Survival Advantage Following TAG-72 Antigen-Directed Cancer Surgery in Patients With Colorectal Carcinoma: Proposed Mechanisms of Action.

Patients with colorectal carcinoma (CRC) continue to have variable clinical outcomes despite undergoing the same surgical procedure with curative intent and having the same pathologic and clinical stage. This problem suggests the need for better techniques to assess the extent of disease during surgery. We began to address this problem 35 years ago by injecting patients with either primary or recurrent CRC with 125I-labeled murine monoclonal antibodies against the tumor-associated glycoprotein-72 (TAG-72) and using a handheld gamma-detecting probe (HGDP) for intraoperative detection and removal of radioactive, i.e., TAG-72-positive, tissue. Data from these studies demonstrated a significant difference in overall survival data (p < 0.005 or better) when no TAG-72-positive tissue remained compared to when TAG-72-positive tissue remained at the completion of surgery. Recent publications indicate that aberrant glycosylation of mucins and their critical role in suppressing tumor-associated immune response help to explain the cellular mechanisms underlying our results. We propose that monoclonal antibodies to TAG-72 recognize and bind to antigenic epitopes on mucins that suppress the tumor-associated immune response in both the tumor and tumor-draining lymph nodes. Complete surgical removal of all TAG-72-positive tissue serves to reverse the escape phase of immunoediting, allowing a resetting of this response that leads to improved overall survival of the patients with either primary or recurrent CRC. Thus, the status of TAG-72 positivity after resection has a significant impact on patient survival.

Coaxial projective imaging system for surgical navigation and telementoring.

A coaxial projective imaging (CPI) module acquires surgical scene images from the local site of surgery, transfers them wirelessly to the remote site, and projects instructive annotations to the surgical field. At the remote site, the surgical scene images are displayed, and the instructive annotations from a surgical specialist are wirelessly transferred back to the local site in order to guide the surgical intervention by a less experienced surgeon. The CPI module achieves seamless imaging of the surgical field and accurate projection of the instructive annotations, by a coaxial optical path design that couples the imaging arm with the projection arm and by a color correction algorithm that recovers the true color of the surgical scene. Our benchtop study of tele-guided intervention verifies that the proposed system has a positional accuracy of better than 1 mm at a working distance ranging from 300 to 500 mm. Our in vivo study of cricothyrotomy in a rabbit model proves the concept of tele-mentored surgical navigation. This is the first report of tele-guided surgery based on CPI. The proposed technique can be potentially used for surgical training and for telementored surgery in resource-limited settings.

Handheld projective imaging device for near-infrared fluorescence imaging and intraoperative guidance of sentinel lymph node resection.

We propose a handheld projective imaging device for orthotopic projection of near-infrared fluorescence images onto target biological tissue at visible wavelengths without any additional visual aid. The device integrates a laser diode light source module, a camera module, a projector, an ultrasonic distance sensor, a Raspberry Pi single-board computer, and a battery module in a rugged handheld unit. It is calibrated at the detected working distance for seamless coregistration between fluorescence emission and projective imaging at the target tissue site. The proposed device is able to achieve a projection resolution higher than 314 µm and a planar projection bias less than 1 mm at a projection field of view of 58 × 108 mm2 and a working distance of 27 cm. Technical feasibility for projective imaging is verified in an ex vivo model of chicken breast tissue using indocyanine green as a fluorescence agent. Clinical utility for image-guided surgery is demonstrated in a clinical trial where sentinel lymph nodes in breast cancer patients are identified and resected under the guidance of projective imaging. Our ex vivo and in vivo experiments imply the clinical utility of deploying the proposed device for image-guided surgical interventions in resource-limited settings.

Linker engineering in anti-TAG-72 antibody fragments optimizes biophysical properties, serum half-life, and high-specificity tumor imaging.

