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Sentinel-lymph-node (SLN) surgery was designed to minimise the side-effects of lymph-node surgery but still offer outcomes equivalent to axillary-lymph-node dissection (ALND). The aims of National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-32 were to establish whether SLN resection in patients with breast cancer achieves the same survival and regional control as ALND, but with fewer side-effects. NSABP B-32 was a randomised controlled phase 3 trial done at 80 centres in Canada and the USA between May 1, 1999, and Feb 29, 2004. Women with invasive breast cancer were randomly assigned to either SLN resection plus ALND (group 1) or to SLN resection alone with ALND only if the SLNs were positive (group 2). Random assignment was done at the NSABP Biostatistical Center (Pittsburgh, PA, USA) with a biased coin minimisation approach in an allocation ratio of 1:1. Stratification variables were age at entry (≤49 years, ≥50 years), clinical tumour size (≤2·0 cm, 2·1–4·0 cm, ≥4·1 cm), and surgical plan (lumpectomy, mastectomy). SLN resection was done with a blue dye and radioactive tracer. Outcome analyses were done in patients who were assessed as having pathologically negative sentinel nodes and for whom follow-up data were available. The primary endpoint was overall survival. Analyses were done on an intention-to-treat basis. All deaths, irrespective of cause, were included. The mean time on study for the SLN-negative patients with follow-up information was 95·6 months (range 70·1–126·7). This study is registered with ClinicalTrials.gov, number NCT00003830. 5611 women were randomly assigned to the treatment groups, 3989 had pathologically negative SLN. 309 deaths were reported in the 3986 SLN-negative patients with follow-up information: 140 of 1975 patients in group 1 and 169 of 2011 in group 2. Log-rank comparison of overall survival in groups 1 and 2 yielded an unadjusted hazard ratio (HR) of 1·20 (95% CI 0·96–1·50; p=0·12). 8-year Kaplan-Meier estimates for overall survival were 91·8% (95% CI 90·4–93·3) in group 1 and 90·3% (88·8–91·8) in group 2. Treatment comparisons for disease-free survival yielded an unadjusted HR of 1·05 (95% CI 0·90–1·22; p=0·54). 8-year Kaplan-Meier estimates for disease-free survival were 82·4% (80·5–84·4) in group 1 and 81·5% (79·6–83·4) in group 2. There were eight regional-node recurrences as first events in group 1 and 14 in group 2 (p=0·22). Patients are continuing follow-up for longer-term assessment of survival and regional control. The most common adverse events were allergic reactions, mostly related to the administration of the blue dye. Overall survival, disease-free survival, and regional control were statistically equivalent between groups. When the SLN is negative, SLN surgery alone with no further ALND is an appropriate, safe, and effective therapy for breast cancer patients with clinically negative lymph nodes. US Public Health Service, National Cancer Institute, and Department of Health and Human Services.

The authors detected isolated tumor-cell clusters in otherwise negative nodes in 16% of women with breast cancer. The 5-year estimates of survival were 94.6% among women with occult nodal spread and 95.8% among those without occult nodal spread. A landmark 1948 article by Saph and Amromin showed that the routine analysis of lymph nodes in breast cancer was insufficient to detect all metastases present. 1 Although the practice of additional pathological analysis was not adopted, the concept of occult metastases (metastases that are not detected initially but are detected with further evaluation) was introduced and has been the subject of considerable research and controversy over the ensuing decades. 2 – 4 The National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-32 was designed to evaluate whether sentinel-lymph-node biopsy alone was equivalent to complete axillary dissection with respect to overall survival . . .

