Is Oncotype DX testing informative for breast cancers with low ER expression? A retrospective review from a biomarker testing referral center.

PURPOSE: It remains unclear whether patients with HER2-negative, low-estrogen receptor (ER-low)-positive early breast cancer (BC) benefit from Oncotype DX® (ODX) testing. METHODS: We conducted a retrospective review of cases referred for ODX testing over a seven-year period from a breast biomarker testing referral center (n = 854). For each case, we recorded the ODX Recurrence Score (RS) along with percentage of ER nuclear positivity and staining intensity on immunohistochemistry. Our criteria for ER-low was defined as ≤10% cells with nuclear positivity and/or weak intensity of staining. Slides from all ER-low cases were reviewed and the reported ODX ER gene scores were recorded. We randomly selected a comparator group of 56 patients with ER > 10% positivity and non-weak staining intensity (ER-high). RESULTS: We identified 27 cases (3.2%) that met our criteria for ER-low. Of these, 92.6% had a high RS (>25), and 7.4% had a RS of 25. All cases with ≤10% ER nuclear positivity had a high RS. Most ER-low cases (85.2%) had ODX quantitative ER gene scores in the negative range, whereas all (100%) ER-high cases had positive ER gene scores. CONCLUSION: ODX does not appear to add significant additional information to inform treatment decisions for most patients with ER-low BC. Incorporating weak ER staining intensity in addition to low percentage of nuclear positivity identifies about twice as many ER-low patients, although with reduced specificity for high RS. Our study supports the contention that most ER-low early BC should be regarded similarly to ER-negative BC.

Barriers and Unequal Access to Timely Molecular Testing Results: Addressing the Inequities in Cancer Care Delays across Canada.

Genomic medicine is a powerful tool to improve diagnosis and outcomes for cancer patients by facilitating the delivery of the right drug at the right dose at the right time for the right patient. In 2023, a Canadian conference brought together leaders with expertise in different tumor types. The objective was to identify challenges and opportunities for change in terms of equitable and timely access to biomarker testing and reporting at the education, delivery, laboratory, patient, and health-system levels in Canada. Challenges identified included: limited patient and clinician awareness of genomic medicine options with need for formal education strategies; failure by clinicians to discuss genomic medicine with patients; delays in or no access to hereditary testing; lack of timely reporting of results; intra- and inter-provincial disparities in access; lack of funding for patients to access testing and for laboratories to provide testing; lack of standardized testing; and impact of social determinants of health. Canada must standardize its approach to biomarker testing across the country, with a view to addressing current inequities, and prioritize access to advanced molecular testing to ensure systems are in place to quickly bring innovation and evidence-based treatments to Canadian cancer patients, regardless of their place of residence or socioeconomic status.

Screening and testing practices for Lynch syndrome in Nova Scotians with endometrial cancer: a descriptive study.

BACKGROUND: Identifying people with Lynch syndrome, a genetic condition predisposing those affected to colorectal, endometrial and other cancers, allows for implementation of risk-reducing strategies for patients and their families. The goal of this study was to describe screening and testing practices for this condition among people with endometrial cancer in Nova Scotia, Canada, and to determine the prevalence of Lynch syndrome in this population. METHODS: All patients diagnosed with endometrial cancer in Nova Scotia between May 1, 2017, and Apr. 30, 2020 were identified through a provincial gynecologic oncology database. Patients from out of province were excluded. We collected age, body mass index, tumour mismatch repair protein immunohistochemistry results, personal and family histories, and germline testing information for all patients. RESULTS: We identified 465 people diagosed with endometrial cancer during the study period. Most were aged 51 years or older, and had obesity and low-grade early-stage endometrioid tumours. Tumour immunohistochemistry testing was performed in 444 cases (95.5%). Based on local criteria, 189 patients were eligible for genetic counselling, of whom 156 (82.5%) were referred to medical genetics. Of the 98 patients who underwent germline testing, 9 (9.2%) were diagnosed with Lynch syndrome. INTERPRETATION: The prevalence of Lynch syndrome was at least 1.9% (9/465) in this population. Our results illustrate successful implementation of universal tumour testing; however, there remains a gap in access to genetic counselling.

Merkel cell carcinoma: an update.

