the change was by one stage up or down in 463 (19.3%), by 2 stages up or down in 401 (16.7%) and by 3 stages up or down in 92 (3.8%). Of the 1,842 patients with hormone receptor (HR)-positive and human epidermal growth factor 2 (HER2)-negative (HR+/HER2-) disease, 40.5% (745/1842) of cases were downstaged, and 0.7% (1/1842) were upstaged. Discussion: The AJCC 8th edition prognostic staging system provided more refined stratification than the anatomic stage. In the Japanese cohort, the proportion of the downstaging rate was higher than the upstaging rate, and the prognostic evaluation of HR+ patients in particular was improved. Citation Format: Miyamoto H, Saita C, Onishi M, Goto R, Iwamoto N, Honda Y, Aruga T. Validation of the AJCC eighth edition prognostic stage compared with the anatomic stage for breast cancer with a Japanese single-institutional cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-16.

Clinical Significance of Molecular Micrometastasis in the Sentinel Lymph Node of Early-stage Non-Small Cell Lung Cancer Patients.

Metastatic affectation of lymph node is the main prognostic factor in localized lung cancer. A pathologic study of the obtained samples, even after adequate lymphadenectomy, showed tumor relapses for 20% of stage I patients after oncological curative surgery. We evaluated the prognostic value of molecular micrometastasis in the sentinel lymph node of patients with early-stage lung cancer. The sentinel node was marked immediately after performing thoracotomy by peritumorally injecting 0.25 mCi of nanocoloid of albumin (Nanocol1) labeled with Tc-99m in 0.3 mL. Guided by a Navigator1 gammagraphic sensor, we proceeded to its resection. The RNA of the tissue was extracted, and the presence of genes CEACAM5, BPIFA1, and CK7 in mRNA was studied. The significant association between the presence of micrometastasis, clinicopathologic characteristics, and patients' outcome was assessed. Eighty-nine stage I-II non-small cell lung cancer patients were included in the study. Of the 89 analyzed sentinel lymph nodes, 44 (49.4%) were positive for CK7, 24 (26.9%) for CEACAM5, and 17 (19.1%) for BPIFA1, whereas 10 (11.2%) were positive for the 3 analyzed genes. A survival analysis showed no significant relation between the presence of molecular micrometastasis in the sentinel node and patients' progression. The molecular analysis of the sentinel node in patients with early-stage lung cancer shows node affectation in cases staged as stage I/II by hematoxylin-eosin or an immunohistochemical analysis. However, this nodal affectation was not apparently related to patients' outcome.

Abstract P3-08-02: Validation of the online prediction tool PREDICT v. 2.0: A large population-based study in the Netherlands