Antibody (Ab) fragments have great clinical potential as cancer therapeutics and diagnostics. Their small size allows for fast clearance from blood, low immunoreactivity, better tumor penetration, and simpler engineering and production. The smallest fragment derived from a full-length IgG that retains binding to its antigen, the single-chain variable fragment (scFV), is engineered by fusing the variable light and variable heavy domains with a peptide linker. Along with switching the domain orientation, altering the length and amino acid sequence of the linker can significantly affect scFV binding, stability, quaternary structure, and other biophysical properties. Comprehensive studies of these attributes in a single scaffold have not been reported, making design and optimization of Ab fragments challenging. Here, we constructed libraries of 3E8, an Ab specific to tumor-associated glycoprotein 72 (TAG-72), a mucinous glycoprotein overexpressed in 80% of adenocarcinomas. We cloned, expressed, and characterized scFVs, diabodies, and higher-order multimer constructs with varying linker compositions, linker lengths, and domain orientations. These constructs dramatically differed in their oligomeric states and stabilities, not only because of linker and orientation but also related to the purification method. For example, protein L-purified constructs tended to have broader distributions and higher oligomeric states than has been reported previously. From this library, we selected an optimal construct, 3E8.G4S, for biodistribution and pharmacokinetic studies and in vivo xenograft mouse PET imaging. These studies revealed significant tumor targeting of 3E8.G4S with a tumor-to-background ratio of 29:1. These analyses validated 3E8.G4S as a fast, accurate, and specific tumor-imaging agent.

A 3E8.scFv.Cys-IR800 Conjugate Targeting TAG-72 in an Orthotopic Colorectal Cancer Model.

PURPOSE: Optical surgical navigation (OSN) will be a potent tool to help surgeons more accurately and efficiently remove tumors. The purpose of this study was to evaluate a novel humanized 3E8 antibody (3E8 MAb) fragment site-specifically conjugated with IR800, 3E8.scFv.Cys-IR800, as a potential OSN agent to target colorectal adenocarcinoma. PROCEDURES: An engineered single-chain variable fragment of 3E8 MAb (targeted to TAG-72), appending a C-terminal cysteine residue (3E8.scFv.Cys), was created and reacted with IRDye800-maleimide. 3E8.scFv.Cys-IR800 identity and purity were verified by MALDI-TOF mass spectra and 800 nm detected size exclusion column HPLC. In vitro human colon adenocarcinoma LS-174 T cells binding and competition assay validated biological functionality. We further evaluated the imaging ability and receptor-specific binding of 3E8.scFv.Cys-IR800 in an orthotopic LS-174 T mouse model. RESULTS: A 1:1 dye to protein conjugate was achieved at greater than 90 % HPLC purity. A 1 nmol dose of 3E8.scFv.Cys-IR800 via intraperitoneal injection administration was sufficient to produce high tumor to background fluorescence contrast. Blocking competition studies both in vitro and in vivo using a different blocking protein, 3E8ΔCH2, demonstrated 3E8.scFv.Cys-IR800 binding specificity for TAG-72 antigen. CONCLUSIONS: 3E8.scFv.Cys-IR800 shows properties useful in a clinically viable OSN agent for colorectal cancer.

A Wearable Goggle Navigation System for Dual-Mode Optical and Ultrasound Localization of Suspicious Lesions: Validation Studies Using Tissue-Simulating Phantoms and an Ex Vivo Human Breast Tissue Model.

Surgical resection remains the primary curative treatment for many early-stage cancers, including breast cancer. The development of intraoperative guidance systems for identifying all sites of disease and improving the likelihood of complete surgical resection is an area of active ongoing research, as this can lead to a decrease in the need of subsequent additional surgical procedures. We develop a wearable goggle navigation system for dual-mode optical and ultrasound imaging of suspicious lesions. The system consists of a light source module, a monochromatic CCD camera, an ultrasound system, a Google Glass, and a host computer. It is tested in tissue-simulating phantoms and an ex vivo human breast tissue model. Our experiments demonstrate that the surgical navigation system provides useful guidance for localization and core needle biopsy of simulated tumor within the tissue-simulating phantom, as well as a core needle biopsy and subsequent excision of Indocyanine Green (ICG)-fluorescing sentinel lymph nodes. Our experiments support the contention that this wearable goggle navigation system can be potentially very useful and fully integrated by the surgeon for optimizing many aspects of oncologic surgery. Further engineering optimization and additional in vivo clinical validation work is necessary before such a surgical navigation system can be fully realized in the everyday clinical setting.

