Role of one-step nucleic acid amplification (OSNA) to detect sentinel lymph node low-volume metastasis in early-stage cervical cancer.

INTRODUCTION: Growing evidence in the literature supports the accuracy of sentinel lymph node (SLN) biopsy in early-stage cervical cancer. One-step nucleic acid amplification (OSNA) is a rapid assay able to detect cytokeratin 19-mRNA in SLNs, and it can be used for intra-operative detection of low-volume metastases. The aim of this study was to evaluate the rate of low-volume metastasis in SLNs detected by OSNA in patients with early-stage cervical cancer. Secondary aims were to define the sensitivity and the negative predictive value of SLN biopsy assessed with OSNA. METHODS: After IRB approval, consecutive patients who underwent surgery for International Federation of Gynecology and Obstetrics stage IA1 with lymph-vascular space involvement to IB1 between November 2017 and July 2019 and had SLN biopsy and pelvic lymphadenectomy were included. SLNs were detected with indocyanine-green cervical injection and sent intra-operatively for OSNA. RESULTS: Eighteen patients underwent SLN assessment with OSNA and systematic pelvic lymphadenectomy in the study period. Four (22.2%) patients had unilateral and 14 (77.8%) had bilateral mapping. OSNA detected micro-metastasis in 6/18 (33.3%) patients. All micro-metastases were detected in patients with bilateral SLN mapping. The sensitivity and negative predictive value of SLN in detecting lymph node metastasis with OSNA calculated per pelvic sidewall were 85.7% and 96.1%, respectively. The false negative rate in mapped sidewalls was 14.3%. DISCUSSION: This is the first series entirely processing SLNs for OSNA in early-stage cervical cancer. OSNA is able to intra-operatively detect low-volume metastases in SLNs. Further studies are necessary to confirm the accuracy of this technique and to assess survival implications of low-volume metastases detected by OSNA.

Standard ultra-staging compared to one-step nucleic acid amplification for the detection of sentinel lymph node metastasis in endometrial cancer patients: a retrospective cohort comparison.

INTRODUCTION: The objective of this study was to compared standard ultra-staging (SU) with one-step nucleic acid amplification (OSNA) for the detection of sentinel lymph node (SLN) metastasis in women with apparent uterine-confined endometrial cancer. METHODS: All women underwent SLN identification with complete surgical staging. All SLNs were cut perpendicular to the long axis and two adjacent 5 µm sections were cut at each of two levels 50 µm apart. At each level, one slide was stained with hematoxylin and eosin and the other with immunohistochemistry using the AE1/AE3 anti-cytokeratin antibody, as well as one negative control slide for a total of five slides per block. For OSNA analysis, the 2 mm sections of the lymph nodes were homogenized to form a lysate. The lysate was then centrifuged and inserted into the RD 100i instrument where the isothermal amplification of CK19 mRNA was executed. RESULTS: Of the 396 patients included in the retrospective analysis, 214 were in the SU group, and 182 in the OSNA group. Overall 869 SLNs were identified (490 SU, 379 OSNA). Sixty patients exhibited SLN metastasis (34 SU, 26 OSNA). Macrometastasis, micrometastases, and isolated tumor cells (ITC) were 5.1%, 4.1%, and 0.2%, respectively, in the US group, and 2.4%, 6.3%, and 0.1%, respectively, in the OSNA group (p=0.022). CONCLUSIONS: The OSNA assay detected a higher rate of micrometastasis and a lower rate of macrometastasis and ITC when compared with SU. The clinical and prognostic impact of ITC is debatable and controversial. Further studies are needed to clarify the respective roles of the OSNA and SU methods, and the possible role of ITC in the prognosis of patients with apparent early-stage endometrial cancer.

Moving a neodymium magnet promotes the migration of a magnetic tracer and increases the monitoring counts on the skin surface of sentinel lymph nodes in breast cancer.

