Lymphoscintigraphy in the Time of COVID-19: Effect of Molybdenum-99 Shortage on Feasibility of Sentinel Node Mapping.

Background: In the current study, we reported our experience on sentinel node mapping of breast cancer patients during the extreme shortage of Mo99-Tc99m generators using Tc-99m phytate. Methods and Results: During the period from March 7, 2019, to April 18, 2020, due to disruption of molybdenum supply chain, we used low specific activity Tc-99m pertechnetate elute (0.5-2 mCi of 99mTcO4 in 5 mL) for each kit preparation. Two or three intradermal periareolar injections were done for each patient (0.02-0.1 mCi/0.2 mL for each injection). Immediately following injection, dynamic lymphoscintigraphy was done. Surgery was done the same day of injection and the axillary sentinel node was sought using a gamma probe. Overall, 35 patients were included in the study. The specific activity of the Tc-99m elute (in 5 mL) used for kit preparation was 2 mCi/10 mg in four, 1.5 mCi/10 mg in eight, 1.25 mCi/10 mg in eight, 1 mCi/10 mg in three, 0.75 mCi/10 mg in five, and 0.5 mCi/10 mg of 99mTc-Phytate in seven patients. For the first four groups of patients, we used two 0.2 mL injections, while in the latter two groups, three 0.2 mL injections were used. At least one sentinel node was detected in all patients but three in whom axilla was involved. Conclusion: Sentinel node biopsy can be achieved with low specific activity of Tc-99m elute at the time of Mo99-Tc-99m generator shortage. If special personal protection is used, sentinel node mapping can be done in nuclear medicine departments with excellent results despite the COVID-19 pandemic and disruption of generator shipment.

Novel Biomarkers Aim at Detecting Metastatic Sentinel Lymph Nodes in Breast Cancer

Background: Intra-operative molecular diagnostic assays are currently used for the detection of lymph node metastases. The objective of this study was to find new biomarkers to improve diagnostic accuracy in the detection of metastatic axillary lymph nodes in breast cancer patients. Methods: We applied an absolute quantitative real-time reverse transcription-PCR to quantitate the expression of CK19, KLK11, and CLEC3A mRNAs in 79 FFPE sentinel lymph nodes (SLNs) from 35 breast cancer patients. The CK19 was confirmed as a standard biomarker, and the level of expression of selected new markers, KLK11 and CLEC3A, was evaluated in pathologically negative and positive SLNs by using absolute quantitative real-time PCR. Results: The overall concordance of the CK19 gene with pathological results was 92.4% (less than 250 copies) in negative SLNs and 85% in positive SLNs (more than 250 copies). The sensitivity and specificity of CK19, which were detected by real-time PCR, was 85% and 46%, respectively. Our results revealed that lower CLEC3A was associated with more lymph node involvement. We could set a cut-off point for CLEC3A with the sensitivity of 78% and specificity of 60%. Also, the mean KLK11 had a statistically significant reverse correlation with tumor grade (p = 0.017). Higher CK19 levels were related to more tumor invasion (p < 0.0001). Conclusion: Regarding the findings, CLEC3A along with CK19 can be used as a promising marker with high sensitivity and specificity for the detection of metastatic SLN.

Assessment of Ultrasound / Radio-guided Occult Lesion Localization in Non-palpable Breast Lesions.