Merkel cell carcinoma (MCC) is an uncommon primary cutaneous neuroendocrine carcinoma associated with an adverse prognosis. In recent years, our understanding of MCC biology has markedly progressed. Since the discovery of the Merkel cell polyomavirus, it has become clear that MCC represents an ontogenetically dichotomous group of neoplasms with overlapping histopathology. Specifically, most MCCs arise secondary to viral oncogenesis, while a smaller subset is the direct result of UV-associated mutations. The distinction of these groups bears relevance in their immunohistochemical and molecular characterization, as well as in disease prognosis. Further recent developments relate to the landmark utilization of immunotherapeutics in MCC, providing optimistic options for the management of this aggressive disease. In this review, we discuss both fundamental and emerging concepts in MCC, with a particular focus on topics of practical relevance to the surgical or dermatopathologist.

RB1-deficient squamous cell carcinoma: the proposed source of combined Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine (NE) carcinoma arising from integration of Merkel cell polyomavirus (MCPyV) DNA into a host cell or from ultraviolet light-induced genetic damage (proportions vary geographically). Tumors in the latter group include those with "pure" NE phenotype and those "combined" with other elements, most often squamous cell carcinoma (SCC). We performed comprehensive genomic profiling (CGP) of MCPyV+ and MCPyV- (pure and combined) tumors, to better understand their mutational profiles and shed light on their pathogenesis. Supplemental immunohistochemistry for Rb expression was also undertaken. After eliminating low quality samples, 37 tumors were successfully analyzed (14 MCPyV+, 8 pure MCPyV- and 15 combined MCPyV-). The SCC and NE components were sequenced separately in 5 combined tumors. Tumor mutational burden was lower in MCPyV+ tumors (mean 1.66 vs. 29.9/Mb, P < 0.0001). MCPyV- tumors featured frequent mutations in TP53 (95.6%), RB1 (87%), and NOTCH family genes (95.6%). No recurrently mutated genes were identified in MCPyV+ tumors. Mutational overlap in the NE and SCC components of combined tumors was substantial ('similarity index' >24% in 4/5 cases). Loss of Rb expression correlated with RB1 mutational (P < 0.0001) and MCPyV- status (P < 0.0001) in MCCs and it was observed more frequently in the SCC component of combined MCC than in a control group of conventional cutaneous SCC (P = 0.0002). Our results (i) support existing evidence that MCPyV+ and MCPyV- MCCs are pathogenetically distinct entities (ii) concur with earlier studies linking the NE and SCC components of combined MCCs via shared genetic profiles and (iii) lend credence to the proposal that an Rb-deficient subset of SCC's is the source of phenotypically divergent combined MCCs.

Morphological patterns of metastases from combined Merkel cell carcinomas: study of an eastern Canadian cohort of cases.

Combined Merkel cell carcinomas are hybrid tumors composed of neuroendocrine and other phenotypic (usually squamous) elements. They form a minority of Merkel cell carcinomas (MCCs) as a whole, are usually Merkel cell polyomavirus-negative, and have rarely been segregated for specific study. Sporadic reports have indicated that metastases from these tumors can show a combined phenotype. We retrospectively studied 38 cases (24 men [63%], 14 women [37%], mean age 78 years [range, 46-99 years]) of combined MCC. Metastases occurred in 20 patients (53%) (at presentation and/or in follow-up [mean 38 months (range, 0.6-185 months)]). Those from 17 individuals (45%) were examined microscopically. These were mainly nodal in distribution. In 12 patients (71%), the secondary deposits were of pure neuroendocrine type, whereas in 5 (29%), combined deposits were identified. Squamous elements were the most common divergent component, in the primary and secondary tumors. The combined metastases varied from obvious squamous nests in a neuroendocrine background to scattered bizarre tumor giant cells expressing CK5/6 on immunohistochemistry. In one case, individual nodes within a single basin displayed purely squamous or purely neuroendocrine deposits. The mean overall survival in the cohort was 48 months (range, 30-67 months) and the mortality was 82%. Our work sheds light on the frequency and patterns of metastases in combined MCCs. In concert with the poor outcome data documented by others, it also raises a question as to the potential prognostic significance of a combined phenotype per se, independent of a virus-negative status and other variables. This issue deserves further study.

PAXgene Fixation for Pancreatic Cancer: Implications for Molecular and Surgical Pathology.

