NADH-diaphorase Staining Research Articles

Polymer-assisted intratumoral delivery of ethanol: Preclinical investigation of safety and efficacy in a murine breast cancer model.

Focal tumor ablation with ethanol could provide benefits in low-resource settings because of its low overall cost, minimal imaging technology requirements, and acceptable clinical outcomes. Unfortunately, ethanol ablation is not commonly utilized because of a lack of predictability of the ablation zone, caused by inefficient retention of ethanol at the injection site. To create a predictable zone of ablation, we have developed a polymer-assisted ablation method using ethyl cellulose (EC) mixed with ethanol. EC is ethanol-soluble and water-insoluble, allowing for EC-ethanol to be injected as a liquid and precipitate into a solid, occluding the leakage of ethanol upon contact with tissue. The aims of this study were to compare the 1) safety, 2) release kinetics, 3) spatial distribution, 4) necrotic volume, and 5) overall survival of EC-ethanol to conventional ethanol ablation in a murine breast tumor model. Non-target tissue damage was monitored through localized adverse events recording, ethanol release kinetics with Raman spectroscopy, injectate distribution with in vivo imaging, target-tissue necrosis with NADH-diaphorase staining, and overall survival by proxy of tumor growth. EC-ethanol exhibited decreased localized adverse events, a slowing of the release rate of ethanol, more compact injection zones, 5-fold increase in target-tissue necrosis, and longer overall survival rates compared to the same volume of pure ethanol. A single 150 μL dose of 6% EC-ethanol achieved a similar survival probability rates to six daily 50 μL doses of pure ethanol used to simulate a slow-release of ethanol over 6 days. Taken together, these results demonstrate that EC-ethanol is safer and more effective than ethanol alone for ablating tumors.

Microwave ablation zones are larger than they macroscopically appear - Reevaluation based on NADH vitality staining ex vivo.

Animal liver is established as an ex vivo model for studies on hepatic microwave ablation (MWA). Macroscopically visible color changes in the ablation zone are used to assess cell destruction and confirm successful ablation ex vivo. Macroscopy and histology of MWA zones regarding cell viability in ex vivo porcine livers were compared in this study. Six MWA were performed in porcine livers post mortem. A 14-G antenna and microwave generator (928 MHz; 9.0 kJ) were used. MWA were cut at the maximum cross section in vertical alignment to the antenna. NADH-diaphorase staining determined cell vitality. Macroscopic and microscopic ablation zones were statistically analyzed. Histology showed two distinct ablation zones: central white zone (WZH) with no cell viability and peripheral red zone (RZH) with partial cell viability. However, the macroscopically visible WZM was significantly smaller than the microscopic WZH with an area difference of 43.1% (p < 0.05) and a radius difference of 21.2% (1.6 mm; p < 0.05). Macroscopy and histology showed a very high correlation for the complete lesion area (WZH/M+RZH/M; r = 0.9; p = 0.001). The avital central zone is significantly larger as the macroscopically visible WZ which is commonly used to assess successful ablation in MWA ex vivo studies. Irreversible cell destruction can be underestimated in macroscopic evaluation.

Phase II study on radiofrequency ablation in stage 0 and I breast cancer without extensive intraductal components.

e12094 Background: We reported about a multi-center registration study on radiofrequency ablation (RFA) in early breast cancer (BC) (ASCO2012 #1119). Although various devices and ablation procedure were used, 5-year’s recurrence-free survival was 96% in 425 cases of T1BC treated with RFA alone. To validate complete pathological ablation in BC, we started a non-randomized phase II study in 2013 (UMIN000013836). Methods: Unilateral BC patients with stage 0 (TisN0M0) or I (T1N0M0 or T1N1miM0) were eligible. Tumor diameter of 2cm or less should be diagnosed by ultrasound and MR mammography. RFA was performed by Cool-tip RF ablation system (Covidien, USA). One month later, ablated tissue was collected by core needle biopsy or vacuum-assisted breast biopsy. Cell viability was determined by central review of independent pathologists using tumor specimens stained with hematoxylin–eosin and nicotinamide adenine dinucleotide (NADH) diaphorase. In case of complete ablation, breast irradiation and adjuvant therapy was planned. In case of incomplete ablation, partial mastectomy was recommended. The primary endpoint is complete ablation rate. The secondary endpoints are breast deformity after RFA, relapse-free survival and overall survival for 10 years. Two-step design consisting of 9 cases at 1st step and 20 cases at 2nd step was used for statistical evaluation. We expected complete ablation rate of 95% and 29 cases were needed to reject the null hypothesis that complete ablation rate would be less than 80% at lower threshold. Thus, 32 cases were required. Results: Thirty-six patients were enrolled between February 2013 and May 2016. The mean tumor size was 1.3 cm measured by MR mammography. Two patients were ineligible because of macrometastasis in a sentinel node. Of 34 eligible patients, one patient had viable cancer cells with NADH diaphorase staining in ablated specimens. Finally, complete ablation rate was 97%. As of January 2017, one patient, who refused adjuvant therapy and breast irradiation, had in-breast tumor recurrence adjacent to previously ablated lesion. Conclusions: Long-term follow-up should be needed, but RFA in early BC is a promising strategy instead of breast-conserving surgery. Clinical trial information: UMIN000013836.