Background Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with advanced-stage disease frequently marked by treatment resistance and recurrence. Tumor heterogeneity, driven by the accumulation of somatic mutations, undermines the efficacy of conventional therapies and limits the long-term success of targeted agents. There is an urgent need for new therapeutic strategies that can exploit, rather than be constrained by, this heterogeneity. Methods We developed a personalized immunotherapeutic pipeline in the syngeneic CT26 murine model of CRC. Briefly, whole exome sequencing identified mutated surface proteins (MSPs) unique to these cells. Of these MSPs, we selected 10 for the generation of MSP-specific polyclonal antibodies (pAbs). These pAbs were tested for specificity and peptide binding to peptides via ELISA, tumor tissue by immunofluorescence, and tumor cells by flow cytometry. Therapeutic efficacy was evaluated in vivo using CT26 tumor-bearing mice treated with the pAb cocktail alone or in combination with anti-PD-1 immune checkpoint blockade. To assess clinical relevance, we analyzed The Cancer Genome Atlas (TCGA) whole exome sequencing data from 100 human CRC patients for MSP prevalence and inter-patient variability. Results The 10-pAb oligoclonal antibody cocktail preparations exhibited additive, high-affinity, tumor-specific binding with minimal reactivity to healthy tissues. In vivo, this pAb cocktail significantly suppressed tumor growth and, when combined with PD-1 blockade, prolonged median survival to over 90 days in treated mice compared to less than 25 days in controls. Whole exome sequence data revealed that the majority of human CRC tumors harbored 10 or more MSPs, with minimal overlap between individuals, highlighting the feasibility and necessity of personalized antibody-based therapies. Conclusion Our findings establish a proof-of-concept for individualized, mutation-guided antibody therapies, supporting further development of this approach to improve outcomes in patients with advanced CRC.

The goals of axillary-lymph-node dissection (ALND) are to maximise survival, provide regional control, and stage the patient. However, this technique has substantial side-effects. The purpose of the B-32 trial is to establish whether sentinel-lymph-node (SLN) resection can achieve the same therapeutic goals as conventional ALND but with decreased side-effects. The aim of this paper is to report the technical success and accuracy of SLN resection plus ALND versus SLN resection alone. 5611 women with invasive breast cancer were randomly assigned to receive either SLN resection followed by immediate conventional ALND (n=2807; group 1) or SLN resection without ALND if SLNs were negative on intraoperative cytology and histological examination (n=2804; group 2) in the B-32 trial. Patients in group 2 underwent ALND if no SLNs were identified or if one or more SLNs were positive on intraoperative cytology or subsequent histological examination. Primary endpoints, including survival, regional control, and morbidity, will be reported later. Secondary endpoints are accuracy and technical success and are reported here. This trial is registered with the Clinical Trial registry, number NCT00003830. Data for technical success were available for 5536 of 5611 patients; 75 declined protocol treatment, had no SLNs removed, or had no SLN resection done. SLNs were successfully removed in 97·2% of patients (5379 of 5536) in both groups combined. Identification of a preincision hot spot was associated with greater SLN removal (98·9% [5072 of 5128]). Only 1·4% (189 of 13171) of SLN specimens were outside of axillary levels I and II. 65·1% (8571 of 13 171) of SLN specimens were both radioactive and blue; a small percentage was identified by palpation only (3·9% [515 of 13 171]). The overall accuracy of SLN resection in patients in group 1 was 97·1% (2544 of 2619; 95% CI 96·4–97·7), with a false-negative rate of 9·8% (75 of 766; 95% CI 7·8–12·2). Differences in tumour location, type of biopsy, and number of SLNs removed significantly affected the false-negative rate. Allergic reactions related to blue dye occurred in 0·7% (37 of 5588) of patients with data on toxic effects. The findings reported here indicate excellent balance in clinical patient characteristics between the two randomised groups and that the success of SLN resection was high. These findings are important because the B-32 trial is the only trial of sufficient size to provide definitive information related to the primary outcome measures of survival and regional control. Removal of more than one SLN and avoidance of excisional biopsy are important variables in reducing the false-negative rate.

Cellular heterogeneity presents a significant challenge to cancer treatment. Antibody therapies targeting individual tumor-associated antigens can be extremely effective but are not suited for all patients and often fail against tumors with heterogeneous expression as tumor cells with low or no antigen expression escape targeting and develop resistance. Simultaneously targeting multiple tumor-specific proteins with multiple antibodies has the potential to overcome this barrier and improve efficacy, but relatively few widely expressed cancer-specific antigens are known. In contrast, neoepitopes, which arise from mutations unique to tumor cells, are considerably more abundant. However, since neoepitopes are not commonly shared between individuals, a patient-customized approach is necessary and motivates efforts to develop an efficient means to identify suitable target mutations and isolate neoepitope-specific monoclonal antibodies. Here, focusing on the latter goal, we use directed evolution in yeast and phage display systems to engineer antibodies from nonimmune, human antibody fragment libraries that are specific for neoepitopes previously reported in the B16F10 melanoma model. We demonstrate proof-of-concept for a pipeline that supports rapid isolation and functional enhancement of multiple neoepitope peptide-targeted monoclonal antibodies and demonstrate their robust binding to B16F10 cells and potent effector functions in vitro. These antibodies were combined and evaluated in vivo for anticancer activity in tumor-bearing mice, where they suppressed B16F10 tumor growth and prolonged survival. These findings emphasize the potential for clinical application of patient-customized antibody cocktails in the treatment of the many cancers poorly addressed by current therapies.

