Melanoma: Pruitt SK

Coit DG, Andtbacka R, Bichakjian CK, Dilawari RA, Dimaio D, Guild V, Halpern AC, Hodi FS, Kashani-Sabet M, Lange JR, Lind A, Martin L, Martini MC, Pruitt SK, Ross MI, Sener SF, Swetter SM, Tanabe KK, Thompson JA, Trisal V, Urist MM, Weber J, Wong MK, Anonymous00048. · No affiliation provided · J Natl Compr Canc Netw. · Pubmed #19401060 No free full text.

This publication has no abstract.

Beasley GM, Petersen RP, Yoo J, McMahon N, Aloia T, Petros W, Sanders G, Cheng TY, Pruitt SK, Seigler H, Tyler DS. · Department of Surgery, Duke University Medical Center, Box 3118, Durham, NC 27710, USA. · Ann Surg Oncol. · Pubmed #18528730 No free full text.

Abstract: BACKGROUND: Isolated limb infusion (ILI) is a recently described minimally invasive technique developed in Australia for delivering regional chemotherapy. This study examined the efficacy and toxicity of ILI, compared to hyperthermic isolated limb perfusion (HILP), in treating extremity in-transit melanoma. METHODS: Variables from a prospective single institution database of 120 regionally treated melanoma patients (1995-2007) were compared using chi-square analysis. This included 61 consecutive ILI treatments in 58 patients and 59 HILP treatments in 54 patients. Response was defined at 3 months using the response evaluation criteria in solid tumors (RECIST). ILI was performed using melphalan (LPAM) and dactinomycin for 30 min after limb temperature reached 37 degrees C. HILP was performed using LPAM for 60 min after limb temperature reached 38.5 degrees C. RESULTS: For ILI (n = 61), the complete response (CR) rate was 30%, the partial response (PR) rate was 14%, and there was no response (NR) in 56% of patients. The median duration of CR was 12 months and 18% of patients experienced (grade >or=3) toxicity. HILP (n = 59) was associated with a better (P < 0.001) response rate (CR 57%, PR 31%, and NR 12%) however, more patients (32%) experienced grade >or=3 toxicity (P = 0.037). The dose of LPAM was corrected for ideal body weight (IBW) in 40 out of 61 ILI procedures, and 13 of 59 HILP procedures. This dosing modification was associated with decreased toxicity (P = 0.024) without diminishing response. CONCLUSION: ILI was found to be a well-tolerated alternative to HILP. While ILI does not appear to be as effective as HILP, it does seem to be associated with less morbidity.

Beasley GM, Caudle A, Petersen RP, McMahon NS, Padussis J, Mosca PJ, Zager JS, Hochwald SN, Grobmyer SR, Delman KA, Andtbacka RH, Noyes RD, Kane JM, Seigler H, Pruitt SK, Ross MI, Tyler DS. · Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA. · J Am Coll Surg. · Pubmed #19476821 No free full text.

Abstract: BACKGROUND: Isolated limb infusion (ILI) is a minimally invasive approach for treating in-transit extremity melanoma, with only two US single-center studies reported. Establishing response and toxicity to ILI as compared with hyperthermic isolated limb perfusion is important for optimizing future regional chemotherapeutic strategies in melanoma. STUDY DESIGN: Patient characteristics and procedural variables were collected retrospectively from 162 ILIs performed at 8 institutions (2001 to 2008) and compared using chi-square and Student's t-test. ILIs were performed for 30 minutes in patients with in-transit melanoma. Melphalan dose was corrected for ideal body weight (IBW) in 42% (n = 68) of procedures. Response was determined at 3 months by Response Evaluation Criteria in Solid Tumors; toxicity was assessed using the Wieberdink Limb Toxicity Scale. RESULTS: In 128 evaluable patients, complete response rate was 31%, partial response rate was 33%, and there was no response in 36% of patients. For all patients (n = 162), 36% had Wieberdink toxicity grade >or=3 with one toxicity-related amputation. On multivariate analysis, smaller limb volumes were associated with better overall response (p = 0.021). Use of papaverine in the circuit to achieve cutaneous vasodilation was associated with better response (p < 0.001) but higher risk of grade >or=3 toxicity (p = 0.001). Correction of melphalan dose for ideal body weight did not alter complete response (p = 0.345), but did lead to marked reduction in toxicity (p < 0.001). CONCLUSIONS: In the first multi-institutional analysis of ILI, a complete response rate of 31% was achieved with acceptable toxicity demonstrating this procedure to be a reasonable alternative to hyperthermic isolated limb perfusion in the management of advanced extremity melanoma.