BACKGROUND: We suspected that moving a small neodymium magnet would promote migration of the magnetic tracer to the sentinel lymph node (SLN). Higher monitoring counts on the skin surface before making an incision help us detect SLNs easily and successfully. The present study evaluated the enhancement of the monitoring count on the skin surface in SLN detection based on the magnet movement in a sentinel lymph node biopsy (SNB) using superparamagnetic iron oxide (SPIO) nanoparticles. METHODS: After induction of general anesthesia, superparamagnetic iron oxide nanoparticles were injected sub-dermally into the subareolar area or peritumorally. The neodymium magnet was moved over the skin from the injection site to the axilla to promote migration of the magnetic tracer without massage. A total of 62 patients were enrolled from February 2018 to November 2018: 13 cases were subjected to magnet movement 20 times (Group A), 8 were subjected to 1-min magnet movement (Group B), 26 were given a short (about 5 min) interval from injection to 1-min magnet movement (Group C), and 15 were given a long (about 25 min) interval before 1-min magnet movement using the magnetometer's head (Group D). In all cases, an SNB was conducted using both the radioisotope (RI) and SPIO methods. The monitoring counts on the skin surface were measured by a handheld magnetometer and compared among the four groups. Changes in the monitoring count by the interval and magnet movement were evaluated. RESULTS: The identification rates of the SPIO and RI methods were 100 and 95.2%, respectively. The mean monitoring counts of Group A, B, C, and D were 2.39 µT, 2.73 µT, 3.15 µT, and 3.92 µT, respectively (p < 0.0001; Kruskal-Wallis test). The monitoring counts were higher with longer magnet movement and with the insertion of an interval. Although there were no relationships between the monitoring count on the skin surface and clinicopathologic factors, magnet movement strongly influenced the monitoring count on the skin surface. CONCLUSION: Moving a small neodymium magnet is effective for promoting migration of a magnetic tracer and increasing monitoring counts on the skin surface. TRIAL REGISTRATION: UMIN, UMIN000029475. Registered 9 October 2017.

Immunohistochemistry for PRAME in the Distinction of Nodal Nevi From Metastatic Melanoma.

The distinction of metastatic melanoma from melanocytic nevi in lymph nodes can on occasion be difficult. As diffuse immunohistochemical (IHC) PRAME (PReferentially expressed Antigen in MElanoma) expression is detected in the majority of primary and metastatic melanomas, but rarely in nevi, we reasoned that PRAME could be a useful adjunct marker for the diagnosis of melanocytes in lymph nodes. In this study, we examined 45 nodal melanocytic deposits comprising 30 nodal nevi and 15 melanoma metastases. The latter were diagnostically not straightforward because they either coexisted with nodal nevi or were present in perinodal fibrous tissue. All nodal nevi (30/30) were negative for PRAME, whereas all melanoma metastases (15/15) were diffusely positive for PRAME IHC. We additionally report the novel use of a PRAME/Melan A dual-label immunostain. Our results show that PRAME IHC may be useful in the assessment of diagnostically challenging nodal melanocytic deposits, such as intraparenchymal nodal nevi, metastases confined to the capsular fibrous tissue, or in the setting of small metastases coexisting with a nodal nevus in the same lymph node.

Prognostic Significance of "Nonsolid" Microscopic Metastasis in Merkel Cell Carcinoma Sentinel Lymph Nodes.