A better understanding of antitumor immune responses is the key to advancing the field of cancer immunotherapy. Endogenous immunity in cancer patients, such as circulating anticancer antibodies or tumor-reactive B cells, has been historically yet incompletely described. Here, we demonstrate that tumor-draining (sentinel) lymph node (SN) is a rich source for tumor-reactive B cells that give rise to systemic IgG anticancer antibodies circulating in the bloodstream of breast cancer patients. Using a synergistic combination of high-throughput B-cell sequencing and quantitative immunoproteomics, we describe the prospective identification of tumor-reactive SN B cells (based on clonal frequency) and also demonstrate an unequivocal link between affinity-matured expanded B-cell clones in the SN and antitumor IgG in the blood. This technology could facilitate the discovery of antitumor antibody therapeutics and conceivably identify novel tumor antigens. Lastly, these findings highlight the unique and specialized niche the SN can fill in the advancement of cancer immunotherapy.

Background Our research is focused on using the vaccine draining lymph node to better understand the immune response to cancer vaccines and as a possible source of anti-cancer reagents. We evaluated vaccine draining lymph nodes archived from a clinical study in melanoma patients and determined the reaction of B cells to the vaccine peptides. Methods Mononuclear cells (MNCs) were recovered from cryopreserved lymph nodes that were directly receiving drainage from multi-peptide melanoma vaccine. The patients were enrolled on a vaccine study (NCT00089219, FDA, BB-IND No. 10825). B cell responses in the vaccine-draining lymph nodes were studied under both stimulated and un-stimulated conditions. Cryopreserved cells were stimulated with CD40L, stained with multiple human cell-surface markers (CD19, CD27, IgM) to identify different categories of B cell sub populations with flow cytometry. Hybridomas were generated from the lymph node cells after CD40L-stimulation. Cells were fused to murine plasmacytoma P3X63.Ag8.653 using Helix electrofusion chamber. ELISA was used to evaluate hybridoma derived antibody binding to vaccine peptides. Results Viable MNCs were satisfactorily recovered from lymph nodes cryopreserved from six vaccine study patients 8–14 years previously. B cell ELISPOT demonstrated responses for each patient to multiple vaccine peptides. CD40L stimulation of lymph node cells increased the proportion of CD19 + CD27 + cells from 12 to 65% of the sample and increased the proportion of class-switched cells. Screening of IgG secreting clones demonstrated binding to melanoma vaccine peptides. Conclusions B cells were successfully recovered and expanded from human cryopreserved vaccine-draining lymph nodes. Individual B cells were identified that secreted antibodies that bound to cancer vaccine peptides. The ability to reliably generate in vitro the same antibodies observed in the blood of vaccinated patients will facilitate research to understand mechanisms of human antibody activity and possibly lead to therapeutic antibodies.

