Radiolocalization of the sentinel lymph node in Merkel cell carcinoma: A clinical analysis of seven cases

Merkel cell carcinoma (MCC) is a rare cutaneous skin lesion with a variable but often aggressive clinical course. Patient survival correlates with nodal status and the presence of distant metastases. The histologic status of the sentinel lymph node consistently correlates with the incidence of regional lymphatic metastases in other dermal malignancies. The technique of radiolocalization and surgical resection of the sentinel lymph node using an intraoperative gamma probe is used to guide clinical management in these patients. We report on seven cases of MCC managed utilizing this technique. Four patients had negative sentinel nodes and no other nodal disease at completion lymphadenectomy (n = 2) or clinical follow-up (n = 2) and currently remain disease free. Two patients had a positive sentinel node but no other positive lymph nodes at completion lymphadenectomy; one of them developed regional recurrence. One patient with a positive sentinel node and six additional positive nodes developed extensive nodal disease and systemic recurrence during radiotherapy and expired of MCC. Our results suggest that the sentinel node was identified and removed successfully using radiolocalization making this technique useful in the staging and therapy of patients with MCC. J. Surg. Oncol. 1998;67:251–254. © 1998 Wiley-Liss, Inc.     

Sentinel lymph node mapping for defining site and extent of elective radiotherapy management of regional nodes in Merkel cell carcinoma: A pilot case series

Merkel cell carcinoma is a rare and aggressive skin malignancy. We discuss sentinel lymph node mapping which is a valuable decision aid for radiotherapy management and planning of treatment volumes as illustrated by four cases.     

Merkel cell carcinoma from 2008 to 2012: Reaching a new level of understanding

Merkel cell carcinoma (MCC) is a rare primary cutaneous carcinoma of the skin who has high aggressiveness, high risk of locoregional and distant spread, a mortality rate considerably higher than that of cutaneous melanoma and a poor survival. Its incidence has increased during the past twenty years. The studies published from 2008 to early 2012 have introduced interesting changes in the understanding of its epidemiology, pathogenesis and consequently in the diagnostic codes and the therapeutic approach. Early and detailed nodal diagnosis with posterior multidisciplinary decision is mandatory. Surgery and Radiotherapy play a fundamental role in the management of this tumor. Both are associated with improved locoregional control and disease free survival; but patients continue to have distant failure because, currently, there is no effective systemic treatment available.     

Prokineticins and Merkel cell polyomavirus infection in Merkel cell carcinoma

Background:

Prokineticin-1 (PROK1) and prokineticin-2 (PROK2) are chemokine-like proteins that may influence cancer growth by regulating host defence and angiogenesis. Their significance in viral infection-associated cancer is incompletely understood. We studied prokineticins in Merkel cell carcinoma (MCC), a skin cancer linked with Merkel cell polyomavirus (MCPyV) infection.

Methods:

Carcinoma cell expression of PROK1 and PROK2 and their receptors (PROKR1 and PROKR2) was investigated with immunohistochemistry, and tumour PROK1 and PROK2 mRNA content with quantitative PCR from 98 MCCs. Subsets of tumour infiltrating leukocytes were identified using immunohistochemistry.

Results:

Merkel cell polyomavirus-positive MCCs had higher than the median PROK2 mRNA content, whereas MCPyV-negative MCCs contained frequently PROK1 mRNA. Cancers with high tumour PROK2 mRNA content had high counts of tumour infiltrating macrophages (CD68+ and CD163+ cells). Patients with higher than the median PROK2 mRNA content had 44.9% 5-year survival compared with 23.5% among those with a smaller content (hazard ratio (HR): 0.53; 95% confidence interval (CI): 0.34–0.84; P=0.005), whereas the presence of PROK1 mRNA in tumour was associated with unfavourable survival (P=0.052).

Conclusions:

The results suggest that prokineticins are associated with MCPyV infection and participate in regulation of the immune response in MCC, and may influence outcome of MCC patients.     

