Identification of tumor-reactive B cells and systemic IgG in breast cancer based on clonal frequency in the sentinel lymph node.

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.

Vaccine-draining lymph nodes of cancer patients for generating anti-cancer antibodies.

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.

Identification of tumor-binding scFv derived from clonally related B cells in tumor and lymph node of a patient with breast cancer.

The purpose of this study was to determine the clonal relationship between B cells within a breast cancer and the B cells in the tumor-draining lymph node (TDLN). We also determined the binding capacity of antibodies derived from these sources to autologous cancer and autologous noncancer breast tissue. Antibody clonality of B cells derived from tumor and lymph node was determined by analyzing heavy and light chain immunoglobulin sequences. The number of shared clonal groups observed between tumor and lymph node antibodies was significant for both heavy (p = 0.004) and light chain (p = 0.012) populations. Panning with phage-displayed single-chain variable fragment libraries derived from the tumor and lymph node B cells resulted in multiple antibodies that bound autologous tumor. Sequence analysis of enriched antibodies recovered after the third round of panning the tumor and TDLN libraries against autologous tumor lysates had a genetic relationship. These results indicate that B cells infiltrating a patient's breast cancer and B cells present in the tumor-draining lymph node are clonally and functionally related.

Effect of occult metastases on survival in node-negative breast cancer.

BACKGROUND: Retrospective and observational analyses suggest that occult lymph-node metastases are an important prognostic factor for disease recurrence or survival among patients with breast cancer. Prospective data on clinical outcomes from randomized trials according to sentinel-node involvement have been lacking. METHODS: We randomly assigned women with breast cancer to sentinel-lymph-node biopsy plus axillary dissection or sentinel-lymph-node biopsy alone. Paraffin-embedded tissue blocks of sentinel lymph nodes obtained from patients with pathologically negative sentinel lymph nodes were centrally evaluated for occult metastases deeper in the blocks. Both routine staining and immunohistochemical staining for cytokeratin were used at two widely spaced additional tissue levels. Treating physicians were unaware of the findings, which were not used for clinical treatment decisions. The initial evaluation at participating sites was designed to detect all macrometastases larger than 2 mm in the greatest dimension. RESULTS: Occult metastases were detected in 15.9% (95% confidence interval [CI], 14.7 to 17.1) of 3887 patients. Log-rank tests indicated a significant difference between patients in whom occult metastases were detected and those in whom no occult metastases were detected with respect to overall survival (P=0.03), disease-free survival (P=0.02), and distant-disease-free interval (P=0.04). The corresponding adjusted hazard ratios for death, any outcome event, and distant disease were 1.40 (95% CI, 1.05 to 1.86), 1.31 (95% CI, 1.07 to 1.60), and 1.30 (95% CI, 1.02 to 1.66), respectively. Five-year Kaplan-Meier estimates of overall survival among patients in whom occult metastases were detected and those without detectable metastases were 94.6% and 95.8%, respectively. CONCLUSIONS: Occult metastases were an independent prognostic variable in patients with sentinel nodes that were negative on initial examination; however, the magnitude of the difference in outcome at 5 years was small (1.2 percentage points). These data do not indicate a clinical benefit of additional evaluation, including immunohistochemical analysis, of initially negative sentinel nodes in patients with breast cancer. (Funded by the National Cancer Institute; ClinicalTrials.gov number, NCT00003830.).

Axillary concordance between superficial and deep sentinel node mapping material injections in breast cancer patients: systematic review and meta-analysis of the literature.

It is still unclear whether the deep and superficial lymphatics of the breast always drain into the same nodes and which route best simulates the spread of breast cancer. In the current study, we systematically searched the available literature to find the studies evaluated the sentinel node locations of deep and superficial injections in the same patients simultaneously or serially. We searched SCOPUS, and PUBMED for relevant studies. Patient basis concordance rate was defined as the ratio of patients with at least one identified axillary sentinel node by both deep and superficial injections to all patients with identified axillary sentinel nodes using either methods. Sentinel node basis concordance was defined as the ratio of the number of axillary sentinel nodes identified by both deep and superficial injections to the sum of all identified axillary sentinel nodes using either methods. Pooled sentinel node detection rates were 94 % [92.1-95.5], 91.2 % [87.1-94.1], and 97.2 % [96-98] for superficial, deep, and combined (superficial and deep) injections. Pooled patient basis and sentinel node basis concordance rates were 90 % [86.7-92.4] and 73 % [63.3-80.9]. Pooled false negative rates were 9.1 % [5.9-14], 8.6 % [3.7-18.8], and 6.5 % [3.4-11.9] for superficial, deep, and combined (superficial and deep) injections, respectively. Axillary lymphatic drainage concordance between superficial and deep sentinel node mapping material in breast cancer patients is fairly high and clinically acceptable. However, both injection techniques can complement each other and the combined superficial/deep injection technique seems to be more successful clinically and can decrease the overall false negative rate.