Abstract OT1-3-01: Phase II study on radiofrequency ablation in stage 0 and I breast cancer without extensive intraductal components

Abstract Background: Screening mammography makes it possible to identify small size of breast cancer (BC) and minimize surgical management. Previously we reported about a multi-center cohort study on radiofrequency ablation (RFA) in early breast cancer (ASCO2012 #1119). Although various devices and ablation procedure were attempted, 5-year’s recurrence-free survival of ipsilateral breast tumor was 96% in 425 cases of T1BC treated with RFA alone (the mean follow-up, 50 months). To validate complete pathological ablation in BC, we started a non-randomized phase II study in 2013 (UMIN000013836). Eligibility criteria: Unilateral BC patients with stage 0 (TisN0M0) or I (T1N0M0 or T1N1miM0) are eligible. Written informed consent is obtained. Tumor diameter of 2cm or less should be diagnosed by ultrasound and MR mammography. BC with diffuse calcification, extensive intraductal components, or multiple tumors is excluded. Methods: RFA is performed by Cool-tip RF ablation system (Covidien, USA). The needle electrode is inserted into the center of referent tumor under ultrasound guidance. The first ablation is started at an initial electrical power level of 5W for 1 min and the power is increased at 10W for 1 min. After then, it is increased in steps of 10W from every minute until rapid elevation of tissue impedance. The second ablation is allowed by physician’s discretion. One month later, ablated tissue is collected by core needle biopsy or vacuum-assisted breast biopsy. Cell viability is examined by central review of independent pathologists using tumor specimens stained with hematoxylin–eosin and nicotinamide adenine dinucleotide (NADH) diaphorase. In case of complete ablation, breast irradiation and adjuvant therapy will be performed. In case of incomplete ablation, partial mastectomy should be recommended. Aims: The primary endpoint is complete ablation rate. The secondary endpoints are deformity after RFA, relapse-free survival and overall survival for 10 years. Two-step design is used for statistical evaluation. Finally, 32 patients will be needed. Present accrual: As of April 2014, 16 patients were enrolled. One patient was ineligible because of macrometastasis in a sentinel node. Of 13 eligible patients who underwent pathological examination, one patient had viable cancer cells with NADH diaphorase staining. We also investigate optimal histological examination of cell viability and adequate image diagnosis after RFA. Citation Format: Shigeru Imoto, Shinji Nagamine, Shunichi Ito, Hitoshi Tsuda, Masayuki Yoshida, Mitsuhiro Tozaki, Satoshi Morita, Takayuki Ueno. Phase II study on radiofrequency ablation in stage 0 and I breast cancer without extensive intraductal components [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-3-01.

Abstract P4-15-05: Long-term outcome of breast cancer patients treated with radiofrequency ablation