OBJECTIVES: Controversy exists about the localization of non-palpable breast lesions. In many countries, the gold standard for the diagnosis of these lesions is needle localization due to its accuracy. This study sought to compare the ultrasound- and radio-guided occult lesion localization (ROLL) as a simple method with the conventional procedures in terms of their diagnostic power. METHODS: This study was conducted on 94 patients with non-palpable breast lesions detected by ultrasonography and localized by the combination of ultrasonography and using radiopharmaceuticals. One to ten hours prior to surgery, 0.1-0.2 ml (equivalent to 0.5-1 mCi) of Tc-99m-phytate was injected to the lesion under the guidance of ultrasonography. Then, the lesion was localized using a hand-held gamma probe, and excision of the lesion was performed according to its radioactivity signal. Data analysis was performed using SPSS, version 16. RESULTS: Benign and malignant pathologic results were observed in 77 (81.9%) and 17(18.1%) of the patients, respectively, and the mean volume of the excised tissue was 26.29±27 mm³. 79 patients had a solitary lesion (84%), 55 in the left breast (58.5%) and 39 in superolateral quadrant (41.5%). The mean size of the lesions was 15.7 mm in diameter (ranging from 4 to 34 mm). Additionally, there was a need to secondary surgery in 3 (3.2%) patients and inappropriate localization in 6 (6.4%) patients (subcutaneous or intra-ductal spread of radiodrug). CONCLUSION: Combination of ultrasound- and radio-guided localization methods for localizing non-palpable breast lesions is a simple and acceptable method for localization with no significant complications. For radio-drug spread and subsequent excessive excised tissue volume, subcutaneous and intra-ductal lesions are not suitable indication for ROLL.

A clinical trial of neoadjuvant concurrent chemoradiotherapy followed by resection for esophageal carcinoma.

BACKGROUND: Esophageal carcinoma is a common malignancy in the North East of Iran. Combined modality treatments have been adopted to improve survival in patients with esophageal carcinoma. In this trial, we evaluated the efficacy and toxicity of a preoperative concurrent chemoradiotherapy protocol in the patients with locally advanced esophageal carcinoma. MATERIALS AND METHODS: Between 2006 and 2011, eligible patients with locally advanced esophageal carcinoma underwent concurrent radiotherapy and chemotherapy and 3-4 weeks later, esophagectomy. Pathologic response, overall survival rate, toxicity, and feasibility were evaluated. RESULTS: One hundred ninety-seven patients with a median age of 59 (range: 27-70) entered the protocol. One hundred ninety-four cases (98.5%) had esophageal squamous cell carcinoma. Grades 3-4 of toxicity in patients undergoing neoadjuvant chemoradotherapy were as follows: Neutropenia in 21% and esophagitis in 2.5% of cases. There were 11 (5.6%) early death probably due to the treatment-related toxicities. One hundred twenty-seven patients underwent surgery with postsurgical mortality of 11%. In these cases, the complete pathological response was shown in 38 cases (29.9%) with a 5-year overall survival rates of 48.2% and median overall survival of 44 months (95% confidence interval, 24.46-63.54). CONCLUSION: The pathological response rate and the overall survival rate are promising in patients who completed the protocol as receiving at least one cycle of chemotherapy. However, the treatment toxicities were relatively high.

Can methylene blue dye be used as an alternative to patent blue dye to find the sentinel lymph node in breast cancer surgery?

BACKGROUND: Sentinel lymph node biopsy (SLNB) is standard care to evaluate axillary involvement in early breast cancer. It has fewer complications than complete lymph node dissection; however, using blue dye in SLNB is controversial. We have evaluated the detection rate and local complications associated with methylene blue dye (MBD) used in SLNB in early breast cancer patients and compared these results to patent blue dye (PBD). MATERIALS AND METHODS: In a cohort prospective study, 312 patients with early breast cancer without axillary lymph node involvement were divided into two groups according to dye type. All of the patients received radiotracer and one type of blue dye. We filled out a checklist for the patients that contained demographic data, size of tumor, stage, detection of sentinel lymph node, and complications and then analyzed the data. RESULTS: Demographic and histopathologic characteristics were not significantly different in both groups. Mean (standard deviation [SD]) tumor size in all patients was 2.4 (0.8) cm. Detection rate in the MBD group was 77.5% with dye alone and 94.2% with dye and radioisotope; and in the PBD group it was 80.1% and 92.9% respectively (P > 0.05). We had blue discoloration of the skin in 23.7% in the PBD and 14.1% in the MBD group (P < 0.05) local inflammation was detected in one patient in the PBD and five in the MBD group (P < 0.05). Skin necrosis and systemic complications were not observed. CONCLUSION: Methylene blue has an acceptable detection rate, which may be a good alternative in SLNB. Complication such as blue discoloration of the skin was also lower with MBD.

Efficacy of high-energy collimator for sentinel node lymphoscintigraphy of early breast cancer patients.