Accuracy of sentinel node biopsy in esophageal carcinoma: a systematic review and meta-analysis of the pertinent literature.

The use of sentinel node surgery for esophageal carcinoma is still under investigation. We evaluated the data available in the literature on this topic, and herein present the results in a systematic review format. PUBMED, SCOPUS, the ISI web of knowledge and the information from the annual meetings of the Japan Esophageal Society were searched using the search terms: "(esophagus OR esophageal) AND sentinel". The outcomes of interest were the detection rate and sensitivity. Overall, 18 studies were included. The pooled detection rate was 89.2% [82.6-93.5]. Patients with T1 and two tumors had a 17% higher detection rate compared to those with T3 and four tumors. The pooled sensitivity was 84% [78-88%]. The sensitivity was higher for adenocarcinoma compared to squamous cell carcinoma (SCC) (91 vs. 81%). In the SCC patients, there was a trend toward decreased sensitivity associated with an increasing tumor depth (T1:88%, T2:76%, T3:50%). Our analysis indicated that sentinel node biopsy is useful in adenocarcinoma patients. For SCC patients, including only cN0 patients (preferably T1 and 2) would increase the detection rate and sensitivity. Due to the limited number of high-quality studies, drawing any more definite conclusions is impossible. Large cohort studies with a standardized and consistent design will be needed in the future.

Intravenous infusion of phage-displayed antibody library in human cancer patients: enrichment and cancer-specificity of tumor-homing phage-antibodies.

Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage-antibodies and a Stage IV cancer status. All patients were intravenously administered 1 × 10(11) TUs/kg of an scFv library 1 to 4 h before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0-5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor-specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2,355) that was found to share a 12-amino-acid-long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis.

Spatial organization of dendritic cells within tumor draining lymph nodes impacts clinical outcome in breast cancer patients.

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.

Relationship between arm morbidity and patient-reported outcomes following surgery in women with node-negative breast cancer: NSABP protocol B-32.

BACKGROUND: The impact of arm morbidity following breast cancer surgery on patient-observed changes in daily functioning and health-related quality of life (HRQoL) has not been well-studied. OBJECTIVE: To examine the association of objective measures such as range of motion (ROM) and lymphedema, with patient-reported outcomes (PROs) in the arm and breast, upper extremity function, activities, and HRQoL. METHODS: The National Surgical Adjuvant Breast and Bowel Project Protocol B-32 was a randomized trial comparing sentinel node resection (SNR) with axillary dissection (AD) in women with node-negative breast cancer. ROM and arm volume were measured objectively. PROs included symptoms; arm function; limitations in social, recreational, occupational, and other regular activities; and a global index of HRQoL. Statistical methods included cross-tabulations and multivariable linear regression models. RESULTS: In all, 744 women provided at least 1 postsurgery assessment. About one-third of the patients experienced arm mobility restrictions. A similar number of patients avoided the use of the arm 6 months after surgery. Limitations in work and other regular activities were reported by about a quarter of the patients. In this multivariable analysis, arm mobility and sensory neuropathy were predictors of patient-reported arm function and overall HRQoL. Predictors for activity limitations also included side of surgery (dominant vs nondominant). Edema was not significant after adjustment for sensory neuropathy and ROM. LIMITATIONS: Arm mobility and edema were measured simultaneously only once during the follow-up (6 months). CONCLUSION: Clinical measures of sensory neuropathy and restrictions in arm mobility following breast cancer surgery are associated with self-reported limitations in activity and reductions in overall HRQoL.

Impact of the COVID-19 pandemic on melanoma diagnosis and presentation: a `review.