Abstract Background: Radiofrequency ablation (RFA) is considered to be the most promising non-surgical ablation technique for the treatment of small breast cancer. In feasibility studies, the ablated tumor was surgically removed after RFA, and the tumor cell viability were assessed by histological and/or immunohistological examinations. However, these assessments of tumor viability do not take the place of long-term follow-up in patients treated with RFA alone, because they do not allow for them to be followed up for recurrence after RFA, or to determine if there were any undesirable complications of this procedure. At present, few data are available regarding long-term follow-up of patients treated with this modality. Methods: Since 2005, we have performed RFA and sentinel lymph node (SLN) biopsy in 19 cases with invasive breast cancer less than 2.0 cm in greatest diameter. After SLN biopsy, a primary electrode (seven-array model 70 Starburst needle electrode; RITA Medical Systems, Mountain View, CA) was inserted into the tumor under real-time US guidance. Then, the prongs of the needle electrode were deployed over a distance of 3 cm in all cases, and the RF generator (RITA model 1500) was activated and set to automatic, with power at 20 W, temperature of 95°C, and an ablation time of 15 min. Axillary lymph node dissection (ALND) was performed in patients with positive SLNs. Several months after RFA therapy, the ablated tumor tissue was excised by multiple mammotome biopsy and examined histologically or immunohistochemically with H&amp;E staining, nicotinamide adenine dinucleotide (NADH)-diaphorase staining, and single-stranded (ss) DNA staining. All cases were followed-up after breast radiation and systemic therapies. Results: The mean tumor size based on the ultrasonographic maximum dimension was 1.3 cm (range: 0.5–2.0 cm). Although complete response was histologically confirmed in only 8 cases, NADH-diaphorase and ssDNA staining did not demonstrate any viable tumor cells in any of the ablated lesions. At a mean follow-up of 60 months (follow-up range, 37–82 months), there were no cases of in-breast recurrence, although one patient died due to hepatic metastases. Cosmesis of the conserved breast was excellent or good in all of the cases, but a hard lump was persistent after RFA in half of the cases. Conclusions: The long-term outcome of patients treated with RFA is encouraging with regard to cosmesis and local control. However, a persisted lump can cause patient discomfort, anxiety and fear. Therefore, further studies are needed to establish the optimal technique. Moreover, a prospective study will be required to determine the equivalency in local recurrence rates between the RFA therapy and conventional breast-conserving treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-15-05.

Quantification and Morphometry of Myenteric Neurones in the Jejunum of Holtzman Rats (Rattus norvegicus)

With 2 figures and 3 tables The morphological pattern of the myenteric plexus (MP) is species-specific, and little is known about this pattern in Holtzman rats. The aim of the current experiment was the morphological and quantitative study of myenteric neurones in the Holtzman rat jejunum. Hematoxylin-Eosin and NADH-diaphorase (NADH-dp) staining were used to assess muscular layer thickness, neurone cell body area (CBA) and nuclei area (NA). Muscular layer thickness was found to be 114.77 ± 14.89 μm. Neuronal densities across the subregions of the jejunum were similar: mesenteric, 11.78 ± 2.89/mm(2) ; intermediate, 12.06 ± 2.69/mm(2) ; and antimesenteric, 10.67 ± 1.89/mm(2) . As expected, there was positive correlation between the CBA and NA of 79.19, 79.26 and 78.5% in the mesenteric, intermediate and antimesenteric subregions of the jejunum, respectively. Medium-sized neurones predominated in the ganglionic arrangement of the MP. These results indicate that the NADH-dp myenteric neurones in the jejunum of Holtzman rats are similar in many aspects to those found in the ileum of Holtzman rats and to those found in the small intestine of Wistar rats, including their location, ganglionic disposition and predominance of medium-sized CBA. However, neuronal density in the jejunum is lower than in the ileum. Based on these results showing morphological similarities to the MP of the Wistar rat, the Holtzman strain can be used to investigate the effects of adverse conditions on the morphology of the MP.

Use of cytokeratin 8 immunohistochemistry for assessing cell death after radiofrequency ablation of breast cancers

The effects of minimally invasive therapies such as radiofrequency ablation (RFA) and laser induced thermal therapy on breast carcinoma lesions usually is assessed by NADH diaphorase enzyme histochemistry for cell viability. NADH staining requires frozen material, however, with its associated poor morphology. We aimed to validate cytokeratin 8 (CK 8) immunohistochemistry as an alternative that works on paraffin sections. RFA was performed ex vivo on 20 breast resections after surgery and in vivo in eight patients who underwent general anesthesia followed by immediate resection. After treatment, specimens were lamellated and the tumors were divided into two equal parts. One part was fixed in neutral buffered formaldehyde for routine histopathological evaluation using hematoxylin and eosin (H & E) staining and CK 8 immunostaining. The other section was snap frozen and stored at −80° C for staining with NADH diaphorase. Both NADH diaphorase and CK 8 immunostaining demonstrated a clear and comparable demarcation between viable and nonviable tissues. The morphology of the CK 8 immunostained slides was much better, and fatty tissues could be judged readily by contrast to the NADH stained frozen sections, which had poor morphology and whose fatty parts were difficult to interpret. CK 8 immunohistochemistry seems to be well suited for assessing cell viability in breast tissue and for assessing the effects of RFA for breast cancer treatment. Because it can be applied to paraffin fixed material, it provides much better morphology than NADH staining and also can be applied to fatty tissues that usually are difficult to work up for frozen sections. Therefore, CK 8 immunohistochemistry may be preferred over NADH diaphorase staining for daily pathology practice for assessing the viability of breast carcinoma cells after RFA treatment.