Abstract Background This study aimed to validate the online prediction tool PREDICT version 2.0 on a large population-based cohort in the Netherlands. Separate analyses were performed to study its validity in specific prognostic subgroups. Methods All women diagnosed with non-metastasised primary invasive breast cancer in 2005, who received surgery as part of their treatment, were selected from the nationwide Netherlands Cancer Registry (NCR). The predicted 5-year and 10-year overall survival (OS) from PREDICT were compared with the observed OS from the NCR, for the overall cohort (separated by ER- and ER+ patients) and for predefined subgroups based on age, T stage, N stage, presence of micrometastases, grade, HER2 status, type of surgery, use and type of adjuvant systemic therapy and generation chemotherapy. Our a priori assumption was that PREDICT accurately predicted OS whenever the differences between predicted and observed outcomes were within a range of 5%, since a difference of 5% or more was considered as clinically relevant. The discriminatory accuracy and goodness-of-fit were determined using the area under the receiver operating characteristic curve (AUC) and the Chi2-test, respectively. Results Our study population consisted of 8,834 patients. Discriminatory accuracy for 5-year OS was good with an AUC of 0.80. For ER- and ER+ patients, the AUCs were 0.75 and 0.79, respectively. Predicted 5-year OS differed from observed by -1.4% (p=0.14) in the entire cohort, by -4.9% (p=0.02) in ER- patients and -0.7% (p=0.53) in ER+ patients. In all predefined subgroups no statistically significant differences between predicted and observed events were observed. Discriminatory accuracy for 10-year OS was good with an AUC of 0.78 for the overall cohort, 0.76 for ER- patients and 0.78 for ER+ patients. Predicted 10-year OS differed from observed by -1.0% (p=0.27) in the entire cohort, by -5.3 (p=0.01) in ER- patients and -0.1% (p=0.92) in ER+ patients. Ten-year OS was overestimated in patients ≥75 years (6.3%, p=0.00), and underestimated in T3 stage (-13.%, p=0.00) and in patients treated with both endocrine therapy and chemotherapy (-6.6%, p=0.02). Conclusion PREDICT accurately predicts 5-year OS in the entire Dutch validation population, and in all predefined subgroups. However, 10-year OS was overestimated for patients ≥75 years, and underestimated for T3 stage and patients treated with both endocrine therapy and chemotherapy. Given PREDICT's intentions to guide treatment decision-making, PREDICT may serve as a reliable prediction tool for the Dutch breast cancer population, but especially in patients ≥75 years, 10-year OS predictions should be interpreted with care. Citation Format: van Maaren MC, van Steekbeek CD, Pharoah PDP, Witteveen A, Sonke GS, Strobbe LJA, Poortmans PMP, Siesling S. Validation of the online prediction tool PREDICT v. 2.0: A large population-based study in the Netherlands [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-08-02.

Lymph node micrometastasis in gallbladder cancer.

Prognosis of gallbladder cancer (GBC) is grim even after curative surgery. Lymph node metastasis is the most important prognostic factor, but distant relapses occurring in their absence point towards additional factor. Lymph node micrometastasis could be one. The present study aimed to evaluate the incidence and clinical significance of lymph node micrometastasis. This is a prospective study of patients undergoing curative surgery for GBC from 1 March 2013 to 30 April 2015, at our institute. All lymph nodes were examined with hematoxylin and eosin and immunohistochemistry against CK7. The incidence of lymph node and its relation to other clinicopathological parameters, recurrence, and survival was evaluated. Out of 589 lymph nodes retrieved from 40 patients, metastasis was seen in 13 (2.20%) nodes from 8 (20%) patients and micrometastasis in 4 (0.68%) nodes from 3 (7.5%) patients. Micrometastases were absent in pT1 tumors (0/10 in pT1 vs. 3/30 in pT2-4) and more common in patients with nodal metastasis (13% vs. 6%). Though the presence of micrometastasis would have upstaged the disease, it did not statistically relate to clinicopathological parameters, recurrence, and survival. Incidence of lymph node micrometastasis in GBC was low and did not correlate with other clinicopathological parameters, recurrence, and survival.

Abstract 4667: mtDNA sequencing in HNSCC as a measurer of intratumoral heterogeneity and lymph node metastasis