The ongoing challenges posed by COVID-19 are concerning for their impact on successful detection and recognition of melanoma as total body skin examinations and skin biopsies are critical for identifying early-stage melanoma and intervening before progression to metastatic disease. A comprehensive electronic search of PubMed/MEDLINE was conducted on or before August 1, 2022, using the search terms ("skin" AND "COVID-19"), (["skin cancer" AND "COVID-19"] OR ["skin cancer" AND "coronavirus"]), (["melanoma" AND "COVID-19"] OR ["melanoma" AND "coronavirus"]), ("dermatology" AND "COVID-19"), and ("cutaneous" AND "COVID-19"). Eight articles representing Belgium, Chile, France, Germany, Spain, the United Kingdom, and the United States were included. Four articles analyzed changes in the proportion of in situ melanoma at diagnosis and consistently reported decreases, with an overall decrease ranging from 7.6 to 40.4%. Five studies analyzed changes in the proportion of melanoma diagnoses by staging, but no clear changes in staging patterns were observed. Five studies analyzed changes in the mean Breslow thickness of melanoma diagnoses and consistently reported increases, with an overall increase ranging from 4.0 to 38%. Disruptions to proper diagnosis and treatment of melanoma are creating undue morbidity, mortality, and healthcare costs as the pandemic continues. Continued research with improved, centralized data collection is needed to better address the COVID-19 pandemic's ongoing challenge to appropriate detection and treatment of melanoma.

GRB7 is required for triple-negative breast cancer cell invasion and survival.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that is usually associated with poor prognosis, and frequently associated with the basal-like breast cancer gene expression profile. There are no targeted therapeutic modalities for this disease, and no useful biomarkers. High GRB7 RNA expression levels are associated with an elevated risk of recurrence in patients with operable TNBC treated with standard adjuvant anthracycline and taxane therapy. To determine whether GRB7 is involved in the pathobiology of TNBC, we evaluated the biological effects of GRB7 inhibition in a panel of triple-negative cell lines-MDA-MB-468, MDA-MB-231, HCC70, and T4-2. We found GRB7 inhibition reduced cell motility and invasion of these cell lines and promoted cell death by apoptosis in 3D culture. These data suggest that GRB7 itself, or GRB7-dependent pathways, may prove to be important therapeutic targets in this disease.

Interaction of the non-phosphorylated peptide G7-18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity.

Src-homology (SH2) domains are an attractive target for the inhibition of specific signalling pathways but pose the challenge of developing a truly specific inhibitor. The G7-18NATE cyclic peptide is reported to specifically inhibit the growth factor receptor bound protein 7 (Grb7) adapter protein, implicated in the progression of several cancer types, via interactions with its SH2 domain. G7-18NATE effectively inhibits the interaction of Grb7 with ErbB3 and focal adhesion kinase in cell lysates and, with the addition of a cell permeability sequence, inhibits the growth and migration of a number of breast cancer cell lines. It is thus a promising lead in the development of therapeutics targeted to Grb7. Here we investigate the degree to which G7-18NATE is specific for the Grb7-SH2 domain compared with closely related SH2 domains including those of Grb10, Grb14, and Grb2 using surface plasmon resonance. We demonstrate that G7-18NATE binds with micromolar binding affinity to Grb7-SH2 domain (K(D) = 4-6 µm) compared with 50-200 times lower affinity for Grb10, Grb14, and Grb2 but that this specificity depends critically on the presence of phosphate in millimolar concentrations. Other differences in buffer composition, including use of Tris or 2-(N-Morpholino)ethanesulfonic acid or varying the pH, do not impact on the interaction. This suggests that under cellular conditions, G7-18NATE binds with highest affinity to Grb7. In addition, our findings demonstrate that the basis of specificity of G7-18NATE binding to the Grb7-SH2 domain is via other than intrinsic structural features of the protein, representing an unexpected mode of molecular recognition.

Targeting of palpable B16-F10 melanoma tumors with polyclonal antibodies on white blood cells.

BACKGROUND: Antibodies and other recognition molecules direct cancer cell death by multiple types of immune cells. Therapy directed at only one target typically results in tumor regrowth because of tumor heterogeneity. Our goal is to direct therapy to multiple targets simultaneously. Our previous studies showed that multiple antibodies targeting mutated tumor proteins inhibited tumor growth when injected subcutaneously near the time of cancer cell implantation. METHODS: A cocktail of rabbit antibodies against B16-F10 cell surface related mutated proteins were generated. Implanted B16-F10 cells were allowed to grow to palpable size before treatment. Antibodies were administered using different routes of exposure. Free antibody was compared to antibody armed on mouse splenic white blood cells (WBCs). Binding of the antibody cocktail was determined for mouse and human WBCs. RESULTS: The antibody cocktail inhibited tumor growth and prolonged survival when administered as free antibody or armed on WBCs. The antibody cocktail armed on WBCs achieved similar tumor inhibition as free antibody but at a dose 1000-fold less. Armed WBCs achieved tumor inhibition by intravenous and subcutaneous administration. The antibody cocktail bound well to human WBCs and saturation dose was defined. Binding was stable under simulated in vivo condition in human plasma at 37 °C. CONCLUSIONS: Antibodies targeting multiple tumor mutated proteins inhibited tumor growth and prolonged survival. Effective antibody dose was reduced 1000-fold by arming WBCs. Rabbit antibodies saturated human WBCs using <1 mg per billion cells. A phase I trial in cancer patients using this strategy has been approved by the FDA.