Molecular analysis of breast sentinel lymph nodes

Lymphatic mapping and sentinel lymph node (SLN) biopsy have become the standard of care for staging the axilla in patients with invasive breast cancer. Current histologic methods for SLN evaluation have limitations, including subjectivity, limited sensitivity, and lack of standardization. The discovery of molecular markers to detect metastases has been reported over the last 2 decades. The authors review the historical development of these markers and the clinical use of one of the molecular platforms in 478 patients at their institution. Controversies and future directions are discussed.     

Histopathologic examination of axillary sentinel lymph nodes in breast carcinoma patients

The axillary sentinel lymph node biopsy (SLNB) has gained increasing popularity as a novel surgical approach for staging patients with breast carcinoma and for guiding the choice of adjuvant therapy with minimal morbidity. Patients with negative SLNB represent a subset of breast carcinoma patients with definitely better prognosis, because their pN0 status is based on a very thorough examination of the sentinel lymph nodes (SLNs), with a very low risk of missing even small micrometastatic deposits, as compared with routine examination of the 20 or 30 lymph nodes obtained by the traditional axillary clearing. The histopathologic examination of the SLNs may be performed after fixation and embedding in paraffin, or intraoperatively on frozen sections. Whatever is the preferred tracing technique and surgical procedure, the histopathologic examination of each SLN must be particularly accurate, to avoid a false-negative diagnosis. Unfortunately, because of the lack of standardised guidelines or protocols for SLN examination, different institutions still adopt their own working protocols, which differ substantially in the number of sections cut and examined, in the cutting intervals (ranging from 50 to more than 250 microm), and in the more or less extensive use of immunohistochemical assays for the detection of micrometastatic disease. Herein, a very stringent protocol for the examination of the axillary SLN is reported, which is applied either to frozen SLN for the intraoperative diagnosis, and to fixed and embedded SLN as well.     

Sentinel lymph node biopsy in Merkel cell carcinoma: The Mayo Clinic experience of 150 patients

Background

Merkel cell carcinoma (MCC) is a rare cutaneous malignancy of neuroendocrine origin with a high propensity for lymph node metastasis. Sentinel lymph node (SLN) status is important for accurate staging; however, the optimal treatment following SLN biopsy, regardless of nodal status, remains unclear.

Interobserver reproducibility of histologic parameters of melanoma deposits in sentinel lymph nodes

BACKGROUND:

Histologic parameters of melanoma deposits in sentinel lymph nodes (SLNs) have been shown to be predictive of clinical outcome and the presence or absence of tumor in non‐SLNs, but assessment of these parameters is prone to interobserver variation.

METHODS:

Histologic sections of 44 SLNs containing metastatic melanoma were examined by 7 pathologists. Parameters assessed included cross‐sectional area of tumor deposits, cross‐sectional area of SLNs, percentage of SLN area involved by tumor calculated from the 2 previous parameters, estimated percentage of SLN area involved by tumor, tumor penetrative depth, location of tumor within the SLN, and presence of extracapsular spread. Levels of interobserver agreement were measured by using intraclass correlation coefficients (ICC).

RESULTS:

There was good to excellent interobserver agreement on measurement of quantitative parameters: maximal size of largest tumor deposits, calculated area of 3 largest tumor deposits, percentage of the area of SLN involved by tumor, and tumor penetrative depth (ICC, 0.88, 0.73, 0.68, and 0.83, respectively). There was moderate agreement on the evaluation of subcapsular versus nonsubcapsular location of tumor deposits (ICC = 0.50). Agreement on assessment of extracapsular spread was fair (ICC = 0.39).

CONCLUSIONS:

Assessment of some of the quantitative parameters was highly reproducible between pathologists. However, evaluation of the location of tumor deposits within SLNs and assessment of extracapsular spread was less reproducible. Clearer definitions and training can be expected to improve the reproducibility of assessment. These results have important implications for reliability and reproducibility of these parameters in staging, prediction of outcome, and clinical management of melanoma patients. Cancer 2009. © 2009 American Cancer Society.     