A histopathological study for evaluation of therapeutic effects of radiofrequency ablation in patients with breast cancer

To reveal the rate of complete therapeutic effect of radiofrequency ablation (RFA) and its correlation with tumor size by the histopathological examination of surgically resected early breast cancers. For 28 patients who received RFA and subsequent surgical therapies for early breast cancer treatment, the effect of RFA was evaluated by both histopathological examination and nicotinamide adenine dinucleotide (NADH)-diaphorase staining of resected tumor specimens according to the criteria described by Seki et al. (this issue). The correlation of 100% RFA effect with tumor parameters including tumor size and the presence of extensive intraductal component (EIC) was examined. The mean size and invasive size of the primary tumors were 2.21 cm (ranging from 0.6 to 5.0 cm) and 1.44 cm (ranging from 0 to 5.0 cm), respectively. By examining hematoxylin-eosin (HE) sections, the effectiveness of RFA was found to be 100% in 16 tumors (57%). However, the effectiveness of RFA was found to be 100% in 22 cases (79%) examined by NADH-diaphorase staining of frozen sections containing part of tumorous and nontumorous tissues. The accuracy of diagnosis of complete RFA effect using NADH-diaphorase staining with reference to HE was 79% (22 of 28) with 100% (16 of 16) sensitivity and 50% (6 of 12) specificity. The rate of 100% RFA effect by HE examination was higher in EIC(-) tumors (13 of 17, 76%) than in EIC(+) tumors (1 of 9, 11%) (P = 0.0022), and was higher in tumors of ≤ 1.5 cm (10 of 11, 91%) than in tumors of >1.5 cm (6 of 17, 35%; P = 0.0034). All five tumors of ≤ 1.0 cm showed 100% RFA effect, but 3 (27%) of 11 tumors of >1.0 and ≤ 2.0 cm and 9 (75%) of 12 tumors of > 2.0 cm showed suboptimal RFA effect by HE. Tumor size of ≤ 1.5 cm, strictly ≤ 1.0 cm, could be an indication for RFA if a complete histological therapeutic effect is mandatory.

Pilot study of radiofrequency ablation therapy without surgical excision for T1 breast cancer: evaluation with MRI and vacuum-assisted core needle biopsy and safety management

There is increasing demand for minimally invasive treatments for small breast cancer mainly because of the desire for better cosmetic results. Although radiofrequency ablation (RFA) is an attractive approach as a local control method for small breast cancer, the problems of histological effectiveness and safety management remain. A total of 29 patients including one patient with bilateral breast cancer were enrolled in this study. The mean tumor size of 30 breasts was 12.8 mm (range 5-19 mm). Under general anesthesia, RFA was performed with a Cool-tip RF system (Valleylab, Boulder, CO, USA) after sentinel lymph node biopsy. Postoperative evaluation with magnetic resonance imaging (MRI) and vacuum-assisted core needle biopsy was done 3-4 weeks after RFA before radiotherapy. Ablated tumors were evaluated with hematoxylin-eosin (H&E) and nicotinamide adenine dinucleotide (NADH)-diaphorase staining. If needed, adjuvant chemo and/or endocrine therapy was performed. All patients except one completed one session of RFA. The mean temperature near the center of the tumors was 89.6°C (range 78-100°C). Postoperative MRI showed the ablated zone clearly in all patients. MRI revealed no hypervascularity of the tumors in the ablated zone. Evaluation with H&E staining of the tumors showed remarkable degenerative changes in only three patients. NADH-diaphorase staining showed no viable tumor tissue in 24 patients out of 26 examined. Three patients received small diameter grade 3 skin burns, two on the outside of the thigh from the grounding pad and one on the breast skin. One patient had a breast lesion like a chronic granulomatous mastitis resulting from overreaction of the ablated zone. RFA therapy appeared relevant and applicable for patients with small breast cancer. Because small skin burns were observed as adverse events, close attention should be paid in the course of the RFA procedure.