Dannull J, Lesher DT, Holzknecht R, Qi W, Hanna G, Seigler H, Tyler DS, Pruitt SK. · Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA. · Blood. · Pubmed #17855630 links to  free full text

Abstract: The process of dendritic cell (DC) maturation, critical for effective DC-based immunotherapy, also alters the proteasome such that peptides presented in the context of HLA class I are generated not by the constitutive proteasome, but by the immunoproteasome. Cytotoxic T lymphocytes (CTLs) induced by such DCs might not optimally recognize tumor cells normally expressing the constitutive proteasome. Using small interfering RNA (siRNA) transfection of DCs to inhibit expression of the 3 inducible immunoproteasome subunits in mature DCs, we found that such DCs expressed increased intracellular levels of constitutive proteasomes and presented an altered repertoire of tumor-antigenic peptides. When DCs generated from the monocytes of 3 patients with melanoma were transfected with immunoproteasome siRNA, induced to mature, and then trans-fected with RNA encoding defined melanoma antigens, these DCs were superior inducers of antigen-specific CTLs against autologous melanoma cells. This alteration of DC proteasome composition, which enhances the ability of mature antigen-loaded DCs to stimulate anti-tumor immune responses, may lead to more effective DC-based tumor immunotherapy.

Yoshimoto Y, Augustine CK, Yoo JS, Zipfel PA, Selim MA, Pruitt SK, Friedman HS, Ali-Osman F, Tyler DS. · Department of Surgery, Duke University Medical Center, Box 3118, Durham, NC 27710, USA. · Mol Cancer Ther. · Pubmed #17483437 links to  free full text

Abstract: Five different human melanoma xenografts were used in a xenograft model of extremity melanoma to evaluate the variability of tumor response to regionally administered melphalan or temozolomide and to determine if various components of pertinent drug resistance pathways for melphalan [glutathione S-transferase (GST)/glutathione] and temozolomide [O(6)-alkylguanine DNA alkyltranferase (AGT)/mismatch repair (MMR)] could be predictive of tumor response. Xenograft-bearing rats underwent regional isolated limb infusion with either melphalan (90 mg/kg) or temozolomide (2,000 mg/kg). The levels of AGT activity, GST activity, glutathione level, and GST/AGT expression were examined in this group of xenografts and found to be quite heterogeneous. No correlation was identified between melphalan sensitivity and the GST/glutathione cellular detoxification pathway. In contrast, a strong correlation between the levels of AGT activity and percentage increase in tumor volume on day 30 (r = 0.88) was noted for tumors treated with temozolomide. Regional therapy with temozolomide was more effective when compared with melphalan for the xenograft with the lowest AGT activity, whereas melphalan was more effective than temozolomide in another xenograft that had the highest AGT activity. In three other xenografts, there was no significant difference in response between the two chemotherapy agents. This study shows that AGT activity may be useful in predicting the utility of temozolomide-based regional therapy for advanced extremity melanoma tumors. Our observations also point out the limited ability of analysis of the GST/glutathione pathway to predict response to chemotherapies like melphalan whose resistance is primarily mediated through a complex mechanism of detoxification.