Development of Clinical-Grade Antibodies against Tumor-Specific Mutations to Target Neuroblastoma.

OBJECTIVE: Tumor heterogeneity is a fundamental problem in treating cancer with monotargeting therapy, including chemical, antibody, and T cell therapies. Our goal is to target multiple mutated peptides found in a patient's cancer to increase antibody therapy effectiveness. METHODS: Tumor samples were derived from patients with neuroblastoma. Whole-exome sequencing was performed of tumor and normal cells. Mutated proteins with missense mutations were selected from the patient tumor. These mutated proteins were further selected for the presence of missense mutations in the outer cell surface. Peptides representing a mutated section of the proteins were used for vaccinating rabbits and generating anti-peptide antibodies. The binding of individual polyclonal antibodies (pAbs) and the mixtures of pAbs were determined against the patient's tumor as cultured neuroblastoma cells and in a murine xenograft model. Antibodies were prepared according to FDA requirements of a phase I clinical protocol. RESULTS: All of the generated rabbit pAbs bound with high affinity to the corresponding peptide used for vaccination. The pAbs also bound to low passage neuroblastoma cells. Mixed as cocktails, the pAbs had substantially increased binding to cells and bound well to the xenograft tissue. No binding was observed to the panel of normal human tissues. Preparation of pAbs by an academic lab to clinical-grade was approved by FDA for phase I clinical trial. CONCLUSION: We describe a new strategy to make customized antibodies for individual cancer patients and present the data required to meet FDA specifications to begin a phase I clinical trial.

Sentinel-lymph-node resection compared with conventional axillary-lymph-node dissection in clinically node-negative patients with breast cancer: overall survival findings from the NSABP B-32 randomised phase 3 trial.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: US Public Health Service, National Cancer Institute, and Department of Health and Human Services.

Preparation of clinical-grade WBCs using leukocyte reduction filters.

OBJECTIVE: Our goal was to develop a simpler and less expensive method of obtaining human clinical-grade WBCs using an alternative method to continuous leukapheresis. Our purpose for the WBCs is to arm them with rabbit anticancer antibodies for a phase I clinical trial. METHODS: Using leukocyte reduction filters (LRFs) discarded from the blood bank, we evaluated multiple variables to maximize recovery of WBCs with the lowest contamination of RBCs. Using an optimized protocol, full-scale runs according to FDA current Good Manufacturing Practice (cGMP) standards were completed with immediate filtration of blood obtained from donors participating in our study. RESULTS: Forward flushing of the filter removed 85% to 95% of residual RBCs and platelets. When backward flushed with 800 mL, 95% of the WBCs recovered were contained in the first 400 mL. The number of recovered WBCs was in the range of 166-211 million/100 mL filtered blood. Subpopulations of WBCs recovered from the LRFs were in the same proportion as the donors' whole blood. Viability of recovered WBCs was 96-99%. Exogenous rabbit antibodies bound well to the recovered WBCs and were retained for at least 5 h without significant reduction. Three full scale runs of WBCs recovered from donor blood filtered through the LRF met all FDA specification of sterility, endotoxin levels, viability and stability. CONCLUSION: Using LRFs, high quality clinical grade WBCs are readily obtained in quantities of 0.2 to 1.2 billion cells from 100 mL to 450 mL (1 unit) of whole blood.

Morbidity results from the NSABP B-32 trial comparing sentinel lymph node dissection versus axillary dissection.