INTRODUCTION: Lymphoscintigraphy is an important part of sentinel node mapping in breast cancer patients. Sometimes star shaped artefacts due to septal penetration can be problematic during imaging. In the current study, we evaluated the possibility of high energy (HE) collimators use for lymphoscintigraphy. PATIENTS AND METHODS: Twenty patients with early breast carcinoma were included. Thirty minutes after radiotracer injection (99mTc-antimony sulphide colloid), anterior and lateral images were acquired using a dual head gamma camera equipped with a parallel hole low energy high resolution (LEHR) collimator on one head and HE collimator on another head. All images were reviewed by two nuclear medicine specialists regarding detectability and number of axillary sentinel nodes and presence of star artefact. RESULTS: All images taken by LEHR collimators showed star artefact of the injection site. No image taken by HE collimator showed this effect. In two patients the sentinel node was visible only by HE collimator. Tumour location in both of these patients was in the upper lateral quadrant and both had history of excisional biopsy. In two patients additional sentinel node was visible adjacent to the first one only on the LEHR images. CONCLUSIONS: HE collimators can be used for sentinel lymph node mapping and lymphoscintigraphy of the breast cancer patients. This collimator can almost eliminate star-shaped artefacts due to septal penetration which can be advantageous in some cases. However, to separate two adjacent sentinel nodes from each other LEHR collimators perform better.

Factors influencing the time of sentinel node visualization in breast cancer patients using intradermal injection of the radiotracer.

BACKGROUND: The objective of our study was to determine the important factors that have influence on the time of sentinel node visualization using intradermal injection of (99m)Tc-antimony sulfide colloid. METHODS: Two hundred fifty consecutive patients with the diagnosis of early-stage breast cancer were evaluated. Anterior and lateral views were acquired in various intervals after intradermal injection of the tracer until 180 minutes or visualization of the sentinel node. The effect of several variables on the time of sentinel node visualization was evaluated by univariate and multivariate analyses. RESULTS: The time of sentinel node visualization was significantly correlated with age, body mass index (BMI), and interval between biopsy and sentinel node mapping. Standardized beta values for these variables were .1, .3, -.55 respectively. CONCLUSIONS: Older age and higher BMI can result in slow sentinel node visualization. Longer interval between biopsy and sentinel node mapping can be associated with rapid sentinel node detection.

Cancer-testis gene expression profiling in esophageal squamous cell carcinoma: identification of specific tumor marker and potential targets for immunotherapy.

Cancer-testis antigens (CTAs) are often specifically expressed in cancer cells and under normal conditions are only considered to be expressed in the germ line cells and the placenta. CTAs are potential targets for cancer immunotherapy and therefore necessitates their expression profiling. The expression profile of LAGE1, MAGE-A4 and NY-ESO1, their possible correlations and interaction, and the clinicopathological associations of each marker were studied. RNA was extracted from fresh esophagectomy tissues of 41 esophageal squamous cell carcinoma (ESCC) patients prior to any other therapeutic intervention. The relative mRNA expression of LAGE1, MAGE-A4 and NY-ESO1 was assessed with the real-time reverse transcription-polymerase chain reaction (RT-PCR) 5' nuclease assay. The overexpression of LAGE1, MAGE-A4 and NY-ESO1 was found in 39, 90.2 and 41.4% of ESCC samples respectively. Of the patients, 97.5% showed an overexpression of at least one CTA. The relative expression of MAGE-A4 was directly associated with lymph node metastasis and the stage of the tumor (p < 0.05). A significant direct correlation was also detected between the MAGE-A4/LAGE1 and MAGE-A4/NY-ESO1 levels of gene expression. MAGE-A4 is identified as a specific biomarker of ESCC with a possible oncogenic role contributing to tumor progression. Interactions between MAGE-A4, LAGE1 and NY-ESO1 and their significant clinical consequences introduce these CTAs as appropriate targets for a polyvalent cancer vaccine.

The efficacy of Tc-99m sestamibi for sentinel node mapping in breast carcinomas: comparison with Tc-99m antimony sulphide colloid.