Ueno T, Ko SH, Grubbs E, Yoshimoto Y, Augustine C, Abdel-Wahab Z, Cheng TY, Abdel-Wahab OI, Pruitt SK, Friedman HS, Tyler DS. · Department of Surgery, Duke University Medical Center, Box 3118, Durham, NC 27710, USA. · Mol Cancer Ther. · Pubmed #16546988 links to  free full text

Abstract: This study investigated whether the therapeutic index of regional melanoma therapy using parenteral temozolomide could be improved by chemomodulation with O6-benzylguanine (O6BG), an inhibitor of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT). Using a nude rat s.c. human melanoma xenograft model of the extremity, tumors were analyzed for AGT level 2 to 3 hours after the i.p. injection of 3.5 to 70.0 mg/kg O6BG to inhibit AGT activity. Survival studies were conducted using animals that were treated with a 15-minute isolated limb infusion with 10% DMSO in PBS (control), temozolomide alone, or temozolomide in conjunction with single or multiple doses of i.p. O6BG. Tumor volume and toxicity level were monitored every other day. Administration of 3.5 mg/kg O6BG depleted tumor AGT activity by 93.5% (P < 0.01). Groups treated with regional temozolomide alone (350 mg/kg), systemic temozolomide with O6BG, or vehicle combined with O6BG showed no significant tumor responses compared with controls. Whereas use of regional temozolomide alone at a higher dose (750 mg/kg) showed some degree of tumor response, regional temozolomide given in conjunction with multiple dosages of O6BG showed a marked (P < 0.01) reduction in tumor growth with minimal toxicity. Our findings suggest that AGT modulation by the administration of O6BG in combination with temozolomide regional chemotherapy leads to a significant improvement in melanoma antitumor responses. Clinical trials using chemotherapy modulation may improve response rates in future regional infusion and perfusion drug trials.

Ko SH, Ueno T, Yoshimoto Y, Yoo JS, Abdel-Wahab OI, Abdel-Wahab Z, Chu E, Pruitt SK, Friedman HS, Dewhirst MW, Tyler DS. · Department of Surgery, Duke University School of Medicine, Durham, North Carolina 27710, USA. · Clin Cancer Res. · Pubmed #16397054 links to  free full text

Abstract: PURPOSE: Previous preclinical studies have shown that regional temozolomide therapy via isolated limb infusion is more effective than melphalan, the current drug of choice for regional chemotherapy for advanced extremity melanoma. The aim of this study was to determine whether hyperthermia could further augment the efficacy of temozolomide, an alkylating agent, against melanoma and improve its therapeutic index in a rat model of isolated limb infusion. EXPERIMENTAL DESIGN: Athymic rats bearing s.c. human melanoma xenografts (DM6) in their hind limbs were randomized to a 15-minute isolated limb infusion procedure with or without temozolomide at room temperature, normothermic (37.5 degrees C), or hyperthermic (43 degrees C) conditions. RESULTS: The concomitant administration of hyperthermia during an infusion with temozolomide led to the greatest increase in tumor growth delay, decreased proliferative index, and increased cell death. Isolated limb infusion treatment with a low dose (350 mg/kg) of temozolomide was ineffective at producing tumor growth delay (P = 0.07). Similarly, temozolomide infusion under normothermia yielded minimal tumor growth delay (P = 0.08). In contrast, the combination of hyperthermia plus temozolomide treatment produced marked tumor growth delay of 10.4 days (P = 0.02) with minimal toxicity. The addition of heat to temozolomide treatment yielded the smallest proliferative index (P = 0.001), while markedly increasing the level of apoptosis 48 hours after isolated limb infusion. CONCLUSION: This study, the first to examine the interaction between hyperthermia and temozolomide, shows a strong, synergistic antitumor effect when hyperthermia is combined with temozolomide for regional treatment of melanoma confined to an extremity. The mechanism of this synergy seems to be through an augmentation, by hyperthermia, of the antiproliferative and proapoptotic effects of temozolomide.