Radiofrequency ablation as local therapy for early breast carcinomas

To evaluate the safety and efficacy of radiofrequency ablation (RFA) as a local therapy for early breast carcinomas, we performed a phase I/II study at our institution. Fifty patients with core-needle biopsy-proven breast carcinoma that was ≤ 3 cm in diameter on ultrasonography were enrolled in this study. Under ultrasound (US) guidance, the tumor and surrounding breast tissue were ablated with a saline-cooled RF electrode followed by immediate surgical resection. Resected specimens were examined by hematoxylin and eosin (H&E) staining and nicotinamide adenine dinucleotide (NADH) diaphorase staining to assess tumor viability. Forty-nine patients completed the treatment. The mean tumor size was 1.70 cm. The mean ablation time was 8.7 min using a mean power of 48.5 W. Of the 49 treated patients, complete ablation was recognized in 30 patients (61%) by H&E staining and/or NADH diaphorase staining. The NADH viability staining was available for 38 patients, and in 29 (76.3%), there was no evidence of viable malignant cells. Of the 29 treated patients with breast carcinomas ≤ 2 cm in diameter examined by pathological examination, complete ablation was achieved in 24 patients (83%). Of the 26 treated patients with breast carcinomas without an extended intraductal component (EIC) according to pathological examination, complete ablation was determined in 22 patients (85%). RFA-related adverse events were observed in five cases: two with skin burn and three with muscle burns. RF ablation is a safe and promising minimally invasive treatment for small breast carcinomas with pathological tumor size ≤ 2 cm in diameter and without EIC.

Radiofrequency Ablation in Breast Cancer.

Abstract Background: Radiofrequency ablation (RFA) of breast tumors is characterized by variable efficiency. Cell viability is principally evaluated by NADH-diaphorase and Hematoxylin-eosin (H&amp;E) staining techniques.Methods: Twenty patients with breast cancer, 20.7 ± 9.14mm in diameter, underwent RFA of the tumor and of the margin of surrounding breast tissue. RFA was based on the radiofrequency impedance-switching algorithm. The algorithm was based on an internally cooled probe, which carried out algorithm-guided deposition of heat in a pulse-cycle fashion and under continuous monitoring of the desiccation status of the ablated site to avoid tissue charring (until its impedance level reached 30Ω above the baseline level or a maximum time interval (tmax) was reached). A minimum time interval of 5 seconds was set as the duration of time that the electrode must be off. If the cycles of power application occurred too rapidly, the power was diverted to the 150Ω resistance load for the remainder of the 5-second interval. Surgical resection was the next step. Sixteen patients underwent quardzantectomy (five with axillary lymph node dissection) and four unilateral total mastectomy. In fifteen patients SLNB was searched and identified (ten negative, four positive and one failure). The ablated tumor tissue was histologically examined and Ki67 immunohistological staining was applied to evaluate cellular proliferation, invasion and survival.Results: The mean time of operation was 119.47 ± 23.35minutes.The mean time of RFA was 12 - 15minutes. One patient had a post-operative complication (trauma infection). All twenty patients had a positive Ki67 immunohistochemical staining mean value of 28.79 ± 21.42, before RFA. The post-RFA Ki67 expression was zero in sixteen patients. In three patients no residual tumor cells were found and in one patient the Ki67 expression was under five percent. Recurrence developed in zero patients, to date (follow up time: 22.5 ± 2.3 months).Conclusion: Ki67 evaluation is a targeting indicator of viability of malignant cells after lesion RFA of breast tumors. RFA is able to provide satisfactory local control in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2106.

Histopathological and immunohistochemical evaluations of breast cancer treated with radiofrequency ablation

Although RF ablation is a promising non-surgical ablation technique for the treatment of breast cancer, assessment of the efficacy requires not only imaging of tumor necrosis but also histological confirmation. Two series of patients were treated with RF ablation. In the first series, 17 patients underwent surgical resection immediately and 17 underwent delayed mammotome excision in the second series. The ablated tumor tissue was examined histologically with H&E staining and NADH-diaphorase staining. Furthermore, ssDNA was immunohistochemically stained in the specimen where tumor cells were histologically detected. In the first series, mild response was histologically seen in 14 of 17 patients (82%), whereas complete response was observed in 8 of 16 patients (50%) in the second series. However, NADH-diaphorase staining did not demonstrate any viable tumor tissues in any patient in either the first or the second series. ssDNA staining was positive (non-viable) in 13 of 16 cases in the first series, but it was positive in all 8 cases tested in the second series. Although NADH-diaphorase staining is essential to evaluate tumor cell viability immediately after RF ablation, ssDNA may be useful for the assessment of cell viability after some interval following RF ablation.