Extracting Infrared Spectra of Protein Secondary Structures Using a Library of Protein Spectra and the Ramachandran Plot.

Infrared (IR) spectra from 1200 to 1800 cm(-1) of the pure α-helix and ß-sheet secondary structures have been extracted using a covariant least-squares procedure which relates a library of 40 infrared (IR) solution protein spectra from the work of Dong, Carpenter, and Caughey and amino acid fractions of the proteins based on assignments by STRIDE (secondary structure identification) of Eisenhaber and Argos. The excitonic splitting of the ß-sheet structures is determined for this library of solution proteins. The method is extended to find a set of spectral basis functions that analyze IR spectra of protein samples for α-helix and ß-sheet content. A rigorous error analysis including covariance, the correlations between the input library spectra, was used to justify the results and avoid less meaningful results. The utility of the results on α-helix and ß-sheet regions is demonstrated by detecting protein changes due to cancer in imaging Fourier transform IR (FTIR) spectra of liver tissue slices. This work ends with a method to extract IR spectra of less prominent torsional angle distributions.

Intraoperative Use of a Portable Large Field of View Gamma Camera and Handheld Gamma Detection Probe for Radioguided Localization and Prediction of Complete Surgical Resection of Gastrinoma: Proof of Concept.

BACKGROUND: Surgical management of Zollinger-Ellison syndrome (ZES) relies on localization and resection of all tumor foci. We describe the benefit of combined intraoperative use of a portable large field of view gamma camera (LFOVGC) and a handheld gamma detection probe (HGDP) for indium-111 ((111)In)-pentetreotide radioguided localization and confirmation of gastrinoma resection in ZES. STUDY DESIGN: Five patients (6 cases) with (111)In-pentetreotide-avid ZES were evaluated. Patients were injected with (111)In-pentetreotide for diagnostic imaging the day before surgery. Intraoperatively, an HGDP and LFOVGC were used to localize (111)In-pentetreotide-avid lesions, guide resection, assess specimens for (111)In-pentetreotide activity, and to verify lack of abnormal post-resection surgical field activity. RESULTS: Large field of view gamma camera imaging and HGDP-assisted detection were helpful for localization and guided resection of tumor and removal of (111)In-pentetreotide-avid tumor foci in all cases. In 3 of 5 patients (3 of 6 cases), these techniques led to detection and resection of additional tumor foci beyond those detected by standard surgical techniques. The (111)In-pentetreotide-positive or-negative specimens correlated with neuroendocrine tumors or benign pathology, respectively. In one patient with mild residual focal activity on post-resection portable LFOVGC imaging, thought to be artifact, had recurrence of disease in the same area 5 months after surgery. CONCLUSIONS: Real-time LFOVGC imaging and HGDP use for surgical management of gastrinoma improve success of localizing and resecting all neuroendocrine tumor-positive tumor foci, providing instantaneous navigational feedback. This approach holds potential for improving long-term patient outcomes in patients with ZES.

Feasibility of a multimodal (18)F-FDG-directed lymph node surgical excisional biopsy approach for appropriate diagnostic tissue sampling in patients with suspected lymphoma.