Ueno T, Ko SH, Grubbs E, Pruitt SK, Friedman HS, Tyler DS. · Department of Surgery, Box 3118 Medical Center, Duke University Medical Center, Durham, NC 27710, USA. · Am J Surg. · Pubmed #15546565 No free full text.

Abstract: BACKGROUND: Regional infusion therapy with melphalan (LPAM) is an accepted treatment for advanced extremity melanoma. However, much room exists for improving the therapeutic index of this type of therapy. METHODS: Isolated limb infusion (ILI) with temozolomide (TMZ), a novel methylating agent, was performed using a nude rat bearing human melanoma xenograft. Additional rats were treated systemically with TMZ, or regionally with LPAM or 10% dimethyl sulfoxide (DMSO; control) using ILI. RESULTS: Rats that received systemic TMZ showed a poor tumor response and no tumor regression. In contrast, intra-arterial TMZ demonstrated a prolongation of tumor growth delay in a dose-responsive manner. In comparison with LPAM of equitoxic dose, TMZ provided both longer tumor growth delay and a greater number of tumor regressions. CONCLUSIONS: These data suggest that ILI with TMZ is an effective treatment for advanced extremity melanoma and may be better than LPAM in this setting.

Grubbs EG, Abdel-Wahab O, Cheng TY, Abdel-Wahab Z, Peterson B, Pruitt SK, Colvin OM, Friedman HS, Tyler DS. · Department of General Surgery, Duke University Medical Center, Durham, NC 27710, USA. · J Am Coll Surg. · Pubmed #15325612 No free full text.

Abstract: BACKGROUND: Regional perfusion treatments for melanoma, using the alkylating agent melphalan, show variable responses in magnitude and duration. Surprisingly, the potential contribution of alkylating-agent resistance mechanisms to diminish tumor responses, especially the crucial cellular detoxifying system formed by glutathione (GSH) and its associated enzyme glutathione-S-transferase (GST), has remained unexplored. Objectives of this study were to characterize GSH levels and GST activity in melanoma of patients undergoing regional perfusion and examine the effect of melphalan concentration in both an in vitro human melanoma cell line and in the extremity melanoma of an in vivo rodent limb infusion model. STUDY DESIGN: Human in-transit melanoma, muscle, subcutaneous tissue, and skin (n = 9) and metastatic regional lymph nodes (n = 7) were evaluated for GSH level and GST activity. Effects of increasing melphalan exposure on GSH and GST were studied in an in vitro human melanoma cell line. A survival human melanoma xenograft model of isolated limb infusion using increasing dosages of melphalan was used, with evaluation of GSH and GST in the recurrent tumor. RESULTS: GSH levels in human in-transit lesions and muscle were significantly higher than that of skin and subcutaneous tissue. Four of 9 patients had tumor-to-muscle GSH ratio > 1. A strong correlation was seen between in vitro melphalan dose and resultant GSH level and GST activity. In vivo recurrent tumor GSH levels correlated with increasing melphalan infusion dose. CONCLUSIONS: A GSH-based resistance pathway may play a role in effecting response and toxicity to regional melphalan perfusion.

Grubbs EG, Ueno T, Abdel-Wahab O, Cheng TY, Pruitt SK, Michael Colvin O, Friedman HS, Tyler DS. · Departments of General Surgery, Internal Medicine, and Neurosurgery, Duke University Medical Center, Durham, NC 27710, USA. · Surgery. · Pubmed #15300182 No free full text.