BACKGROUND: To study the value of periareolar intra-dermal injection of Tc-99m sestamibi (MIBI) for sentinel node mapping in breast carcinoma. MATERIAL AND METHODS: Fifty patients with early-stage breast cancer were included in our study. 17.5 MBq Tc-99m-MIBI was injected intradermally to 25 patients and the remainders were injected with the same dose of Tc-99m-antimony sulphide colloid. Anterior and lateral static images were taken at 2 minutes. If sentinel lymph node was not detected, delayed imaging by up to 180 minutes was carried out. The patients were operated on 2-4 hours post-injection. Sentinel lymph node biopsy was performed by the aid of gamma probe and blue dye during surgery. RESULTS: In the Tc-99m-MIBI group, 23 patients had lymph nodes on scintigraphy images, and sentinel nodes were detected during surgery in all 23 patients. In the Tc-99m-antimony sulphide colloid group, 24 patients had lymph nodes on scintigraphy images, and sentinel nodes were identified during surgery in 24 patients. CONCLUSIONS: We concluded that 99mTc-MIBI is a suitable radiopharmaceutical for sentinel node detection.

The effect of excisional biopsy on the accuracy of sentinel lymph node mapping in early stage breast cancer: comparison with core needle biopsy.

Despite the successful application of sentinel node mapping in breast cancer patients, its use in patients with a history of previous excisional biopsy of the breast tumors is a matter of controversy. In the present study we evaluated the accuracy of sentinel node biopsy in this group of patients and compared the results with those in whom the diagnosis of breast cancer was established by core needle biopsy. Eighty patients with early stage breast carcinoma were included into our study. Forty patients had a history of previous excisional biopsy and the remainder 40 had undergone core needle biopsy. Intradermal injections of 99mTc-antimony sulfide colloid as well as patent blue were both used for sentinel node mapping. Sentinel nodes were harvested during surgery with the aid of surgical gamma probe. All patients underwent standard axillary lymph node dissection subsequently. Detection rate was 97.5 per cent for both groups of the study. Number of detected sentinel node during surgery was not significantly different between groups. False negative rate was 0 per cent for both groups of the study. In conclusion sentinel node biopsy is reliable in patients with previous history of excisional biopsy of the breast tumors and has a low false negative rate.

Induction of cytotoxic T lymphocytes primed with tumor RNA-loaded dendritic cells in esophageal squamous cell carcinoma: preliminary step for DC vaccine design.

BACKGROUND: Dendritic cells (DC) are potent antigen presenting cells with the ability to prime naïve T cells and convert them to cytotoxic T-lymphocytes (CTL). We evaluated the capability of autologous DCs transfected with total tumor and normal RNA to induce cytotoxic CTL as the preliminary step to design a DC-based vaccine in the esophageal squamous cell carcinoma (ESCC). METHODS: Monocytes-derived DCs were electroporated with either total tumor RNA or normal RNA. T cells were then primed with tumor RNA transfected DCs and lytic effects of the generated CTL were measured with Cytotoxicity assay and IFN-gamma Release Elispot assay. RESULTS: Cytotoxicity was induced against DCs loaded with tumoral RNA (%24.8 +/- 5.2 SEM) while in normal RNA-loaded DCs, it was minimal (%6.1 +/- 2.4 SEM) and significantly lower (p < 0.05). INF-gamma secretion was more than 2-folds higher in tumoral RNA-loaded DCs when compared with normal RNA-loaded DCs (p < 0.05). CONCLUSION: Electroporating DCs with tumor RNA generated tumor antigen presenting cells which in turn enhanced cytotoxic effects of the T cells against ESCC. This may be a useful autologous ex vivo screening tool for confirming the lytic effects of primed T cells on tumors and evaluate probable further adverse effects on noncancerous tissues. These data provide crucial preliminary information to establish a total tumor RNA-pulsed DC vaccine therapy of ESCC.

Comparison of early and delayed lymphoscintigraphy images of early breast cancer patients undergoing sentinel node mapping.