BACKGROUND: F-FDG PET/CT imaging is widely utilized in the clinical evaluation of patients with suspected or documented lymphoma. The aim was to describe our cumulative experience with a multimodal (18)F-FDG-directed lymph node surgical excisional biopsy approach in patients with suspected lymphoma. METHODS: Thirteen patients (mean age 51 (± 16;22-76) years), with suspected new or suspected recurrent lymphoma suggested by (18)F-FDG-avid lesions seen on prior diagnostic whole-body PET/CT imaging, were injected IV with (18)F-FDG prior to undergoing same-day diagnostic lymph node surgical excisional biopsy in the operating room. Various (18)F-FDG detection strategies were used on the day of surgery, including, (1) same-day pre-resection patient PET/CT; (2) intraoperative gamma probe assessment; (3) clinical scanner specimen PET/CT imaging of whole surgically excised tissue specimens; (4) specimen gamma well counts; and/or (5) same-day post-resection patient PET/CT. RESULTS: Same-day (18)F-FDG injection dose was 14.8 (± 2.4;12.5-20.6) millicuries or 548 (± 89;463-762) megabecquerels. Sites of (18)F-FDG-avid lesions were 4 inguinal, 3 cervical, 3 abdominal/retroperitoneal, 2 axillary, and 1 gluteal region subcutaneous tissue. Same-day pre-resection patient PET/CT was performed on 6 patients. Intraoperative gamma probe assessment was performed on 13 patients. Clinical scanner PET/CT imaging of whole surgically excised tissue specimens was performed in 10 cases. Specimen gamma well counts were performed in 6 cases. Same-day post-resection patient PET/CT imaging was performed on 8 patients. Time from (18)F-FDG injection to same-day pre-resection patient PET/CT, intraoperative gamma probe assessment, and same-day post-resection patient PET/CT were 76 (± 8;64-84), 240 (± 63;168-304), and 487 (± 104;331-599) minutes, respectively. Time from (18)F-FDG injection to clinical scanner PET/CT of whole surgically excised tissue specimens was 363 (± 60;272-446) minutes. Time from (18)F-FDG injection to specimen gamma well counts was 591 (± 96;420-689) minutes. Intraoperative gamma probe assessment successfully identified (18)F-FDG-avid lesions in 12/13 patients. Histopathologic evaluation confirmed lymphoma in 12/13 patients and benign disease in 1/13 patients. CONCLUSIONS: A multimodal approach to (18)F-FDG-directed lymph node surgical excisional biopsy for suspected lymphoma is technically feasible for guiding appropriate diagnostic tissue sampling of lymph nodes seen as (18)F-FDG-avid lesions on diagnostic (18)F-FDG PET/CT imaging.

Intraoperative scintigraphy using a large field-of-view portable gamma camera for primary hyperparathyroidism: initial experience.

BACKGROUND: We investigated a novel technique, intraoperative (99 m)Tc-Sestamibi (MIBI) imaging (neck and excised specimen (ES)), using a large field-of-view portable gamma camera (LFOVGC), for expediting confirmation of MIBI-avid parathyroid adenoma removal. METHODS: Twenty patients with MIBI-avid parathyroid adenomas were preoperatively administered MIBI and intraoperatively imaged prior to incision (neck) and immediately following resection (neck and/or ES). Preoperative and intraoperative serum parathyroid hormone monitoring (IOPTH) and pathology (path) were also performed. RESULTS: MIBI neck activity was absent and specimen activity was present in 13/20 with imaging after initial ES removal. In the remaining 7/20 cases, residual neck activity and/or absent ES activity prompted excision of additional tissue, ultimately leading to complete hyperfunctioning tissue excision. Postexcision LFOVGC ES imaging confirmed parathyroid adenoma resection 100% when postresection imaging qualitatively had activity (ES) and/or no activity (neck). The mean ± SEM time saving using intraoperative LFOVGC data to confirm resection versus first IOPTH or path result would have been 22.0 ± 2 minutes (specimen imaging) and 26.0 ± 3 minutes (neck imaging). CONCLUSION: Utilization of a novel real-time intraoperative LFOVGC imaging approach can provide confirmation of MIBI-avid parathyroid adenoma removal appreciably faster than IOPTH and/or path and may provide a valuable adjunct to parathyroid surgery.

Complex chronic conditions in Rhode Island's pediatric populace: implications for palliative and hospice services, 2000-2012.