Abstract Head and neck squamous cell carcinoma (HNSCC), accounts for 650,000 new cases worldwide. It is notorious for poor prognosis, which reflects its propensity to present as metastatic disease upon diagnosis. The prognosis and treatment regiments vary dramatically upon the manifestation of lymph node (LN) metastasis. Detection of regional LN metastasis is usually based on the clinical examination and imaging, followed by post-operational histological analysis. Unfortunately, current tools for detection of tumor cells in surgically resected LN may, in some cases, miss the presence of micrometastasis. Novel strategies for detection of micrometastasis based on the analysis of tumor-derived genetic aberrations by next generation sequencing (NGS) offer new hope for improved risk assessment and better selection of the treatment regimen. However, detection of mutations in minuscule metastatic lesions comprised of only few neoplastic cells may still fall below the detection threshold of these highly sensitive techniques. By virtue of their clonal nature, higher mutation rate and copy number, assessing tumor-specific mitochondrial DNA (mtDNA) mutations in histologically clean LN may provide a more sensitive diagnostic tool and eventually reduce the false negative rate in patients. Additionally, due to its circular configuration, mtDNA thought to be more stable than genomic DNA, and may also be suitable for sequencing formalin-fixed paraffin-embedded (FFPE) -derived genomic material, an invaluable resource for clinical research. However, current methods for library preparation are still imperfect for detection of low prevalence variants and heteroplasmy with high depth of coverage, especially in highly degraded samples. To this end, we aimed to set up a novel NGS assay for mtDNA analysis in primary and metastatic samples. Amplicon-based NGS library preparation approach, consisting of 148 primers pairs that cover the entire mitochondrial genome (with a dual coverage of 86.18%) was designed in collaboration with Fluidigm to be used on Access Array™ platform. To enhance the coverage of degraded material the amplicon size was limited to ~170 base pairs. Primers for three nuclear housekeeping genes were added to the panel to allow quantitative copy number analysis. To address mitochondrial mutational patterns, heteroplasmy and heterogeneity, we have used this novel high-throughput enrichment technology to sequence mtDNA from different areas of 16 primary HNSCC tumors (fresh frozen and FFPE), and multiple matched metastatic or histologically clean lymph-nodes. We obtained over 99% coverage with a median average depth of 3000X and no difference between fresh frozen and FFPE samples. This method allows obtaining a snapshot on the extent of mitochondrial heterogeneity of these lesions and may be used for detection of low frequency tumor-associated mtDNA mutations as a measurer of possible metastatic processes in histologically clean LN. Citation Format: Adrian D. Schubert, Evgeny Izumchenko, William H. Westra, Aditi Chatterjee, Wayne M. Koch, Mohammad O. Hoque, David Sidransky. mtDNA sequencing in HNSCC as a measurer of intratumoral heterogeneity and lymph node metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4667. doi:10.1158/1538-7445.AM2017-4667

Micrometastasis volume in stage II colorectal cancer: A prospective study by Clinical Study Group of Osaka University (CSGO).

597 Background: We reported in a retrospective study that the presence of micrometastasis in lymph nodes (LNs), when assessed by CEA-specific RT-PCR, is a significant prognostic factor in stage II colorectal cancer (CRC). The aim of this study was to clarify the clinical value of micrometastasis in a prospective multicenter trial. Methods: From November 2001 to December 2005, a total of 419 CRC cases were preoperatively registered at a central data center. Of them, 315 node-negative stage II CRC were enrolled. After RNA quality check, 304 CRC cases were analyzed for CEA mRNA in LNs by both conventional RT-PCR (a band method) and quantitative RT-PCR. Long-term prognosis of the patients was determined by each method. Results: A positive band for CEA mRNA was detected in 73 (24.0%) of 304 patients. Post-operative adjuvant chemotherapy was applied in 31 CEA band-positive cases with an oral 5-FU derivative HCFU (1-hexylcarbamoyl-5-fluorouracil) for one year, while chemotherapy was not administered to CEA band-negative group. Multivariate Cox regression analyses revealed that a high micrometastasis volume (High-MMV, n = 95) was an independent poor prognostic factor for 5-year DFS ( P= 0.001) and 5-year OS ( P= 0.016). Conclusions: This prospective clinical trial demonstrates that micrometastasis volume is a useful marker in identifying patients who are at high or low risk for recurrence of stage II CRC.