Feasibility study on radiofrequency ablation followed by partial mastectomy for stage I breast cancer patients

To evaluate the safety and reliability of thermal ablation therapy instead of breast-conserving surgery (BCS), we performed radiofrequency ablation (RFA) for clinical stage I breast cancer patients. Subjects were T1N0 breast cancer patients with no extensive intraductal components. Under general anesthesia, sentinel node biopsy was performed, followed by RFA and BCS. Resected specimens were examined at 5-mm intervals by hematoxylin–eosin (H&E) staining and nicotinamide adenine dinucleotide (NADH) diaphorase staining. Thirty of the 34 eligible patients were enrolled. RFA-related adverse events were observed in nine patients: two with skin burn and seven with muscle burn. Twenty-six patients (87%) showed pathological degenerative changes in tumor specimens with H&E staining. In 24 of the 26 cases (92%) examined by NADH diaphorase staining, tumor cell viability was diagnosed as negative. RFA proved to be reliable and feasible in clinical stage I breast cancer, with no extensive intraductal components. Randomized clinical trials are needed to compare RFA with BCS.

Radiofrequency ablation treatment for small breast cancer.

Abstract Abstract #5155 Aims: There is increasing demand for minimally invasive and non-surgical treatment in breast cancer. Although radiofrequency (RF) ablation seems the most promising non-surgical treatment for small breast cancer, its relevance has been controversial. This study was performed to determine the feasibility and the safety of treating small breast cancer with RF ablation. Methods: In this study, all patients had a localized breast cancer of 2.0 cm or less in greatest diameter. The tumors were confirmed to be localized lesions by ultrasonography, mammography, and enhanced MRI or CT. Before treatment, core needle or mammotome biopsy was performed to obtain the tumor tissue for establishing the histological diagnosis and the status of hormonal receptors and HER-2 expression. A model 15000 generator with a seven-array Starbusrt XL needle-electrode Model 70 (RITA Medical System) was used for RF ablation. In the first series, 17 patients underwent RF ablation and sentinel lymph node (SLN) biopsy followed immediately by wide resection (n=13) or total mastectomy (n=4). Axillary dissection was performed in 5 patients with positive SLN. On the other hand, 15 patients underwent RF ablation and SLN biopsy in the second series. Axillary dissection was performed in 3 patients with positive SLN. The ablated lesion was excised by mammotome after several months later. The ablated tumor tissue was examined histologically with H&amp;E staining and NADH-diaphorase staining to assess tumor cell viability. Patients who underwent wide resection or RF ablation alone received breast irradiation, and all of the patients were treated with systemic therapy according to the St. Gallen recommendation. Results: Histological examination with H&amp;E staining revealed a spectrum of changes ranging from complete coagulation necrosis to normal-appearing tumor cells, but NADH-diaphorase staining revealed no viable tumor cells in the either series. No patient developed to local or breast recurrence in the first series with a median follow-up period of 46 months (range: 34-61 months), and in the second series with a median follow-up period of 23 months (range: 12-32 months). However, one patient in the first series and another in the second series developed to the distant metastases, although they are alive with systemic treatment. The cosmesis of conserved breast was excellent in the second series more than in the first series, and no adverse effect such as skin burn or persistent hard lump was observed in the second series. Conclusions: RF ablation is feasible for small breast cancer. To achieve wide acceptance, however, further studies are needed to determine whether the use of RF ablation for small breast cancer can provide local control and survival rates equivalent to those of conventional breast-conserving treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5155.