Our recent work regarding Merkel cell carcinoma sentinel lymph node (SLN) metastasis found that "solid" pattern microscopic metastasis conferred worse prognosis than the "nonsolid" ones. The goals of the present study were to (1) compare the prognostic significance/outcomes of 2 diagnostic groups-patients with a nonsolid pattern of SLN metastasis and those with diagnostically negative SLN biopsies (SLNB), and (2) evaluate the durability of SLN metastasis after extensive sectioning. Five-level, step-wise sectioning at 250-µm intervals was performed in all SLN blocks with an immunohistochemical stain for CK20 on all levels. The presence and pattern of metastases were recorded and analyzed as were corresponding patient and tumor parameters. Median follow-up durations for all patients (n=38), positive SLNB (n=16) and negative SLNB (n=22) groups were 56.3, 50.4, and 66.8 months, respectively. Overall survival (OS) and disease-specific survival (DSS) did not differ between the 2 diagnostic groups (OS P=0.65, DSS P=0.37) but did differ by immune status (immunocompetent vs. immunosuppressed, OS P=0.03, DSS P=0.005) and primary tumor category (OS P<0.0001, DSS P=0.001). On deeper sectioning, all 16 diagnostically positive SLNB continued to show nonsolid microscopic metastasis, and 32% (7/22) diagnostically negative SLNB revealed nonsolid metastasis. DSS was worse for sinusoidal-pattern metastasis versus all others (P=0.02). Five of 38 patients (13%) died of disease; the only immunocompetent patient had sinusoidal-pattern metastasis discovered in a diagnostically negative SLNB. Our data suggest that outcome for nonsolid metastasis is similar to that of negative SLNB with the exception of the sinusoidal pattern, which was associated with worse outcome. Larger studies are warranted to quantify and compare microscopic metastatic tumor burden by pattern and confirm whether the sinusoidal pattern confers an intermediate prognostic risk between solid and other nonsolid microscopic metastases.

Small and Isolated Immunohistochemistry-positive Cells in Melanoma Sentinel Lymph Nodes Are Associated With Disease-specific and Recurrence-free Survival Comparable to that of Sentinel Lymph Nodes Negative for Melanoma.

Although immunohistochemistry (IHC) has improved our ability to detect melanoma metastases in sentinel lymph nodes (SLN), the American Joint Committee on Cancer (AJCC) does not provide a lower threshold for determining if a SLN is positive for metastasis. Existing literature suggests that even a small aggregate or an enlarged, abnormal cell detectable by IHC can be associated with an adverse outcome. In our experience, however, some SLNs contain small solitary cells the size of neighboring lymphocytes demonstrable only by IHC. We sought to determine their clinical significance. A total of 821 patients underwent a SLN biopsy at our institution over a 12-year period. In all, 639 (77.8%) were SLN-negative, 125 (15.2%) were SLN-positive, and 57 (6.9%) had rare IHC-positive cells of undetermined clinical significance with no disease progression over a mean 59-month follow-up. Kaplan-Meier method with pair-wise comparisons revealed no significant difference in disease-specific survival and recurrence-free survival between SLN-negative and rare IHC-positive groups. There were significant differences in survival and recurrence between patients in the rare IHC-positive group and those with melanoma metastases, including those with solitary melanoma cells and those with tumor burdens ≤0.2 mm. While the lower diagnostic threshold for metastatic melanoma on IHC-stained sections needs to be studied further, our data suggest that rare IHC-positive cells lacking cytomorphologic features of overt malignancy are equivocal for melanoma and could impart a similar prognosis as patients with no evidence of SLN involvement.

Prognostic relevance of an epigenetic biomarker panel in sentinel lymph nodes from colon cancer patients.

BACKGROUND: Patients with early colorectal cancer (stages I-II) generally have a good prognosis, but a subgroup of 15-20% experiences relapse and eventually die of disease. Occult metastases have been suggested as a marker for increased risk of recurrence in patients with node-negative disease. Using a previously identified, highly accurate epigenetic biomarker panel for early detection of colorectal tumors, we aimed at evaluating the prognostic value of occult metastases in sentinel lymph nodes of colon cancer patients. RESULTS: The biomarker panel was analyzed by quantitative methylation-specific PCR in primary tumors and 783 sentinel lymph nodes from 201 patients. The panel status in sentinel lymph nodes showed a strong association with lymph node stage (P = 8.2E-17). Compared with routine lymph node diagnostics, the biomarker panel had a sensitivity of 79% (31/39). Interestingly, among 162 patients with negative lymph nodes from routine diagnostics, 13 (8%) were positive for the biomarker panel. Colon cancer patients with high sentinel lymph node methylation had an inferior prognosis (5-year overall survival P = 3.0E-4; time to recurrence P = 3.1E-4), although not significant. The same trend was observed in multivariate analyses (P = 1.4E-1 and P = 6.7E-2, respectively). Occult sentinel lymph node metastases were not detected in early stage (I-II) colon cancer patients who experienced relapse. CONCLUSIONS: Colon cancer patients with high sentinel lymph node methylation of the analyzed epigenetic biomarker panel had an inferior prognosis, although not significant in multivariate analyses. Occult metastases in TNM stage II patients that experienced relapse were not detected.