BACKGROUND: Pediatric patients with complex chronic conditions (CCC) can benefit from pediatric palliative and hospice care (PP/HC) services. PP/HC can be delivered in a variety of health care settings and for a multitude of conditions, but data on hospitalization patterns and on secondary illnesses in pediatric CCC patients remains scant. OBJECTIVE: The study objective was to describe mortality trends for Rhode Island resident children aged 0-17 years, along with the demographics, subtypes, sites of death, and comorbidities of those with CCC. METHODS: This was a retrospective cohort study using demographic, hospitalization, and clinical data from all Rhode Island Department of Health death certificates from 2000 to 2012. RESULTS: Among the 1422 Rhode Island children aged 0-17 years old who died from 2000 to 2012, CCCs accounted for 27% (279/1049) of medically related deaths and 62% (145/233) of such deaths after infancy. CCC deaths were more likely at home (OR 5.202, 95% CI 2.984-9.203, p<0.001) and to have had a secondary cause of death documented (OR 3.032, 95% CI 2.259-4.067, p<0.001) than were other medically related deaths. Infants with CCCs were more likely to die in an inpatient setting (OR 5.141, 95% CI 2.718-10.026, p<0.001), whereas 1-17 year-olds with CCCs were more likely to die at home (OR 5.346, 95% CI 2.200-14.811, p<0.001) or in an emergency department (OR 3.281, 95% CI 1.363-8.721, p<0.040). CONCLUSIONS: CCCs constitute a significant proportion of medically related pediatric deaths in Rhode Island and are associated with both secondary comorbidities and death at home. Specialized, multidisciplinary services are warranted and PP/HC is crucial for patient and family support.

Comparison of two threshold detection criteria methodologies for determination of probe positivity for intraoperative in situ identification of presumed abnormal 18F-FDG-avid tissue sites during radioguided oncologic surgery.

BACKGROUND: Intraoperative in situ identification of (18)F-FDG-avid tissue sites during radioguided oncologic surgery remains a significant challenge for surgeons. The purpose of our study was to evaluate the 1.5-to-1 ratiometric threshold criteria method versus the three-sigma statistical threshold criteria method for determination of gamma detection probe positivity for intraoperative in situ identification of presumed abnormal (18)F-FDG-avid tissue sites in a manner that was independent of the specific type of gamma detection probe used. METHODS: From among 52 patients undergoing appropriate in situ evaluation of presumed abnormal (18)F-FDG-avid tissue sites during (18)F-FDG-directed surgery using 6 available gamma detection probe systems, a total of 401 intraoperative gamma detection probe measurement sets of in situ counts per second measurements were cumulatively taken. RESULTS: For the 401 intraoperative gamma detection probe measurement sets, probe positivity was successfully met by the 1.5-to-1 ratiometric threshold criteria method in 150/401 instances (37.4%) and by the three-sigma statistical threshold criteria method in 259/401 instances (64.6%) (P < 0.001). Likewise, the three-sigma statistical threshold criteria method detected true positive results at target-to-background ratios much lower than the 1.5-to-1 target-to-background ratio of the 1.5-to-1 ratiometric threshold criteria method. CONCLUSIONS: The three-sigma statistical threshold criteria method was significantly better than the 1.5-to-1 ratiometric threshold criteria method for determination of gamma detection probe positivity for intraoperative in situ detection of presumed abnormal (18)F-FDG-avid tissue sites during radioguided oncologic surgery. This finding may be extremely important for reshaping the ongoing and future research and development of gamma detection probe systems that are necessary for optimizing the in situ detection of radioisotopes of higher-energy gamma photon emissions used during radioguided oncologic surgery.

18F-FDG PET/CT oncologic imaging at extended injection-to-scan acquisition time intervals derived from a single-institution 18F-FDG-directed surgery experience: feasibility and quantification of 18F-FDG accumulation within 18F-FDG-avid lesions and background tissues.