Background Dendritic cells (DCs) are important mediators of anti-tumor immune responses. We hypothesized that an in-depth analysis of dendritic cells and their spatial relationships to each other as well as to other immune cells within tumor draining lymph nodes (TDLNs) could provide a better understanding of immune function and dysregulation in cancer. Methods We analyzed immune cells within TDLNs from 59 breast cancer patients with at least 5 years of clinical follow-up using immunohistochemical staining with a novel quantitative image analysis system. We developed algorithms to analyze spatial distribution patterns of immune cells in cancer versus healthy intra-mammary lymph nodes (HLNs) to derive information about possible mechanisms underlying immune-dysregulation in breast cancer. We used the non-parametric Mann–Whitney test for inter-group comparisons, Wilcoxon Matched-Pairs Signed Ranks test for intra-group comparisons and log-rank (Mantel-Cox) test for Kaplan Maier analyses. Results Degree of clustering of DCs (in terms of spatial proximity of the cells to each other) was reduced in TDLNs compared to HLNs. While there were more numerous DC clusters in TDLNs compared to HLNs,DC clusters within TDLNs tended to have fewer member DCs and also consisted of fewer cells displaying the DC maturity marker CD83. The average number of T cells within a standardized radius of a clustered DC was increased compared to that of an unclustered DC, suggesting that DC clustering was associated with T cell interaction. Furthermore, the number of T cells within the radius of a clustered DC was reduced in tumor-positive TDLNs compared to HLNs. Importantly, clinical outcome analysis revealed that DC clustering in tumor-positive TDLNs correlated with the duration of disease-free survival in breast cancer patients. Conclusions These findings are the first to describe the spatial organization of DCs within TDLNs and their association with survival outcome. In addition, we characterized specific changes in number, size, maturity, and T cell co-localization of such clusters. Strategies to enhance DC function in-vivo, including maturation and clustering, may provide additional tools for developing more efficacious DC cancer vaccines.

Results from randomized trials indicate a 5.4% survival advantage associated with axillary dissection. To gain insight on survival outcomes when less than an axillary dissection is performed, we performed a retrospective analysis to determine survival outcome for node-negative and node-positive breast cancer patients when a variable number of nodes were excised. The data analyzed in this paper are from the Surveillance, Epidemiology, and End Results (SEER) database, from which 72,102 patients were selected whose breast cancer had been diagnosed in 1988 or later and who were aged 40-79 years at diagnosis, had a single primary lesion, and had 0 to 3 positive lymph nodes. Cases were separated into age groups (40 to 49 and 50 to 79 years), and node-negative cases were separated from those with one to three positive nodes. This analysis indicates that even when all regional lymph nodes are pathologically negative, the number of nodes removed is associated with survival. In the group of breast cancer patients who had one to three pathologically positive nodes, as with the node-negative group, the higher the number of nodes removed, the greater the survival. The hazard rate for death in the node-negative group was roughly 5% less for each additional five nodes removed. For the node-positive group, the hazard rate for death was between 8% and 9% less for each additional five nodes removed. This retrospective study supports the notion that removal of regional nodes, even when such nodes are interpreted as pathologically negative, is important for the long-term survival of breast cancer patients.

Objectives B cell‐mediated immunity can be associated with favorable clinical outcomes in cancer patients. However, the mechanism and features of anti‐tumor immune responses are not well understood. In particular, how B cells expressing tumor‐specific antibodies are distributed (in the tumor vs. the circulation) has not been well defined. Methods We performed an in‐depth analysis of B cell antibody repertoires derived from the tumor, sentinel lymph nodes and peripheral blood of three treatment‐naïve patients with breast cancer. We integrated transcriptional analysis, next‐generation sequencing of immunoglobulin heavy‐chain gene rearrangements and phage display to define B cell responses and clonal architecture in the tumor microenvironment, and to identify antibodies against autologous tumor tissues. Results B cell clonal lineage mapping across sequencing libraries generated from the tumor, sentinel lymph node and blood revealed that some expanded B cell clones overlap between tumor and lymph node, and fewer clones overlapped with the peripheral blood. Notably, tumor‐associated or tumor‐binding clones recovered in the phage panning and tracked back through the tissues harboured extensive somatic hypermutations in both the tumor and the lymph node. Conclusions These findings suggest an evolving humoral immune response that targets the tumor and the possibility of monitoring B cell clones of interest in blood after identifying them in tumor or lymph node samples. Studying the dynamics and specificity of B cell responses may provide insights into the characteristics of successful anti‐tumor immunity, provide a means for monitoring therapy and yield novel targets for personalised therapies. This study combined antibody phage display and specificity analysis with immune repertoire and transcriptional profiling to provide an in‐depth analysis of B cell clonal networks and specificities in three treatment‐naïve patients with breast cancer. Phage panning and binding studies against autologous tumors allowed us to identify tumor‐binding antibodies and tracked back through the tissues harboured extensive somatic hypermutations in both the tumor and the lymph node.