OBJECTIVE: We compared early and delayed lymphoscintigraphy images using intradermal injection of (99m)Tc-antimony sulfide colloid, which has small particles. METHODS: Eighty patients with early-stage breast cancer were included into the study. Intradermal injection of (99m)Tc-antimony sulfide colloid was used for sentinel node mapping. After radiotracer injection, 30 min and 20 h later, lymphoscitigraphy images were obtained in lateral and anterior views. After the completion of each image sets, the location of the visible nodes in the axilla was marked on the skin. Two nuclear medicine specialists reviewed the images independently and the number and location of detected nodes were recorded. RESULTS: At least one hotspot was detected in the axillary region in 78 (97.5%) and 79 (98.75%) patients on the early and delayed images, respectively. No extra-axillary drainage was noted in the patients. The number and location of detected hot spots were the same in 77 patients on both image sets. In one patient the early image did not show any axillary hot spot despite its visualization on the delayed image set and in one patient no hot spot was noted on either images. In one patient an additional axillary hot spot was noted on the delayed image, which was not apparent on the early image. CONCLUSION: Our study showed that a delay of up to 20 h in sentinel lymph node biopsy using intradermal injection of (99m)Tc-antimony sulfide colloid does not result in washout of the tracer from the true sentinel node or migration of the radiotracer into second-echelon nodes.

The value of touch imprint cytology and frozen section for intra-operative evaluation of axillary sentinel lymph nodes.

BACKGROUND: Sentinel lymph node (SLN) biopsy is a procedure of choice to determine the axillary involvement in breast cancer. The most important intraoperative evaluation methods are touch imprint cytology (TIC) and frozen section (FS). Each of these techniques has its own drawbacks and it is still unclear which is preferred. MATERIAL AND METHODS: 97 consecutive patients with recently diagnosed breast cancer and no clinical evidence of lymph node involvement were included in the study. The SLN was determined with lymphoscintigraphy. Touch imprint cytology and FS were performed and their results were compared with permanent histopathological examination. RESULTS: Using a permanent section as the gold standard TIC showed sensitivity of 71.4%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 88.7%. These figures were 87.5%, 98.5%, 96.5%, and 95% for FS, respectively. CONCLUSIONS: Frozen section is a reliable method for SN assessment during surgery. Using the frozen section during surgery can give the surgeon an opportunity to avoid the second surgery. Touch imprint cytology is also a reliable method for intra-operative SN evaluation. Due to its high predictive value, TIC can be used first in the surgery room and FS can be saved for cases with negative results of TIC.

How long the lymphoscintigraphy imaging should be continued for sentinel lymph node mapping?

OBJECTIVE: We assessed the need for delayed lymphoscintigraphy imaging for sentinel lymph node (SLN) biopsy in stage I and II breast cancer patients using intradermal injection of (99m)Tc-antimony sulfide colloid. METHOD: Seventy-five patients with early-stage breast cancer were included in our study. Periareolar intradermal injections of 0.5 mCi/0.2 mL (99m)Tc-antimony sulfide colloid was used for the patients without previous excisional biopsy (45 patients). Two intradermal injection of 0.5 mCi/0.2 mL (99m)Tc-antimony sulfide colloid was used on each side of the dermal incision in patients with the history of excisional biopsy (30 patients). Anterior and lateral static images were taken at 2 min. If SLN was not visualized, delayed imaging at 5, 10, 30, 60, 90, 150, and 180 min was done (till the visualization of the SLN or 180 min). SLN was performed by the combination of gamma probe and blue dye during surgery. RESULTS: Sentinel lymph node detection rate was 96% (72/75). SLNs were detected on the immediate (2 min) images in 55 (73.33%) patients. In the remaining patients, the SLNs were detected first on the 5-, 10-, and 30-min images in 10 (13/33%), 5 (6.66%), and 2 (2.66%) patients, respectively. In three patients (4%), SLN was not detected by lymphoscintigraphy even on 180-min images. During surgery, SLN was not detected in these three patients either. CONCLUSION: Considering the rapid flow of (99m)Tc-antimony sulfide colloid in our study, lymphoscintigraphy imaging can be completed in the nuclear medicine department without any delay in sending the patient back to the surgery department. Thirty minutes after radiotracer injection seems to be the optimal time for lymphoscintigraphy and delayed imaging beyond 30 min would not be necessary.