Radiofrequency Thermal Ablation of Breast Cancer Local Recurrence: A Phase II Clinical Trial

The role of radiofrequency (RF) ablation to treat local recurrence of breast cancer is unknown. We conducted a two-stage phase II clinical trial. Eligible patients had a histologically confirmed noninflammatory and < or =3 cm ipsilateral breast tumor recurrence. The tumor site was identified by intraoperative sonography. A LeVeen needle electrode (RadioTherapeutics Corp, Mountain View, Calif) was inserted into a single site within the tumor and radiofrequency ablation was performed using a RF-2000 generator (RadioTherapeutics Corp). After completion of radiofrequency, a mastectomy was performed. Conventional staining and nicotinamide adenine dinucleotide-diaphorase (NADH-diaphorase) cell viability staining were performed. During the first stage, procedures were uneventful. Conventional, cytokeratin, and NADH-diaphorase staining identified persistent viable tumor cells in the RF-ablated region in three patients. This phase II trial was stopped after completion of the first stage because of insufficient efficacy. We demonstrate in this study that RF ablation is a potential technique to destroy local recurrence of breast tumors but the technique we tested in this phase II clinical trial had insufficient efficacy to recommend its use in routine.

Time Course of Nicotinamide Adenine Dinucleotide Diaphorase Staining after Renal Radiofrequency Ablation Influences Viability Assessment

Nicotinamide adenine dinucleotide (NADH) diaphorase staining has been used to confirm cell viability or death after radiofrequency ablation (RFA) of renal tissue. The time course over which NADH staining status converts from viable to non-viable after a lethal insult has not been defined for renal RFA, but the change may not be immediate. Our objective was to assess porcine renal tissue for viability using NADH diaphorase staining at various times after RFA. Seven pigs underwent monopolar RFA of both kidneys followed by needle biopsy of the ablation zone before and immediately after ablation and at 15-minute intervals thereafter. Initially, a single kidney was treated, and the contralateral kidney was treated 2 weeks later. Biopsies were taken from untreated renal parenchyma in a similar time course after nephrectomy to examine the effect of ischemia. All biopsy specimens, as well as representative sections of the ablation zone, were subjected to NADH staining and reviewed by a pathologist who was blinded to the tissue treatment. Most of the post-RFA biopsy specimens (86%) showed non-viable tissue. However, 14% of the specimens revealed viable tissue as late as 150 minutes after RFA. Therefore, none were positive. In the nephrectomy parenchyma, 92% of the biopsy specimens showed viable tissue as late as 4 hours after the onset of ischemia. Staining for NADH can establish tissue non-viability after RFA, but the timing of staining after treatment must be considered when interpreting results to avoid false positive tests. Tissue that is apparently viable by NADH staining within 2.5 hours of RFA may in fact have been ablated.

Radiofrequency ablation therapy for small breast cancer followed by immediate surgical resection or delayed mammotome excision

There is increasing demand for minimally invasive treatment approaches. Although radiofrequency (RF) ablation therapy is promising for the local treatment of small, well-localized breast cancer, the problem of determining tumor cell death after RF ablation remains. In the first series of this study, 17 patients underwent surgical resection immediately after RF ablation and 7 patients received delayed mammotome excision in the second series. The ablated tumor tissue was examined histologically with hematoxylin-eosin (H&E) staining and nicotinamide adenine dinucleotide (NADH)-diaphorase staining to assess tumor cell viability. Histological examination with H&E staining revealed a spectrum of changes ranging from complete coagulation necrosis to normal-appearing tumor cells, although the degenerative changes were more remarkable in the second than in the first series. However, NADH-diaphorase staining revealed no viable tumor cells in the ablated lesion in either series. NADH-diaphorase staining is essential to assess the effects of RF ablation. However, further studies are needed to determine whether RF ablation may provide equivalent local control and survival compared with conventional BCT for patients with small breast cancer.