OA07.03 Prognostic Significance of Micrometastases in Mediastinal Lymph Nodes of Patients With Radically Resected Non-Small Cell Lung Cancer

Recurrence occurs in 30-50 % of patients operated for early stage non-small cell lung cancer (NSCLC), what suggests the existence of occult metastases at the time of surgery. Preoperative detection of occult micrometastases in mediastinal lymph nodes could contribute to better selection of patients appropriate for surgery. This retrospective study was undertaken to determine the prognostic significance of preoperatively detected mediastinal lymph node (LN) micrometastases in patients treated with radical surgical resection for stage I and II NSCLC. From January 2007 to December 2010, 82 patients with stage I and 67 patients with stage II NSCLC underwent transcervical extended mediastinal lymphadenectomy (TEMLA) and subsequent radical pulmonary resection. A total of 4841 mediastinal lymph nodes resected during TEMLA procedure and determined as metastases-free by hematoxylin and eosin staining were labelled to detect occult micrometastases (dual immunohistochemical staining with AE1/AE3 and BerEP4 antibodies). Micrometastases were detected in mediastinal LN of 16 patients (9,7%). 11 patients had only one LN station affected (68,8%). Subcarinal LN were most frequently affected station (11 patients, 68,8%). There was significant correlation between the presence of micrometastases and tumor size. 5-year total survival was significantly better for stage I (64,1%, p=0.0001) and stage II (44,4%, p<0.05) patients without micrometastases comparing to those with micrometastases (18,8%). By multivariate analysis, only the presence of micrometastases was demonstrated to be a significant prognostic factor for 5-year total survival. Presence of micrometastases in mediastinal LN of patients with radically resected stage I and II NSCL is associated with significantly reduced 5-year total survival. Preoprative detection of micrometastases with immunohistochemical staining of mediastinal LN resected during TEMLA procedure improves staging and may contribute to better patient selection for curative surgery.

Triple negative breast cancer at the Jos University Teaching Hospital

Background: Breast cancer remains the leading cause of cancer morbidity and mortality in women worldwide. The treatment of breast cancer has drifted from radical mastectomy, to conservative treatment owing to the presence of micro metastasis at even earlier stages of the disease. These novel medical treatments are hinged on characterizing breast cancer at the molecular level. Estrogen receptor (ER), Progesterone receptor (PR), and Human epidermal growth factor 2 receptor (HER2) are the commonest molecular-markers used. Failure to stain for these three receptors, earns a breast cancer the triple negative designation, an aggressive type of the disease with the worst prognosis. Materials and Methods: All histologically confirmed cases of breast cancer at Jos University Teaching Hospital, between 2010 and 2012, were retrieved, and their histological type and grade documented. Using the Avidin-biotin method, the status of each case was established for ER, PR, and HER2. Cases with negative staining for these three antibodies were further stained with CK5/6, a basal biomarker for Triple Negative Breast Cancer (TNBC). Statistical softwares were used to analyse the data and present in tables. Results: Sixty-three cases of female breast cancers met the inclusion criteria. The age range was 28-74 years with mean age of 46.7±11.1 years. Invasive carcinoma (no special type) was the majority of histological type of breast cancer, accounting for 54(85.7%) cases. Histological grades 1, 2 and 3, recorded 18(28.6%), 29(46.0%), and 16(25.4%), cases respectively. TNBC accounted for 26 (41.3%) cases. Fifteen (57.7%) cases of TNBC stained positively for CK 5/6. Conclusion: Indicators of worse prognosis which includes lower hormone receptor status and high percentage of TNBC, as well as greater number of basal subtype of TNBC was exhibited by the study population.

The impact of inguinal lymph node micrometastases in patients with vulvar cancer.