Abstract: BACKGROUND: The presence of resistance to chemotherapy is associated with poor tumor response and patient survival in a variety of tumors. Attempts to modulate resistance in conjunction with systemic chemotherapy have been limited by the toxicity of combined therapy, particularly gastrointestinal or hematopoetic toxicity. This study explored systemic modulation of resistance in conjunction with intra-arterial regional therapy to determine if tumor responses to melphalan could be improved with acceptable toxicity. METHODS: Using a nude rat human xenograft model of extremity melanoma,we analyzed tumors for glutathione (GSH), the main protein in the melphalan resistance pathway. Modulation of GSH was performed with intraperitoneal buthionine sulfoximine (BSO). In parallel, BSO-modulated and nonmodulated animals underwent survival studies after regional intra-arterial perfusion with melphalan or saline. Rats were monitored daily for tumor growth and toxicity. RESULTS: BSO depleted tumor GSH levels by 71.8% with minimal toxicity. Survival studies using increasing melphalan concentrations demonstrated similar tumor growth. The combined use of modulator and chemotherapeutic agent showed a significant tumor growth delay as compared to control and drug-alone group without enhanced toxicity. CONCLUSIONS: Modulation of resistance in conjunction with regional chemotherapy allows for improved tumor responses with minimal toxicity. These results demonstrate that BSO can potentiate the cytotoxic effects of regional melphalan therapy in the setting of extremity melanoma.

Cisco RM, Abdel-Wahab Z, Dannull J, Nair S, Tyler DS, Gilboa E, Vieweg J, Daaka Y, Pruitt SK. · Department of Surgery, Duke University and Durham Veterans Affairs Medical Centers, Durham, NC 27710, USA. · J Immunol. · Pubmed #15153540 links to  free full text

Abstract: Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-alpha secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4(+) T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-alpha, IL-6, IL-1beta, and PGE(2)). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.

Kalady MF, Onaitis MW, Emani S, Abdel-Wahab Z, Tyler DS, Pruitt SK. · Department of Surgery, Duke University Medical Center and Durham VA Medical Center, Durham, North Carolina 27710, USA. · J Surg Res. · Pubmed #14732346 No free full text.

Abstract: BACKGROUND: Despite the increasing use of dendritic cells (DCs) in clinical trials, questions regarding the optimal means of DC preparation, in particular how to achieve optimal maturation, remain unanswered. We hypothesized that delivering two separate sequential maturation signals to DC in vitro, mimicking the process of DC maturation that occurs in vivo, would enhance the ability of DCs to generate antigen-specific effector T cells in an experimental in vitro antimelanoma model. MATERIALS AND METHODS: Human monocyte-derived DCs were transfected with mRNA encoding melanoma-associated antigen Mart-1 (MART) or influenza M1 matrix protein (M1). After mRNA transfection, DCs were left untreated or exposed to different maturation stimuli either added simultaneously or delivered sequentially 18 h after first stimulation. Phenotypic DC cell-surface marker changes and IL-12 secretion were analyzed. Specific antigen presentation by DCs was measured by IFN-gamma release Elispot assay using a CD8(+) MART peptide-specific T cell clone. RNA-transfected and treated DCs were cultured with autologous naive T cells and the induction of antigen-specific effector T cells were assessed by IFN-gamma release Elispot assay. RESULTS: DCs transfected and matured had increased cell-surface expression of CD40 and costimulatory molecules CD80, and CD86. DCs matured and further treated by soluble CD40 ligand (sCD40L) had a 10- and 2-fold increase in MART antigen presentation compared to untreated (immature) DCs and DCs treated only with a first maturation signal, respectively (Elispot P = 0.02). Delivery of sequential maturation stimuli resulted in maximal DC IL-12 secretion compared to simultaneous stimuli. Last, generation of antigen-specific effector T cells more than doubled with the sequential addition of sCD40L to mature DC stimulators (Elispot P = 0.009). CONCLUSIONS: Maturation of DCs following mRNA transfection increases expression of cell-surface costimulatory molecules. Delivery of a second sequential maturation stimulus enhances antigen presentation, increases IL-12 secretion, and augments immunogenicity as evidenced by generation of tumor antigen-specific effector T cells. This strategy should be considered in the future development of RNA-based DC vaccine strategies for the treatment of cancer.