An intact retinoblastoma protein-binding site in Merkel cell polyomavirus large T antigen is required for promoting growth of Merkel cell carcinoma cells

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer that frequently harbours Merkel cell polyomavirus (MCV) DNA integrated in the genome of the tumor cells. In our study, we elaborate our recent finding that MCV-positive MCC cell lines require the expression of the viral T antigens (TA). Indeed, in a xeno-transplantation model, we prove that TA expression is essential also in an in vivo situation, as knock down of TA leads to tumor regression. Moreover, rescuing TA short hairpin RNA (shRNA)-treated MCV-positive MCC cells by ectopic expression of shRNA-insensitive TAs clearly demonstrates that the observed effect is caused by TA knockdown. Notably, introduction of a mutation in the LTA protein interfering with LTA binding to the retinoblastoma protein (RB) ablated this rescue. The importance of this interaction was further confirmed as LTA-specific knockdown leads to explicit cell growth inhibition. In summary, the presented data demonstrate that established MCV-positive MCC tumors critically depend on TA expression, in particular the LTA and RB interaction, for sustained tumor growth. Consequently, interference with LTA/RB interaction appears as promising strategy to treat MCC.     

Micrometastases and isolated tumour cells in sentinel lymph nodes in oral and oropharyngeal squamous cell carcinoma

Background

The occurrence of micrometastases (MMs) and isolated tumour cells (ITCs) in oral sentinel lymph node (SLN) biopsy is poorly known, and the definitions and clinical significance of MMs and ITCs in SLN biopsy are controversial. We compared the UICC/TNM definitions of MMs and ITCs with our previously published sentinel node protocol to assess how the adoption of the UICC/TNM criteria would affect the staging of nodal micrometastatic disease.

Methods

Of 107 patients who had a SLN biopsy and pathology at 150 μm intervals, 35 with metastatic tumour were included. Eighty-six SLNs were reassessed using the UICC/TNM definitions for MMs and ITCs. Findings were linked to the final pathology in the subsequent neck dissection.

Results

Initial H&E sections showed metastases in 24 patients (in 34 out of 61 SLN), 8 of whom (9 SLNs) had MMs. Additional step serial sections revealed metastatic deposits in a further 11 patients (15 out of 25 SLNs were positive) which were reassessed as MMs (6 patients) or ITCs (5 patients). Subsequent neck dissection revealed additional metastases in 46% of patients with MM, whilst one of the ITC patients had subsequent neck metastases (20%).

Conclusion

Despite some limitations, the UICC/TNM classification provides an objective, uniform method of detecting MMs and ITC's. Unlike in cases with ITC, metastases in other non-SLNs were common when a micrometastasis was detected in a SLN, indicating need for further treatment of the neck.     

Human Merkel cell polyomavirus infection I. MCV T antigen expression in Merkel cell carcinoma,lymphoid tissues and lymphoid tumors

Merkel cell polyomavirus (MCV) is a recently discovered human virus closely related to African green monkey lymphotropic polyomavirus. MCV DNA is integrated in ～80% of Merkel cell carcinomas (MCC), a neuroendocrine skin cancer linked to lymphoid malignancies such as chronic lymphocytic leukemia (CLL). To assess MCV infection and its association with human diseases, we developed a monoclonal antibody that specifically recognizes endogenous and transfected MCV large T (LT) antigen. We show expression of MCV LT protein localized to nuclei of tumor cells from MCC having PCR quantified MCV genome at an average of 5.2 (range 0.8–14.3) T antigen DNA copies per cell. Expression of this putative viral oncoprotein in tumor cells provides the mechanistic underpinning supporting the notion that MCV causes a subset of MCC. In contrast, although 2.2% of 325 hematolymphoid malignancies surveyed also showed evidence for MCV infection by DNA PCR, none were positive at high viral copy numbers, and none of 173 lymphoid malignancies examined on tissue microarrays expressed MCV LT protein in tumor cells. As with some of the other human polyomaviruses, lymphocytes may serve as a tissue reservoir for MCV infection, but hematolymphoid malignancies associated with MCC are unlikely to be caused by MCV. © 2009 UICC     

The Merkel cell carcinoma challenge

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin that occurs primarily in elderly or immunocompromised patients. For this report, the authors reviewed the diagnostic challenges associated with MCC encountered on their fine‐needle aspiration (FNA) service and also conducted an in‐depth review of the literature on MCC. A computer search for patients who were diagnosed with MCC by FNA at the authors' institution from 2006 to 2010 was conducted, and 5 patients were selected for cytologic and immunochemical analyses based on their varied and diagnostically challenging clinical presentations. The 5 selected patients had clinical findings commonly associated with MCC, including advanced age (4 of the 5 patients were ages 75‐85 years) and a history of previous malignancies (3 of the 5 patients had a history of previous malignancy), and 1 patient was diagnosed with a concomitant low‐grade lymphoma. The patients and their disease illustrated the protean clinical presentation of MCC and the clinical and cytologic challenges associated with this neoplasm. The current findings indicate the need for cytopathologists to be aware of the deceptive presentation of this neoplasm and its cytologic and immunochemical features to correctly diagnose this insidious neoplasm. Cancer (Cancer Cytopathol) 2013;121:179–188. © 2012 American Cancer Society.     

Merkel cell polyomavirus–negative Merkel cell carcinoma is associated with JAK‐STAT and MEK‐ERK pathway activation

Merkel cell polyomavirus (MCPyV) is monoclonally integrated into the genomes of approximately 80% of Merkel cell carcinomas (MCCs). While the presence of MCPyV affects the clinicopathological features of MCC, the molecular mechanisms of MCC pathogenesis after MCPyV infection are unclear. This study investigates the association between MCPyV infection and activation of the MEK‐ERK and JAK‐STAT signaling pathways in MCC to identify new molecular targets for MCC treatment. The clinicopathological characteristics of 30 MCPyV‐positive and 20 MCPyV‐negative MCC cases were analyzed. The phosphorylation status of MEK, ERK, JAK, and STAT was determined by immunohistochemical analysis. The activation status of the MEK‐ERK and JAK‐STAT pathways and the effects of a JAK inhibitor (ruxolitinib) was analyzed in MCC cell lines. Immunohistochemically, the expression of pJAK2 (P = .038) and pERK1/2 (P = .019) was significantly higher in MCPyV‐negative than in MCPyV‐positive MCCs. Male gender (hazard ratio [HR] 2.882, P = .039), older age (HR 1.137, P < .001), negative MCPyV status (HR 0.324, P = .013), and advanced cancer stage (HR 2.672, P = .041) were identified as unfavorable prognostic factors; however, the phosphorylation states of JAK2, STAT3, MEK1/2, and ERK1/2 were unrelated to the prognosis. The inhibition of cell proliferation by ruxolitinib was greater in MCPyV‐negative MCC cell lines than in an MCPyV‐positive MCC cell line. The expression of pERK1/2 and pMEK was higher in MCPyV‐negative than in MCPyV‐positive cell lines. These results suggest that activation of the JAK2 and MEK‐ERK pathways was more prevalent in MCPyV‐negative than in MCPyV‐positive MCC and the JAK inhibitor ruxolitinib inhibited MEK‐ERK pathway activation. Consequently, the JAK‐STAT and MEK‐ERK signaling pathways may be potential targets for MCPyV‐negative MCC treatment.     

Characterization of functional domains in the Merkel cell polyoma virus Large T antigen

Merkel cell polyomavirus (MCPyV)‐positive Merkel cell carcinoma (MCC) tumor cell growth is dependent on the expression of a viral Large T antigen (LT) with an intact retinoblastoma protein (RB)‐binding site. This RB‐binding domain in MCPyV‐LT is—in contrast to other polyomavirus LTs (e.g., SV40)—embedded between two large MCPyV unique regions (MUR1 and MUR2). To identify elements of the MCPyV‐LT necessary for tumor cell growth, we analyzed the rescue activity of LT variants following knockdown of the endogenous LT in MCC cells. These experiments demonstrate that nuclear localization is essential for LT function, but that a motif previously described to be a nuclear localization sequence is neither required for nuclear accumulation of truncated MCPyV‐LT nor for promotion of MCC cell proliferation. Furthermore, large parts of the MURs distal to the RB binding domain as well as ALTO—a second protein encoded by an alternative reading frame in the MCPyV‐LT mRNA—are completely dispensable for MCPyV‐driven tumor cell proliferation. Notably, even MCPyV‐LTs in which the entire MURs have been removed are still able to promote MCC cellular growth although rescue activity is reduced which may be due to MUR1 being required for stable LT expression in MCC cells. Finally, we provide evidence implying that—while binding to Vam6p is not essential—HSC‐70 interaction is significantly involved in mediating MCPyV‐LT function in MCC cells including growth promotion and induction of E2F target genes.     

Lymphoscintigraphic drainage of acral limb skin to interval sentinel lymph nodes in healthy subjects

BACKGROUND AND OBJECTIVES: In the literature, drainage to epitrochlear and popliteal sentinel lymph nodes (SLN) are analyzed for whole or distal extremity (below elbow or knee) melanomas that are not topographically homogeneous with respect to tendency of drainage to interval SLNs. We hypothesize that acral (hand and foot) skin has a uniform frequency of drainage to interval SLNs, which is higher than reported for distal extremity melanomas. METHODS: One hundred healthy subjects were enrolled. Fifty subjects had standard four extremity lymphoscintigraphies by radiocolloid injection into an interdigital web space as in lymphodynamic studies. On another 50 subjects, either targeted upper (n = 25) or lower (n = 25) extremity lymphoscintigraphies were performed utilizing injection sites that likely drain to interval SLNs. Acral skin drainage to interval SLNs was analyzed for interindividual variability and injection site dependence. RESULTS: There was considerable interindividual variability in drainage of each injection site to interval SLNs. Hand skin had a uniform 50% frequency of drainage to epitrochlear-midhumeral SLNs with both injection sites. This frequency was higher than the epitroclear SLN frequencies reported for distal extremity melanomas. Foot skin had 10% and 90% frequencies of drainage to popliteal SLNs from standard and targeted injection sites, respectively. Foot skin largely simulates the tendency of drainage reported for distal extremity melanomas while lateral heel represents a limited zone that almost uniformly drains to popliteal SLNs. CONCLUSIONS: Despite dissimilarities between hand and foot, acral skin drainage to interval SLNs is high enough to obligate a thorough interval SLN exploration in acral primaries.     

Mapping of sentinel lymph node micrometastasis in Dukes-B colorectal carcinoma and its clinical significance

OBJECTIVE To investigate the detection of sentinel lymph node （SLN） micrometastasis （MM） and the clinical significance in patients with Dukes-B colorectal carcinoma （CRC） using H＆E stained slides. METHODS In a prospective study of 64 patients with Dukes-B CRC undergoing radical surgery, routine H＆E staining combined with the immunohistochemical SP method was used to detect the expression of the cytokeratin 20 （CK20） and telomerase （TLMA） in 122 SLNs from 61 of the 64 cases. During a 3-year follow up, the patients＇ clinicopathologic parameters data, survival and their relationship were collected and analyzed. RESULTS i） In 6 out of the 61 cases with successful mapping, 9 SLNs were positive after routine detection using H＆E staining. ii） In the other 55 with negative results from H＆E staining, the positive rate of CK20 expression was 27.3% （15/55）, and the positive rate of TLMA 21.8% （12/55）, after immunohistochemical examination （IHC） on the SLN. iii） Detection by combining the 2 methods showed that the micrometastasis rate was 38.2% （21/55）. iv） The recurrence or metastasis rate of CRC was significantly higher in the Dukes-B patients with sentinel lymph node micrometastasis （SLNMM） （＋） than in those with SLNMM （-） （P 〈 0.05）, and simultaneously, the survival rate decreased by a large margin in the patients with SLNMM （＋）, compared to those with SLNMM （-） （P 〈 0.05）. There were significant differences in the recurrence or metastasis rate of cancer and the survival rate between the Dukes-B patients with SLNMM （-） and the Dukes-C patients （P 〈 0.05）. CONCLUSION Examination of anti-CK20 and anti-TLMA using immunohistochemical staining can be used to detect SLNMM, and the combined examination of the 2 can increase the detection rate. The detection of SLNMM can precisely determine the Dukes stage of colorectal carcinoma, which is of help in directing postoperative adjunctive therapy and in assessing prognosis.