BACKGROUND: 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) is a well-established imaging modality for a wide variety of solid malignancies. Currently, only limited data exists regarding the utility of PET/CT imaging at very extended injection-to-scan acquisition times. The current retrospective data analysis assessed the feasibility and quantification of diagnostic (18)F-FDG PET/CT oncologic imaging at extended injection-to-scan acquisition time intervals. METHODS: (18)F-FDG-avid lesions (not surgically manipulated or altered during (18)F-FDG-directed surgery, and visualized both on preoperative and postoperative (18)F-FDG PET/CT imaging) and corresponding background tissues were assessed for (18)F-FDG accumulation on same-day preoperative and postoperative (18)F-FDG PET/CT imaging. Multiple patient variables and (18)F-FDG-avid lesion variables were examined. RESULTS: For the 32 (18)F-FDG-avid lesions making up the final (18)F-FDG-avid lesion data set (from among 7 patients), the mean injection-to-scan times of the preoperative and postoperative (18)F-FDG PET/CT scans were 73 (± 3, 70-78) and 530 (± 79, 413-739) minutes, respectively (P < 0.001). The preoperative and postoperative mean (18)F-FDG-avid lesion SUV(max) values were 7.7 (± 4.0, 3.6-19.5) and 11.3 (± 6.0, 4.1-29.2), respectively (P < 0.001). The preoperative and postoperative mean background SUV(max) values were 2.3 (± 0.6, 1.0-3.2) and 2.1 (± 0.6, 1.0-3.3), respectively (P = 0.017). The preoperative and postoperative mean lesion-to-background SUV(max) ratios were 3.7 (± 2.3, 1.5-9.8) and 5.8 (± 3.6, 1.6-16.2), respectively, (P < 0.001). CONCLUSIONS: (18)F-FDG PET/CT oncologic imaging can be successfully performed at extended injection-to-scan acquisition time intervals of up to approximately 5 half-lives for (18)F-FDG while maintaining good/adequate diagnostic image quality. The resultant increase in the (18)F-FDG-avid lesion SUV(max) values, decreased background SUV(max) values, and increased lesion-to-background SUV(max) ratios seen from preoperative to postoperative (18)F-FDG PET/CT imaging have great potential for allowing for the integrated, real-time use of (18)F-FDG PET/CT imaging in conjunction with (18)F-FDG-directed interventional radiology biopsy and ablation procedures and (18)F-FDG-directed surgical procedures, as well as have far-reaching impact on potentially re-shaping future thinking regarding the "most optimal" injection-to-scan acquisition time interval for all routine diagnostic (18)F-FDG PET/CT oncologic imaging.

Pain, depression, and fatigue: loneliness as a longitudinal risk factor.

OBJECTIVE: Pain, depression, and fatigue function as a symptom cluster and thus may share common risk factors. Interpersonal relationships clearly influence health, suggesting that loneliness may promote the development of the pain, depression, and fatigue symptom cluster. We hypothesized that loneliness would be related to concurrent symptom cluster levels and increases in symptom cluster levels over time. METHOD: We utilized two observational studies with distinct longitudinal samples. Study 1 was a sample of cancer survivors and benign controls (N = 115) assessed annually for 2 years. Study 2 was a sample of older adults caring for a spouse with dementia (caregivers) and non-caregiver controls (N = 229) assessed annually for 4 years. Participants completed annual measures assessing loneliness, pain, depression, and fatigue. RESULTS: Across both samples, lonelier participants experienced more concurrent pain, depression, and fatigue and larger increases in symptom cluster levels from one year to the next than less lonely participants. Sleep quality did not mediate the results in either study. All analyses were adjusted for relevant demographic and health variables. CONCLUSIONS: Two longitudinal studies with different populations demonstrated that loneliness was a risk factor for the development of the pain, depression, and fatigue symptom cluster over time. The current research helps identify people most at risk for pain, depression, and fatigue, and lays the groundwork for research about their diagnosis and treatment. These data also highlight the health risks of loneliness; pain, depression, and fatigue often accompany serious illness and place people at risk for poor health and mortality.

The goals of care conversation: a tool to improve patient care in the nursing home at the end of life.

Patients residing in nursing homes may undergo burdensome transitions in care during the final months of life. They may get care they do not want and are unlikely to benefit from. Patients and families may not understand prognosis or the potential benefits of treatment. A "goals of care" conversation can be the critical first step in identifying a patient's wishes and then developing a plan of care that honors those wishes. When the goal of care is to focus on comfort, hospice can be accessed. Hospice can help ensure that the patient's final time is spent in comfort and that the family's needs are attended to both before and after the patient dies.

Site specific discrete PEGylation of (124)I-labeled mCC49 Fab' fragments improves tumor MicroPET/CT imaging in mice.

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2-4 kDa discrete, branched PEGylation reagents on mCC49 Fab' (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab' (Fab'-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG 12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 ((124)I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab' with Mal-dPEG-A (Fab'-A) reduced the binding affinity of the non PEGylated Fab' by 30%; however, in vivo, Fab'-A significantly lengthened the blood retention vs Fab'-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab'-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.