Abdel-Wahab Z, Kalady MF, Emani S, Onaitis MW, Abdel-Wahab OI, Cisco R, Wheless L, Cheng TY, Tyler DS, Pruitt SK. · Department of Surgery, Duke University Medical Center, P.O. Box 2624 MSRB, Durham, NC 27710, USA. · Cell Immunol. · Pubmed #14609574 No free full text.

Abstract: Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.

Onaitis MW, Kalady MF, Emani S, Abdel-Wahab Z, Tyler DS, Pruitt SK. · Department of Surgery, Duke University and Durham VA Medical Centers, Durham, NC 27710, USA. · Surgery. · Pubmed #12947333 No free full text.

Abstract: BACKGROUND: Dendritic cell (DC)-based immunotherapy is a promising form of adjuvant therapy for high-risk tumors. DCs transfected with tumor-associated antigens are capable of stimulating antigen-specific T cells, but cytolytic responses have been disappointing. Activation of DC surface CD40 influences DC cytokine production, particularly that of interleukin (IL)-12, which favors a Th1 (cytotoxic) helper T cell response. This study evaluated the effects of exogenous soluble CD40 ligand (sCD40L) on RNA-transfected DC preparations and their subsequent ability to generate antimelanoma cytolytic T cells. METHODS: Human monocyte-derived DCs were cultured and transfected with mRNA encoding full-length melanoma-associated antigen, Mart-1, and matured with and without sCD40L. DC IL-12 secretion and the ability to stimulate naïve T cells were assessed by enzyme-linked immunosorbent assay (ELISA), tetramer analysis, Elispot, and (51)Cr release assay. RESULTS: Mature DCs stimulated with sCD40L secreted higher levels of IL-12 compared with immature DCs and DCs matured without sCD40L (P <.001). DCs treated with sCD40L generated a greater number of antigen-specific T cells (P <.05) by tetramer and Elispot analyses, and yielded specific T cells with significant cytotoxicity against HLA-matched melanoma cell lines. CONCLUSIONS: CD40L augments DC IL-12 secretion and is essential to potentiate specific antimelanoma cytolytic responses stimulated by the Mart-1 antigen. sCD40L should be considered a crucial adjuvant in DC preparations for RNA-based DC vaccine therapies.

White DC, Schuler FR, Pruitt SK, Culhane DK, Seigler HF, Coleman RE, Tyler D. · Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA. · Surgery. · Pubmed #10455878 No free full text.

Abstract: BACKGROUND: Sentinel lymph node (SLN) mapping is an effective technique for staging patients with melanoma. In an attempt to avoid reinjection of radiolabeled colloid and facilitate SLN mapping at the time of surgery, we examined whether residual radioactivity from preoperative lymphoscintigraphy could be used to accurately identify SLNs during surgery 18 to 24 hours later. METHODS: Forty-six patients with newly diagnosed melanoma underwent injection of 0.22-micron filtered technetium 99m-labeled sulfur colloid followed by lymphoscintigraphy. Patients returned the next day for SLN biopsy with Isosulfan blue dye and the hand-held gamma-probe to identify SLNs. Thirty of 46 patients underwent repeat imaging before operation. No patient had reinjection of radiocolloid. RESULTS: Ninety-five SLNs were identified on initial lymphoscintigraphy, and repeat imaging on the day of surgery confirmed all SLNs previously identified. A total of 122 SLNs (2.65 per patient) were resected from 58 basins. Eighty-four (69%) of 122 SLNs stained blue, and 118 (97%) of 122 SLNs had in vivo gamma-counts greater than 4 times background. Microscopic metastases were present in 13 (10.7%) of 122 SLNs in 12 (26.1%) of 46 patients. There have been no recurrences over a mean follow-up time of 320 days. CONCLUSIONS: Intraoperative gamma-probe detection combined with blue dye injection is highly effective in identifying SLNs 18 to 24 hours after injection of 0.22-micron filtered 99mTc-sulfur colloid. Reinjection of radiocolloid is not required. This technique avoids radiopharmaceutical administration in the operating room, minimizes radiation exposure, and increases scheduling flexibility.