BACKGROUND AND OBJECTIVES: Three year post-surgical morbidity levels were compared between patients with negative sentinel lymph node dissection alone (SLND) and those with negative sentinel node dissection and negative axillary lymph node dissection (ALND) in the NSABP B-32 trial. METHODS: A total of 1,975 ALND and 2,008 SLND node negative breast cancer patients had shoulder range of motion and arm volumes assessed along with self reports of arm tingling and numbness. Relative shoulder abduction deficits and relative arm volume differences between ipsilateral and contralateral arms were calculated. RESULTS: Shoulder abduction deficits >or=10% peaked at 1 week for the ALND (75%) and SLND (41%) groups. Arm volume differences >or=10% at 36 months were evident for the ALND (14%) and SLND (8%) groups. Numbness and tingling peaked at 6 months for the ALND (49%, 23%) and SLND (15%, 10%) groups. Logistic regression correlates of residual morbidity included treatment group, age, handedness, tumor size, systemic chemotherapy, and radiation to the axilla. CONCLUSIONS: Although residual morbidity for both treatment groups was evident, the results of the NSABP B-32 study indicate the superiority of the SLND compared to the ALND treatment approach relative to post-surgical morbidity outcomes over a 3-year follow-up period.

A cocktail of polyclonal affinity enriched antibodies against melanoma mutations increases binding and inhibits tumor growth.

BACKGROUND: Antibodies that target a single tumor antigen fail to cure stage IV cancer patients due to tumor heterogeneity and variable expression of antigen. Tumor cells with insufficient binding of antibody will not undergo antibody induced cytotoxicity. We describe targeting multiple tumor-specific antigens that resulted in homogeneous dense binding to mouse melanoma cells and significant tumor growth inhibition. METHODS: Surface-related tumor-specific mutations on B16-F10 cells were identified. Peptides containing the single amino acid mutation were synthesized for 9 different neoantigens. Rabbits were vaccinated with each of these peptides and high affinity polyclonal antibodies to each peptide were obtained. The 9 antibodies were combined as a cocktail and mice with implanted B16-F10 cells were treated with and without PD1 inhibitor. RESULTS: Even a single dose of the antibody cocktail inhibited tumor growth and prolonged survival. PD1 inhibitor alone had little effect on tumor growth. The antibody cocktail plus PD1 inhibition increased tumor response and 4 doses of the cocktail completely prevented tumor growth in 50% of the mice. Complete responses were durable. The complete responders were highly resistant to tumor re-challenge at 6 months. No adverse events were identified in the antibody treated mice. CONCLUSIONS: Multiple tumor-specific cell surface-related neoantigens were abundant in B16-F10 cells. Antibodies to 9 of these neoantigens had variable binding but when combined had dense homogeneous binding. Even one dose of this cocktail of 9 antibodies improved survival and when multiple doses were combined with PD1 inhibition 50% of the mice were rendered permanently tumor free.

Developing bifunctional beta-lactamase molecules with built-in target-recognizing module for prodrug therapy: identification of Enterobacter Cloacae P99 cephalosporinase loops suitable for randomization and phage-display selection.

This study was focused on developing catalytically active beta-lactamase enzyme molecules that have target-recognizing sites built within their scaffold. Using phage-display approach, nine libraries were constructed by inserting the randomized linear or cysteine-constrained heptapeptides in the five different loops on the outer surface of P99 beta-lactamase molecule. The pIII signal peptide of Sec-pathway was employed for a periplasmic translocation of the beta-lactamase fusion protein, which we found more efficient than the DsbA signal peptide of SRP-pathway. The randomized heptapeptide loops replaced native amino acids between positions (34)Y-(37)K, (238)M-(246)A, (275)N-(280)A, (305)A-(311)S, or (329)I-(334)I of the P99 beta-lactamase molecules for generating the loop-1 to -5 libraries, respectively. The diversity of each loop library was judged by counting the primary and beta-lactamase-active clones. The linear peptide inserts in the loop-2 library showed the maximum number of the beta-lactamase-active clones, followed by the loop-5, loop-3, and loop-4. The insertion of the cysteine-constrained loops exhibited a dramatic loss of the enzyme-active beta-lactamase clones. The complexity of the loop-2 linear library, as determined by the frequency and diversity of amino acid distributions in the randomized region, appears consistent with the standards of other types of phage display library systems. The selection of the loop-2 linear library on streptavidin protein as a test target identified several beta-lactamase clones that specifically bound to streptavidin. In conclusion, this study identified the suitability of the loop-2 of P99 beta-lactamase for constructing a phage-display library of the beta-lactamase enzyme-active molecules that can be selected against a target. This is an enabling step in our long-term goal of developing bifunctional beta-lactamase molecules against cancer-specific targets for enzyme prodrug therapy of cancer.