Overexpression and interactions of interleukin-10, transforming growth factor beta, and vascular endothelial growth factor in esophageal squamous cell carcinoma.

BACKGROUND: Sharing the role of immune suppression, interleukin-10 (IL-10), transforming growth factor beta (TGF-beta), and vascular endothelial growth factor (VEGF) are critical genes in several aspects of tumorigenesis. To elucidate the role of these cytokines in esophageal squamous cell carcinoma (ESCC), their relative mRNA expression in tumoral tissue compared with corresponding tumor-free tissue was evaluated. METHODS: A total of 49 patients with histologically confirmed ESCC were included in the study prior to any therapeutic interventions. Quantitative analysis of the mRNA expression was performed by real-time reverse transcription-polymerase chain reaction and the clinicopathologic associations were assessed. RESULTS: The mRNA of IL-10, VEGF, and TGF-beta was frequently overexpressed in 53.2%, 44.9%, and 37.5% of ESCC patients, respectively. TGF-beta was significantly co-expressed with IL-10 and with VEGF. Although VEGF was not independently associated with increased tumor size (p = 0.065), concomitant overexpression of VEGF with TGF-beta was significantly correlated with increased size of the tumor (p < 0.05). CONCLUSIONS: Overexpression of IL-10, TGF-beta, and VEGF plays an important role in ESCC and consequently leads to the frequent event of immune evasion in ESCC. TGF-beta is concomitantly overexpressed with IL-10 and with VEGF in ESCC. A stimulatory signal from TGF-beta to VEGF is necessary for VEGF to promote tumor progression.

Comparison of pre-operative lymphoscintigraphy with inter-operative gamma probe and dye technique regarding the number of detected sentinel lymph nodes.

Sentinel lymph node (SLN) identification by lymphoscintigraphy (LS) and biopsy are the standard method for axillary lymph node staging in low stage breast cancer patients. Many previous studies did not compare the number of SLN on LS with SLN detected during surgery. We aimed to study the accuracy of pre-operative LS for the prediction of the number of SLN detected by surgical gamma probe and the dye technique during surgery. Sixty patients were included in our study. SLN biopsy was performed using the combined radioactive and blue dye methods. Patients without previous excisional biopsy of the tumor (45 patients) received periareolar intra-dermal injections of 17.5MBq/0.2mL technetium-99m-antimony sulfide colloid ((99m)Tc ASC). The remainder of the patients, with the history of excisional biopsy of the tumor (15 patients); received two intra-dermal injections of 17.5MBq/0.2mL (99m)Tc-ASC in both ends of the surgical incision. All injections were done 2-4 h before surgery and gentle massage was applied to the injection site. Results showed that the number of SLN was correctly detected by LS in 58 patients. Eighty SLN were totally detected during surgery. All these SLN were radioactive and could be identified by surgical gamma probe. No SLN was detected only by the blue dye. Of the 80 detected SLN, 60 (75%) were both radioactive and colored. Pre-operative LS correctly predicted the number of harvested SLN during surgery in 77.5% of the patients. Only 78.7% (63/80) of the total harvested SLN were detected by pre-operative LS. We conclude that pre- operative LS identifies 78.7% and the blue dye technique can identify 75% of the SLN found by the gamma probe during surgery. The pre-operative LS technique can correctly identify the number of SLN in 77.5% of the patients.

p16 promoter hypermethylation: a useful serum marker for early detection of gastric cancer.

AIM: To determine p16 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on p16-protein expression, and correlation with clinical and histological features. METHODS: Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction (MSP) was used to evaluate methylation status of p16 promoter. p16-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections. RESULTS: Methylation was detected in 44.2% (23/52) of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients (14/52). Methylation was not detected in tissue and sera of control samples. p16-protein expression was decreased in 61.5% of cases (32/52), and was significantly associated with promoter hypermethylation (P < 0.001). Methylation was significantly more frequent in higher pathological grades (P < 0.05). Methylation was not associated with other clinicopathological features and environmental factors including H pylori infection and smoking. CONCLUSION: p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer.