Loss of heterozygosity of chromosome 13 in Merkel cell carcinoma

We have examined a series of 24 Merkel cell carcinoma (MCC) DNAs for loss of heterozygosity (LOH) at eight loci on chromosome 13. All patients were heterozygous for at least one locus. Overall, 18 of 24 (75%) patients showed LOH, among whom 10 patients demonstrated LOH at all informative loci. A single common region of loss was identified in all cases and included the marker D13S233 (13q14.3), which maps close to the retinoblastoma susceptibility gene RB1. The RB1 protein was not detected by Western blot analysis in any of nine MCC cell lines tested. These data indicate that 13q losses are the most common chromosomal losses observed to date in MCC and the likely target of these deletions is the RB1 locus. Genes Chromosom. Cancer 20:93–97, 1997. © 1997 Wiley-Liss, Inc.     

Merkel cell carcinoma of the skin: pathological and molecular evidence for a causative role of MCV in oncogenesis

Merkel cell carcinoma (MCC), a skin tumour with neuroendocrine features, was recently found to be associated with a new type of human polyomavirus, called Merkel cell virus (MCV). We investigated the specificity of this association as well as a causal role of MCV in oncogenesis. DNA and RNA from ten cases of MCC were analysed using PCR and RT‐PCR. DNA from 1241 specimens of a wide range of human tumours was also analysed. The DIPS technique was used to identify the integration locus of viral DNA sequences. Array CGH was performed to analyse structural alterations of the cell genome. MCV DNA sequences were found in all ten cases of MCC and in none of the 1241 specimens of other tumour types. Clonal integration of MCV into the host genome was seen in all MCC cases and was checked by FISH in one case. A recurrent pattern of conserved viral sequences which encompassed the replication origin, the small tumour (ST), and the 5′ part of the large tumour (LT) antigen DNA sequences was observed. Both ST and LT viral sequences were found to be significantly expressed in all MCCs. Neither recurrent site of integration nor alteration of cellular genes located near the viral sequences was observed. The tight association of MCV with MCC, the clonal pattern of MCV integration, and the expression of the viral oncoproteins strongly support a causative role for MCV in the tumour process. This information will help the development of novel approaches for the assessment and therapy of MCC and biologically related tumours. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

RB1 gene mutations are a distinct predictive factor in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that tends to show local recurrence and metastasis. Typically, MCC is polyomavirus (MCPyV)-associated and cytokeratin 20 (CK20) positive. However, little is known about this tumor and its origins. Here, we aimed to determine the developmental origins of MCC and to identify prognostic clinicopathologic factors. Initial examinations revealed that CK20 and MCPyV expression (CK20+, MCPyV+ (60%); CK20+, MCPyV− (10%); CK20−, and MCPyV− (30%)) did not affect overall survival. With RB1 gene sequencing of FFPE specimens, which covered an entire exon, all RB1 mutation-positive cases showed positive regional lymph node and/or distant metastases (8/8 cases, 100%), whereas the frequency of the metastasis was statistically significantly lower in RB1 mutation-negative cases, (10/16 cases, 62%, P = 0.033). The results were also confirmed with immunohistochemistry, and either RB1 alterations, entire exon sequencing, or immunohistochemistry was associated with the metastasis (P = 0.007). RB1 alterations may be used to access the aggressive clinical course of MCC.     

Homo- and heterotypic cell-cell contacts in Merkel cells and Merkel cell carcinomas: heterogeneity and indications for cadherin switching

Werling A M, Doerflinger Y, Brandner J M, Fuchs F, Becker J C, Schrama D, Kurzen H, Goerdt S & Peitsch W K
(2011) Histopathology 58, 286–303
Homo‐ and heterotypic cell–cell contacts in Merkel cells and Merkel cell carcinomas: heterogeneity and indications for cadherin switching Aims: Merkel cell carcinomas (MCCs) are rare but aggressive tumours associated recently with Merkel cell polyomavirus (MCV). As development and progression of several types of carcinomas can be promoted by changes in cell adhesion proteins, the aim of this study was to examine homo‐ and heterotypic cell contacts of Merkel cells and MCCs. Methods and results: Merkel cells of healthy glabrous epidermis and 52 MCCs were analysed by double‐label immunostaining, immunofluorescence and confocal microscopy. Merkel cells were connected to keratinocytes by E‐ and P‐cadherin, desmoglein 2 and desmocollin 2. In contrast, the vast majority of MCCs (90%) contained N‐cadherin, but only 67% and 65% contained E‐ and P‐cadherin, respectively. Interestingly, P‐cadherin was absent significantly more frequently in lymph node metastases than in primary tumours and by trend in more advanced clinical stages. Moreover, major subsets of MCCs synthesized desmoglein 2 and, surprisingly, tight junction proteins. No significant differences were observed upon stratification for MCV DNA, detected in 84% of tumours by real‐time polymerase chain reaction. Conclusions: Assuming that MCCs originate from Merkel cells, our data indicate a switch from E‐ and P‐cadherin to N‐cadherin during tumorigenesis. Whether the unexpected heterogeneity of junctional proteins can be exploited for prognostic and therapeutic purposes will need to be examined.     

Merkel cell carcinoma: a review and update on current concepts

Merkel cell carcinoma (MCC) is a malignant, cutaneous neuroendocrine tumour of the elderly with an increasing worldwide incidence. Clinical presentation is generally characterized by a rapidly-evolving dermal tumour on sun-exposed skin of the head, neck or extremities. Histologically, there are sheets and cords of uniform, small cells with hyperchromatic nuclei and multiple small nucleoli. Mitoses and apoptotic bodies are widespread and lymphovascular involvement is commonly present. Aggressive surgical treatment of localized primary lesions followed by radiotherapy remains the mainstay of treatment. Lymph node metastases, local recurrences, and widespread dissemination are commonly seen. The 10-year survival rates for MCC are 71%, 48%, and 20% for localized, regional, and distant disease, respectively. Merkel cell polyomavirus (MCV) has been implicated as a contributing factor in the pathogenesis of MCC with approximately 80% of tumours showing positivity for the virus. This review provides an up-to-date overview of the clinicopathologic features, current knowledge of MCV, and recent advances in diagnosis, prognostication, and management of MCC.     

Alterations in the RB1 pathway in low-grade diffuse gliomas lacking common genetic alterations

We recently reported that the vast majority (>90%) of low‐grade diffuse gliomas (diffuse astrocytoma, oligoastrocytoma and oligodendroglioma) carry at least one of the following genetic alterations: IDH1/2 mutation, TP53 mutation or 1p/19q loss. Only 7% of cases were triple‐negative (ie, lacking any of these alterations). In the present study, array comparative genomic hybridization (CGH) in 15 triple‐negative WHO grade II gliomas (eight diffuse astrocytomas and seven oligodendrogliomas) showed loss at 9p21 (p14^ARF, p15^INK4b, p16^INK4a loci) and 13q14–13q32 (containing the RB1 locus) in three and two cases, respectively. Further analyses in 31 triple‐negative cases as well as a total of 160 non‐triple‐negative cases revealed that alterations in the RB1 pathway (homozygous deletion and promoter methylation of the p15^INK4b, p16^INK4a and RB1 genes) were significantly more frequent in triple‐negative (26%) than in non‐triple‐negative cases (11%; P = 0.0371). Multivariate analysis after adjustment for age, histology and treatment showed that RB1 pathway alterations were significantly associated with unfavorable outcome for patients with low‐grade diffuse glioma [hazard ratio, 3.024 (1.279–6.631); P = 0.0057]. These results suggest that a fraction of low‐grade diffuse gliomas lacking common genetic alterations may develop through a distinct genetic pathway, which may include loss of cell‐cycle control regulated by the RB1 pathway.     

Uterine cervical squamous cell carcinoma without p16 (CDKN2A) expression: Heterogeneous causes of an unusual immunophenotype

Immunohistochemically p16 (CDKN2A)‐negative uterine cervical squamous cell carcinoma (SCC) is uncommon, and there are few reports about its pathological features. This study explored the causes of p16 negativity in such cases. We analyzed diagnostic tissue samples of five cases of p16‐negative cervical SCC among 107 patients who underwent hysterectomy at Kyoto University Hospital between January 2010 and December 2015. The samples were subjected to immunohistochemical staining, in situ hybridization and a genetic analysis. Two of five cases were positive for human papilloma virus (HPV) by genotyping. One was positive for HPV56 with promoter hypermethylation of CDKN2A and co‐existing Epstein–Barr virus infection. Another was positive for HPV6 categorized as low‐risk HPV with condylomatous morphology. Among the remaining three cases, one had amplification of the L1 gene of HPV with promoter hypermethylation of CDKN2A and TP53 mutation, and one of the other two HPV‐negative cases had a homozygous CDKN2A deletion, while the other was positive for p53 and CK7. p16‐negativity of cervical SCC is often associated with an unusual virus infection status and CDKN2A gene abnormality.     

Immunohistochemistry for Merkel cell polyomavirus is highly specific but not sensitive for the diagnosis of Merkel cell carcinoma in the Australian population

Recent studies have demonstrated a high frequency of detection of Merkel cell polyomavirus in Merkel cell carcinoma. However, most of these studies are from European or North American centers that have relatively low sun exposure and may have a higher incidence of virus-driven oncogenesis compared with the highly sun-exposed but predominantly fair-skinned Australian population. We performed immunohistochemistry for Merkel cell polyomavirus on 104 cases of Merkel cell carcinoma and 74 cases of noncutaneous small cell–undifferentiated carcinoma from 3 major Australian centers. Nineteen (18.3%) cases of Merkel cell carcinoma showed positive staining for Merkel cell polyomavirus versus 1 (1.3%) of small cell–undifferentiated carcinoma. All 15 cases (14.3%) of Merkel cell carcinoma with areas of mixed squamous differentiation showed negative staining. We found positive staining in only 3 (7.7%) of 39 Merkel cell carcinoma from the head and neck (the most sun-exposed area) versus 16 (24.6%) of 65 of tumors from other sites (P < .05). Our findings support the concept of a Merkel cell polyomavirus–driven and a non-Merkel cell polyomavirus–driven (primarily sun-dependent) pathway in Merkel cell carcinoma carcinogenesis, with the latter being significantly more frequent in Australia and in mixed squamous–Merkel cell carcinoma (which is also more frequent in Australia). Although immunohistochemistry for Merkel cell polyomavirus seems to be highly specific in all populations, the low incidence of Merkel cell polyomavirus–positive Merkel cell carcinoma in a highly sun-exposed population limits its diagnostic utility in this setting.     

High‐resolution genomic analysis suggests the absence of recurrent genomic alterations other than SMARCB1 aberrations in atypical teratoid/rhabdoid tumors

Atypical teratoid/rhabdoid tumor (AT/RT) is a rare malignant pediatric brain tumor characterized by genetic alterations affecting the SMARCB1 (hSNF5/INI1) locus in chromosome band 22q11.2. To identify potential additional genetic alterations, high‐resolution genome‐wide analysis was performed using a molecular inversion probe single‐nucleotide polymorphism (MIP SNP) assay (Affymetrix OncoScan formalin‐fixed paraffin‐embedded express) on DNA isolated from 18 formalin‐fixed paraffin‐embedded archival samples. Alterations affecting the SMARCB1 locus could be demonstrated by MIP SNP in 15 out of 16 evaluable cases (94%). These comprised five tumors with homozygous deletions, six tumors with heterozygous deletions, and four tumors with copy number neutral loss of heterozygosity (LOH) involving chromosome band 22q11.2. Remarkably, MIB SNP analysis did not yield any further recurrent chromosomal gains, losses, or copy neutral LOH. On MIP SNP screening for somatic mutations, the presence of a SMARCB1 mutation (c.472C>T p.R158X) was confirmed, but no recurrent mutations of other cancer relevant genes could be identified. Results of fluorescence in situ hybridization, multiplex ligation‐dependent probe amplification, and SMARCB1 sequencing were highly congruent with that of the MIP SNP assay. In conclusion, these data further suggest the absence of recurrent genomic alterations other than SMARCB1 in AT/RT. © 2012 Wiley Periodicals, Inc.     

Alterations in the tumor suppressor gene p16(INK4A) are associated with aggressive behavior of penile carcinomas

Alterations in the p16/cyclinD1/Rb and ARF/Mdm2/p53 pathways are frequent events in the pathogenesis of squamous cell carcinomas. Different mechanisms of p16 regulation have been described for penile carcinomas so far. Therefore, expression of p16 and p53 was immunohistochemically detected with monoclonal antibodies in 52 primary invasive penile squamous cell carcinomas. The carcinomas were analyzed for allelic loss (LOH) in p16 ^INK4A and p53, as well as for mutations in the p16 ^INK4A and the p53 gene. In addition, we examined the promoter status of p16 ^INK4A by methylation-specific PCR. The presence of human papilloma virus (HPV) 6/11, HPV 16 and HPV 18 DNA was analyzed by PCR. Data were compared to clinical data. Concerning p16, 26 (50%) tumors showed positive immunohistochemistry, 32 (62%) tumors showed allelic loss and 22 tumors (42%) showed promoter hypermethylation. All tumors with negative p16 immunohistochemistry showed LOH near the p16 ^INK4A locus and/or hypermethylation of the p16 ^INK4A promoter. HPV 16 DNA was detected in 17 tumors, ten of them with positive p16 immunostaining. The remaining seven tumors with negative p16 staining showed allelic loss and/or promoter hypermethylation. Evidence of lymph node metastasis was significantly associated with negative p16 immunohistochemistry as well as with combined LOH and promoter hypermethylation (p = 0.003 and p = 0.018, respectively). Allelic loss around p53 was found in 22 tumors (42%), and seven mutations of the p53 gene could be demonstrated in our tumors. No correlations could be found between any p53 alteration and clinical parameters.     

High load of Merkel cell polyomavirus DNA detected in the normal skin of Japanese patients with Merkel cell carcinoma

BackgroundAlthough Merkel cell polyomavirus (MCPyV) has the potential to cause Merkel cell carcinoma (MCC), it is also found in the normal skin of healthy individuals. However, the mechanism for transformation of MCPyV to an oncogenic form is unknown.ObjectivesTo investigate the levels of MCPyV infection in the normal skin patients with MCC compared with those in a control cohort.Study designWe studied a total of six Japanese patients with cutaneous MCC. Sun-exposed and sun-unexposed skin swabs were obtained and analyzed for MCPyV loads using quantitative real-time polymerase chain reaction.ResultsAt first, we found a patient with MCC carrying an extremely high load of MCPyV DNA in normal skin. This unique case prompted us to further explore the levels of MCPyV as skin microbiota in patients with MCC. We showed that MCPyV DNA levels were significantly higher in swabs obtained from normal skin samples of six patients with MCC compared with those from 30 age-matched healthy individuals and 19 patients with other cutaneous cancers. Whereas MCPyV strains obtained from the normal skin of patients with MCC had gene sequences without structural alterations, sequences of the tumor-derived strains showed truncating mutations or deletions.ConclusionsAlthough the number of patients with MCC studied was small, our findings suggest that MCC may occur with a background of high MCPyV load in the skin, and are expected to stimulate further studies on whether such skin virome levels could be one of predictive markers for the development of MCC.     

Phosphohistone‐H3 (PHH3) is prognostic relevant in Merkel cell carcinomas but Merkel cell polyomavirus is a more powerful prognostic factor than AJCC clinical stage,PHH3, Ki‐67 or mitotic indices

Merkel cell carcinomas (MCCs) associated with Merkel cell polyomavirus (MCPyV) have better prognosis than those without MCPyV. The relationship between mitotic index (MI) and MCC outcome has remained elusive because of the difficulty in differentiating mitotic cells from apoptotic ones. We evaluated the role of phosphohistone‐H3 (PHH3) (Ser10), a new mitotic count biomarker, in MCPyV‐positive or ‐negative MCC patients, and assessed its prognostic value in comparison to Ki‐67 labeling index or MI using hematoxylin and eosin (HE) staining. We compared the prognostic value of PHH3 mitotic index with that of MI by HE in 19 MCPyV‐positive and 9 MCPyV‐negative MCC patients. PHH3‐positive immunoreactivity was mostly observed in mitotic figures. Multivariate analysis significantly showed that MCPyV status (HR, 0.004; 95% CI 0.0003–0.058) and the American Joint Committee of Cancer (AJCC) stage (HR, 5.02; 95% CI 1.23–20.51) were observed as significantly independent prognostic factors for OS. PHH3‐positive cell counts/10 HPF was a slightly significant independent prognostic factor for OS (HR, 4.96; 95% CI 0.93–26.55). PHH3‐positive MI and MCPyV status in MCC patients are useful in prognostication, although MCPyV‐infection is a more powerful prognostic factor in MCCs than the AJCC scheme on proliferation or mitotic indices.     

The diagnostic utility of Merkel cell polyomavirus immunohistochemistry in a fine needle aspirate of metastatic Merkel cell carcinoma of unknown primary to the pancreas

Merkel cell carcinoma (MCC) is an aggressive skin tumor with a high tendency for metastases. We report a case of MCC initially presenting as axillary and pancreatic metastases. A 33‐year‐old HIV‐positive Hispanic male presented with a history of a rapidly growing axillary mass. A needle core biopsy demonstrated an epithelioid neoplasm composed of small to medium‐sized cells with high nuclear‐cytoplasmic ratio, nuclear molding, and frequent mitotic figures. A subsequent PET scan revealed a 1.5 cm FDG avid mass in the pancreas. Endoscopic ultrasound‐guided FNA of the pancreatic mass showed neoplastic cells with similar morphology to those of the axillary mass. The tumor cells were positive with pancytokeratin AE1/AE3, CK20, CD56, synatophysin, chromogranin, and Merkel cell polyomavirus (MCPyV). This case of MCC most likely originated from a resolved primary skin lesion drained by the involved axillary lymph node with subsequent metastases to the pancreas and distant lymph nodes.     

An update on diagnostic features of Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive primary cutaneous neuroendocrine carcinoma that predominantly affects the elderly and immunosuppressed. Although rare, the incidence of MCC is increasing. Evidence suggests that the pathogenesis of MCC is related to Merkel cell polyomavirus infection or ultraviolet mutagenesis. Clinically, MCC typically presents as an asymptomatic violaceous papule on the head and neck. Histologically MCC is a small round blue cell tumor that must be differentiated from other small cell tumors, therefore immunohistochemistry is necessary for diagnosis of MCC. However, recent evidence suggests that some standard diagnostic markers may be less reliable in virus-negative MCC. MCC may develop local and distant metastases, with poor prognosis for advanced disease. Immunotherapy has recently been shown to be effective for many advanced cases. Here, we review diagnostic features of MCC including potential diagnostic and prognostic differences between virus-positive and virus-negative tumors.     

Promoter hypermethylation of the RB1 gene in glioblastomas

Loss of expression of the retinoblastoma gene (RB1) has been shown to occur in up to 25% of glioblastomas (WHO Grade IV). To elucidate the underlying mechanism, we assessed RB1 promoter hypermethylation using methylation-specific polymerase chain reaction and RB1 expression by immunohistochemistry in 35 primary (de novo) glioblastomas and in 21 secondary glioblastomas that had progressed from low-grade diffuse astrocytoma (WHO Grade II) or anaplastic astrocytoma (WHO Grade III). Promoter hypermethylation was significantly more frequent in secondary (9 of 21, 43%) than in primary glioblastomas (5 of 35, 14%; p = 0.0258). There was a clear correlation between loss of RB1 expression and promoter hypermethylation. In the majority of glioblastomas with loss of RB1 expression, there was promoter hypermethylation (11 of 13, 85%), whereas 93% of tumors with RB1 expression had a normal RB1 gene status (p < 0.0001). In three glioblastomas, areas with and without RB1 expression were microdissected; promoter hypermethylation was detected only in areas lacking RB1 expression. In patients with multiple biopsies, methylation of the RB1 promoter was not detectable in the less malignant precursor lesions, ie, low-grade diffuse and anaplastic astrocytoma. These results indicate that promoter hypermethylation is a late event during astrocytoma progression and is the major mechanism underlying loss of RB1 expression in glioblastomas.     

Meiosis error and subsequent genetic and epigenetic alterations invoke the malignant transformation of germ cell tumor

Germ cell tumors (GCTs) are thought to arise from primordial germ cells (PGCs) that undergo epigenetic reprogramming. To explore the mechanisms of GCT formation, we analyzed single‐nucleotide polymorphism array comparative genomic hybridization patterns and the methylation status of 15 tumor suppressor genes (TSGs) and differentially methylated regions (DMRs) of two imprinted genes, H19 and SNRPN, in 28 children with GCTs. Three GCTs with 25–26 segmental uniparental disomies (UPDs), heterozygous centromeric regions, and a highly methylated SNRPN DMR may have occurred through meiosis I error. Three other GCTs with whole UPD and homozygous centromeric regions of all chromosomes may have occurred through endoreduplication of a haploid set in an ovum or testis. The other 22 GCTs had heterozygous centromeric regions of all chromosomes and no or a small number of segmental or whole UPDs and may have developed from premeiotic PGCs before imprint erasure or a reestablishment of imprinting. Gain and amplification of 3p24‐p22 and 20q13‐q13, and loss and UPD of 1p36‐p35, 4q21‐q21, 5q11‐q13, and 6q26‐qter were found in five or more tumors. 1p36‐p35 loss was frequent, and found in 19 tumors; RUNX3 residing at 1p36 was methylated in the promoter regions of 16 tumors. Two yolk sac tumors with many segmental UPDs or whole UPD of all chromosomes had gain of 20q13‐q13 and loss of 1p36‐p35, and seven or eight methylated TSGs. These genetic and epigenetic alterations may have caused malignant transformation because they were rarely found in teratomas with segmental or whole UPDs. © 2012 Wiley Periodicals, Inc.     

Variability in retinoblastoma genome stability is driven by age and not heritability

Retinoblastoma (RB) is a childhood intraocular cancer initiated by biallelic inactivation of the RB tumor suppressor gene (RB1^?/?). RB can be hereditary (germline RB1 pathogenic allele is present) or non‐hereditary. Somatic copy number alterations (SCNAs) contribute to subsequent tumorigenesis. Previous studies of only enucleated RB eyes have reported associations between heritability status and the prevalence of SCNAs. Herein, we use an aqueous humor (AH) liquid biopsy to investigate RB genomic profiles in the context of germline RB1 status, age, and International Intraocular Retinoblastoma Classification (IIRC) clinical grouping for both enucleated and salvaged eyes. Between 2014 and 2019, AH was sampled from a total of 54 eyes of 50 patients. Germline RB1 status was determined from clinical blood testing, and cell‐free DNA from AH was analyzed for SCNAs. Of the 50 patients, 23 (46.0%; 27 eyes) had hereditary RB, and 27 (54.0%, 27 eyes) had non‐hereditary RB. Median age at diagnosis was comparable between hereditary (13 ± 10 months) and non‐hereditary (13 ± 8 months) eyes (P = 0.818). There was no significant difference in the prevalence or number of SCNAs based on (1) hereditary status (P > 0.56) or (2) IIRC grouping (P > 0.47). There was, however, a significant correlation between patient age at diagnosis, and (1) number of total SCNAs (r[52] = 0.672, P < 0.00001) and (2) number of highly‐recurrent RB SCNAs (r[52] = 0.616, P < 0.00001). This evidence does not support the theory that specific molecular or genomic subtypes exist between hereditary and non‐hereditary RB; rather, the prevalence of genomic alterations in RB eyes is strongly related to patient age at diagnosis.