Sentinel Lymph Nodes in Classic Invasive Lobular Carcinoma of the Breast: Cytokeratin Immunostain Ensures Detection, and Precise Determination of Extent, of Involvement.

The assessment of sentinel lymph nodes (SLN) on hematoxylin and eosin (H&E)-stained sections in cases of classic type of invasive lobular carcinoma (cILC) is considered unreliable, particularly in cases with minimal involvement, that is by either isolated tumor cells (pN0i+) or micrometastases (pN1mi). Although the impact of minimal SLN involvement has been shown to be insignificant in most clinical trials (even though cILC was either under-represented or not separated in the respective cohorts), the results of MIRROR trial did emphasize the need for additional therapy in cases with minimally involved SLN to ensure improved disease-free survival. We sought to study the role of cytokeratin immunohistochemistry (CK-IHC) in evaluating SLN in cILC. A total of 582 cILC cases with SLN diagnosed over a 12-year period (2005 to 2016) were reviewed. In all, 394/582 (68%) cases had H&E(-)/CK(-) SLN. In total, 188/582 (32%) cases showed some degree of SLN involvement of which 143/582 (25%) cases had readily identifiable SLN involvement on H&E slides. Overall, 45/582 (7.7%) cases had H&E(-)/CK(+) SLN. The following data relate to the latter subset of 45 cases. Mean age of patients: 61 y (range: 32 to 86 y); right: 24 (53%), left: 21 (47%); multifocal and/or multicentric: 22 (49%); mean size: 2.0 cm (range: 0.25 to 4.4 cm); mean number of SLN: 2.5; mean number of involved SLN: 1.2; and cases with prior needle core or excisional biopsy: 45 (100%). CK(+) cells were identified in isolation or in loose clusters, either in subcapsular sinuses or nodal cortex or both. Overall, 30/45 (67%) showed ≤200 CK(+) cells (ie, pN0i+), and 15/45 (33%) showed >200 CK(+) cells (ie, pN1mi). In total, 15/45 (33%) cases underwent axillary lymph node dissection, of which 4/45 (9%) cases were positive. cILC recurred in 3/45 (7%) cases. On statistical analyses, the number of CK(+) cells (≤/>200) did not correlate with either axillary lymph node-positivity or with recurrence. Number of CK(+) cells (≤/>200) readily distinguished pN0i+ from pN1mi based on AJCC's numerical criteria. CK(+) cells could be quantified in linear terms (ie, AJCC's size criteria of pN0i+ and pN1mi was applicable) in only 2 cases. On the basis of these findings, the use of CK-IHC staining should be considered for SLN in cases of cILC to ensure detection, and precise determination of extent, of involvement; however, the prognostic significance of this procedure would have to await results of additional studies with long-term follow-up.

In vivo biodistribution studies and ex vivo lymph node imaging using heavy metal-free quantum dots.

Quantum dots (QDs) are attractive photoluminescence probes for biomedical imaging due to their unique photophysical properties. However, the potential toxicity of QDs has remained a major obstacle to their clinical use because they commonly incorporate the toxic heavy metal cadmium within the core of the QDs. In this work, we have evaluated a novel type of heavy metal-free/cadmium-free and biocompatible QD nanoparticles (bio CFQD(®) nanoparticles) with a good photoluminescence quantum yield. Sentinel lymph node mapping is an increasingly important treatment option in the management of breast cancer. We have demonstrated their potential for lymph node mapping by ex vivo imaging of regional lymph nodes after subcutaneous injection in the paw of rats. Using photoluminescence imaging and chemical extraction measurements based on elemental analysis by inductively coupled plasma mass spectroscopy, the quantum dots are shown to accumulate quickly and selectively in the axillary and thoracic regional lymph nodes. In addition, lifetime imaging microscopy of the QD photoluminescence indicates minimal perturbation to their photoluminescence properties in biological systems.