IMPACT OF BLOOD FLOW OCCLUSION ON LIVER NECROSIS FOLLOWING THERMAL ABLATION

Laser, radiofrequency and microwave are common techniques for local destruction of liver tumours by thermal ablation. The main limitation of thermal ablation treatment is the volume of necrosis that can be achieved. Blood flow occlusion is commonly advocated as an adjunct to thermal ablation to increase the volume of tissue necrosis based on macroscopic and histological assessment of immediate or direct thermal injury. This study examines the impact of blood flow occlusion on direct and indirect laser induced thermal liver injury in a murine model using histochemical methods to assess tissue vitality. Thermal ablation produced by neodymium yttrium-aluminium-garnet laser (wavelength 1064 nm) was applied to the liver of inbred male CBA strain mice at 2 W for 50 s (100 J). Treatment was performed with and without temporary portal vein and hepatic artery blood flow occlusion. Animals were killed upon completion of the procedure to assess direct thermal injury or at 24, 48 and 72 h to assess the progression of tissue damage. The maximum diameter of necrosis was assessed by vital staining for nicotinamide adenine dinucleotide (NADH) diaphorase. Microvascular changes were assessed by laser Doppler flowmetry, confocal in vivo microscopy and scanning electron microscopy. The direct thermal injury (mean SE) assessed by NADH diaphorase staining was significantly greater following thermal ablation treatment without blood flow occlusion than with blood flow occlusion (3.3 (0.4) mm vs 2.9 (0.3) mm; P = 0.005). Tissue disruption, cracking and vacuolization was more pronounced adjacent to the fibre insertion site in the group treated with thermal ablation combined with blood flow occlusion. There was an equivalent increase in the extent of injury following therapy in both groups that reached a peak at 48 h. The maximum diameter of necrosis in the thermal ablation alone group at 48 h was significantly greater than the thermal ablation combined with blood flow occlusion group (5.8 (0.4) mm vs 5.3 (0.3) mm; P = 0.011). The patterns of microvascular injury were similar in both groups, varying in extent. Temporary blood flow inflow occlusion appears to decrease the extent of initial injury measured by vital staining techniques and does not alter the time sequence of progressive tissue injury following thermal ablation therapy.

Focal Hyperthermia Produces Progressive Tumor Necrosis Independent of the Initial Thermal Effects

Focal hyperthermia, produced using laser, radio frequency, and microwave, is used to treat liver tumors. The exact mechanisms of tissue destruction using focal hyperthermia are, however, unknown. Clinical and experimental studies suggest a progression of injury after cessation of the initial heat stimulus. This study investigates the mechanisms and time sequence of progressive tissue necrosis induced using focal hyperthermia in a murine model of colorectal liver metastases. Focal hyperthermia produced using a neodymium-yttrium aluminum garnet (Nd-YAG) laser source was applied to the normal liver and colorectal cancer liver metastases in inbred male CBA strain mice. The extent of direct lethal thermal injury was assessed histochemically using vital stain for nicotinamide adenine dinucleotide (NADH) diaphorase immediately after laser application. Tissue injury at subsequent time points was assessed using both NADH diaphorase staining and routine histology to determine the temporal relationship between tissue necrosis and time. Thermal injury occurring immediately after the application of 100 joules of energy was greater in the tumor tissue than in the normal liver (mean [standard error of the mean (SEM)]), measuring 23.5 (3.4) and 16.3 (2.6) mm(3), respectively (P=0.046), despite similar tissue temperature profiles. There was a significant increase in tissue necrosis after initial injury that was greater in the normal liver than in the tumor tissue. In the normal liver, the peak volume of necrosis was 137.4 (9.8) mm(3) and occurred at 3 days, whereas in the tumor tissue the peak was 49.0 (3.5) mm(3) at 4.5 days (P < 0.001). Focal hyperthermia produces tissue necrosis that occurs in two phases. The first phase is caused by the direct lethal thermal injury followed by a second phase involving a progression of necrosis beyond the initial thermal effects. The normal liver and the tumor tissue responded differently to focal hyperthermia. In the tumor tissue, the direct injury is more pronounced, whereas the progression of injury is more rapid and extensive in the normal liver.

Initial experience with radiofrequency ablation for hepatic tumours in the Netherlands

Aims: The aim of this study is to report initial experience with radiofrequency thermoablation (RFA) of malignant hepatic tumours. Methods: From June 1999 to November 2001, a seven centre study of 50 patients who had undergone RFA, included 102 primary or metastatic tumours. Multimodality therapy, complications, disease free survival and location of recurrence were recorded. Histopathologic examination was performed in a number of tumours treated with RFA and subsequently resected to determine the effect of RFA on the tissue level. Results: 11 tumours (seven patients) were resected after prior treatment with RFA; NADH-diaphorase staining in all these tumours demonstrated non-viable tumour. Postoperative morbidity and mortality occurred in 14 patients and one patient, respectively, in three cases related to the RFA procedure. Median follow-up of 41 patients with non-resected RF ablated tumours was 11 months (range 1–24 months). 26 patients developed a recurrence of which three at the RFA site after 6–12 months. Conclusions: RFA provides a simple method for local treatment of liver tumours. The introduction of this technique nevertheless involves morbidity and mortality.