Inguinofemoral metastases are a major determinant of vulvar cancer relapse. Until today, the impact of micrometastases of inguinal nodes on local recurrence rates of patients with vulvar cancer remains unknown. The purpose of this retrospective study is to evaluate the rates of micrometastases in a series of patients with vulvar cancer treated with radical vulvectomy and inguinofemoral LND and to assess the probability of cancer relapse among this specific category. We conducted a retrospective observational study on patients with vulvar cancer who attended the gynaecological department of Anticancer Hospital of St. Savvas between January 1989 and January 2007. Ultra-staging of lymph nodes for micrometastases was performed after cutting the remaining specimens with a microtome in multiple slices of 3μm. Subsequently they were stained with traditional hematoxylin and eosin and CK AE1/AE3 antibodies for immunohistochemichal analysis. Ninety-two patients with primary vulvar malignancies were included in the present retrospective study. Ultrastaging of the lymph nodes revealed micrometastases in five patients (5.4%). Neither the duration of the procedure, nor the number of retrieved lymph nodes was directly associated with the presence of micrometastases. The patients were followed up for more than 5years. Sixteen recurrences (17.4%) occurred during this period. The presence of micrometastases did not influence the recurrence rate (OR 3.57, 95% CI 0.55-23.36, p=0.184). Ultrastaging of inguinal nodes does not seem to add any benefit in the prediction of local recurrence rates. Future multicenter studies are needed in the field to corroborate our findings.

Micrometastasis in colorectal cancer: does it affect staging?

Micrometastasis (MM) in colorectal cancer (CRC) is not considered in tumour-node-metastasis (TNM) staging currently and also the prognostic significance of these occult tumoural cells is not proven yet. This study was designed to find whether MM to bone marrow (BM) and/or liver cause upstaging of the tumour according to conventional staging system and if it may alter the survival rate. Thirty-eight CRC patients who were candidates of surgical procedure were enrolled in our cross-sectional study. Liver and BM biopsy were obtained during the surgery to be looked for tissue-specific marker of CRC, CK20. CK20 expression in BM and liver was assessed by the two methods of reverse transcriptase polymerase chain reaction and immunohistochemistry. Among the 38 submitted patients, a total number of 14 MM, including 10 BM (23%) and seven hepatic (18%) were detected. There was no significant difference in clinicopathological aspects including size, location, stage, grade, local invasion to vessels and neurons, TNM staging and relapse rate of tumour among patients with/without disseminated tumoural cells. The result did not demonstrate a survival difference between patients receiving neoadjuvant chemotherapy compared to other patients. Our study did not confirm that the presence of MM influences TNM staging and overall survival of the patients. Additional clinical investigations with longer follow-up period and larger number of cases are required to decide on administration of neoadjuvant therapy in CRC patients.

Micrometastasis Volume in Lymph Nodes Determines Disease Recurrence Rate of Stage II Colorectal Cancer: A Prospective Multicenter Trial.

We reported in a retrospective study that the presence of micrometastasis in lymph nodes, when assessed by carcinoembryonic antigen (CEA)-specific RT-PCR, is a significant prognostic factor in stage II colorectal cancer. The aim of this study was to clarify the clinical value of micrometastasis in a prospective multicenter trial. From November 2001 to December 2005, a total of 419 colorectal cancer cases were preoperatively registered at a central data center. Of them, 315 node-negative stage II colorectal cancer cases were enrolled. After RNA quality check, 304 colorectal cancer cases were analyzed for CEA mRNA in lymph nodes by both conventional RT-PCR (a band method) and quantitative RT-PCR. Long-term prognosis of the patients was determined by each method. A positive band for CEA mRNA was detected in 73 (24.0%) of 304 patients. Postoperative adjuvant chemotherapy was applied in 31 CEA band-positive cases with an oral 5-fluorouracil derivative HCFU (1-hexylcarbamoyl-5-fluorouracil) for 1 year, whereas chemotherapy was not administered to CEA band-negative group. Multivariate Cox regression analyses revealed that a high micrometastasis volume (high MMV, n = 95) was an independent poor prognostic factor for 5-year disease-free survival (DFS; P = 0.001) and 5-year overall survival (OS; P = 0.016). This prospective clinical trial demonstrates that micrometastasis volume is a useful marker in identifying patients who are at high or low risk for recurrence of stage II colorectal cancer. Clin Cancer Res; 22(13); 3201-8. ©2016 AACR.