Genomic profiling of pancreatic cancer using small core biopsies has taken an increasingly prominent role in precision medicine. However, if not appropriately preserved, nucleic acids (NA) from pancreatic tissues are known to be susceptible to degradation due to high intrinsic levels of nucleases. PAXgene fixation (PreAnalytix, Switzerland) represents a novel formalin-free tissue preservation method. We sought to compare the NA and histomorphological preservation of pancreatic cancer tissues preserved with PAXgene-fixed paraffin-embedding (PFPE) and formalin-fixed paraffin-embedding (FFPE). Tissues from 19 patients were obtained prospectively from pancreaticoduodenectomy specimens and evaluated by four gastrointestinal pathologists. The extracted NA were quantified by Nanodrop and Qubit and assessed for quality by qPCR, targeted next-generation sequencing (NGS) assay, and RNA-sequencing. Our results demonstrated that, when assessed blindly for morphological quality, the four pathologists deemed the PFPE slides adequate for diagnostic purposes. PFPE tissues enable greater yields of less fragmented and more amplifiable DNA. PFPE tissues demonstrated significantly improved quality control (QC) metrics in a targeted NGS assay including Median Absolute Pair-wise Difference (MAPD) scores. Our results support the use of PAXgene fixative for the processing of specimens from pancreatic cancers with the potential benefits of improved yields for more amplifiable DNA in low-yield biopsy specimens and its ideal use for amplicon-based NGS assays.

Optimizing Shape Complementarity Enables the Discovery of Potent Tricyclic BCL6 Inhibitors.

To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.

Alzheimer's disease as an autoimmune disorder of innate immunity endogenously modulated by tryptophan metabolites.

Introduction: Alzheimer's disease (AD) is characterized by neurotoxic immuno-inflammation concomitant with cytotoxic oligomerization of amyloid beta (Aß) and tau, culminating in concurrent, interdependent immunopathic and proteopathic pathogeneses. Methods: We performed a comprehensive series of in silico, in vitro, and in vivo studies explicitly evaluating the atomistic-molecular mechanisms of cytokine-mediated and Aß-mediated neurotoxicities in AD.  Next, 471 new chemical entities were designed and synthesized to probe the pathways identified by these molecular mechanism studies and to provide prototypic starting points in the development of small-molecule therapeutics for AD. Results: In response to various stimuli (e.g., infection, trauma, ischemia, air pollution, depression), Aß is released as an early responder immunopeptide triggering an innate immunity cascade in which Aß exhibits both immunomodulatory and antimicrobial properties (whether bacteria are present, or not), resulting in a misdirected attack upon "self" neurons, arising from analogous electronegative surface topologies between neurons and bacteria, and rendering them similarly susceptible to membrane-penetrating attack by antimicrobial peptides (AMPs) such as Aß. After this self-attack, the resulting necrotic (but not apoptotic) neuronal breakdown products diffuse to adjacent neurons eliciting further release of Aß, leading to a chronic self-perpetuating autoimmune cycle.  AD thus emerges as a brain-centric autoimmune disorder of innate immunity. Based upon the hypothesis that autoimmune processes are susceptible to endogenous regulatory processes, a subsequent comprehensive screening program of 1137 small molecules normally present in human brain identified tryptophan metabolism as a regulator of brain innate immunity and a source of potential endogenous anti-AD molecules capable of chemical modification into multi-site therapeutic modulators targeting AD's complex immunopathic-proteopathic pathogenesis. Discussion:  Conceptualizing AD as an autoimmune disease, identifying endogenous regulators of this autoimmunity, and designing small molecule drug-like analogues of these endogenous regulators represents a novel therapeutic approach for AD.

CAP-ACP Workload Model for Advanced Diagnostics in Precision Medicine.

OBJECTIVES: In precision medicine, where oncologic management is tailored to the individual's clinical and genetic profiles, advanced diagnostic testing provides prognostic information and guides management in a growing number of malignancies. There is a need to capture the work pathologists perform to meet this demand by providing medically relevant, timely, and accurate testing results. This work includes not only direct patient consults (interpretation of results and issuing reports) but the administrative and medical oversight as well as the research needed to provide the necessary quality assurance, quality control, direction, and framework for the laboratory. METHODS: An expert panel of Canadian pathologists involved in advanced diagnostics was convened to establish and beta test a model for workload assessment in advanced diagnostics. RESULTS: All aspects of the advanced diagnostics workload were detailed and applied to models based on members' experience, including medical oversight, administration, and the introduction of new testing and platforms. Models for biomarker testing were developed for simple and complex or multiplexed assays, and a detailed model was developed to assess the workload for next-generation sequencing-based assays. CONCLUSIONS: This paper provides the first detailed proposal for capturing an advanced diagnostic workload to enable appropriate pathologist allotment for performing all the steps required to run an advanced diagnostic service.

Relationship between p63 and p53 expression in Merkel cell carcinoma and corresponding abnormalities in TP63 and TP53: a study and a proposal.

Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous neuroendocrine carcinoma. Oncogenesis occurs via Merkel cell polyomavirus-mediated (MCPyV+) and/or ultraviolet radiation-associated (MCPyV-) pathways. Advanced clinical stage and an MCPyV- status are important adverse prognostic indicators. There is mounting evidence that p63 expression is a negative prognostic indicator in MCC and that it correlates with MCPyV- status. p63 is a member of the p53 family of proteins among which complex interactions occur. It has two main isoforms (proapoptotic TAp63 and oncogenic ΔNp63). Paradoxically, TAp63 predominates in MCC. To explore this quandary, we examined relationships between p63 and p53 expression and corresponding abnormalities in the TP63 and TP53 genes in MCC. A cohort of 26 MCCs (12 MCPyV+ and 14 MCPyV-) was studied. Comparative immunohistochemical expression of p63 and p53 was evaluated semiquantitatively (H scores) and qualitatively (aberrant patterns). The results were compared with genetic abnormalities in TP63 and TP53 via next-generation sequencing. p63 was positive in 73% of cases. p53 showed "wild-type" expression in 69%, with "aberrant" staining in 31%. TP63 mutations (predominantly low-level copy gains; 23% of cases) and mainly pathogenic mutations in TP53 (50% of cases) featured in the MCPyV- subset of cases. p63 expression correlated quantitatively with p53 expression and qualitatively with aberrant patterns of the latter. Increased expression of p63 and p53 and aberrant p53 staining correlated best with TP53 mutation. We propose that p63 expression (ie, proapoptotic TAp63) in MCC is most likely functionally driven as a compensatory response to defective p53 tumor suppressor activity.

Independent primary cutaneous and mammary apocrine carcinomas with neuroendocrine differentiation: Report of a case and literature review.

Cutaneous apocrine carcinomas share common features with their counterparts in the breast; hence, metastatic mammary carcinoma must be excluded before such lesions can be designated primary cutaneous neoplasms. Primary tumors from either source rarely exhibit neuroendocrine differentiation. We report a case of a 72-year-old female with a painless 1.2-cm scalp nodule. An incisional biopsy revealed dermal involvement by an invasive apocrine carcinoma juxtaposed to a benign apocrine cystic lesion. Immunohistochemically, the carcinoma expressed neuroendocrine proteins including synaptophysin, chromogranin, and CD56. A primary cutaneous apocrine carcinoma with neuroendocrine differentiation was favored, but additional investigations to exclude breast origin were recommended. These revealed a 1.1-cm nodule in the right breast, which proved to be an invasive ductal carcinoma, morphologically and immunophenotypically similar to the scalp lesion. This confounded the case, yet factors militating against metastatic breast carcinoma to skin included (a) the small size of the mammary tumor, (b) absence of other metastatic disease, and (c) juxtaposition of the scalp carcinoma to a putative benign precursor. Molecular studies were undertaken to resolve the diagnostic quandary. Single nucleotide polymorphism microarray analysis revealed distinct patterns of chromosomal copy number alterations in the two tumors, supporting the concept of synchronous and unusual primary neoplasms.

DNA copy number changes correlate with clinical behavior in melanocytic neoplasms: proposal of an algorithmic approach.

Increasingly, molecular methods are being utilized in the workup of melanocytic neoplasms. To this end, we sought to correlate data from a single nucleotide polymorphism (SNP) array platform based on molecular inversion probes with clinical data. Copy number variation (CNV) data were obtained on 95 melanocytic tumors (6 ordinary nevi, 15 atypical nevi, 34 ambiguous neoplasms, and 40 melanomas) from 92 patients. The average number of significant CNVs was 0 for nevi, 0.6 for atypical nevi (range 0-3), 2.8 for ambiguous neoplasms (range 0-17), and 18.1 for melanomas (range 0-69). Clinical follow-up data were available in 57 of 95 lesions (56 of 92 patients). Tumors from patients with adverse events demonstrated an average number of CNVs of 24.5 (range 6-69) as compared with 7.9 (range 0-35) among tumors without an associated adverse event (p ≤ 0.001). No adverse events were observed in nevi including atypical nevi. Adverse events were found in 2 of 19 ambiguous neoplasms and 10 of 32 melanomas with follow up. In these two latter groups of neoplasms, the correlation between adverse events and the average number of CNVs remained statistically significant even when controlled for Breslow depth (21.5 versus 8.7, p value = 0.036). No neoplasm with adverse events had ≤3 CNVs. These results provide further evidence that SNP array testing for CNVs may be helpful in the classification and prognostication of ambiguous neoplasms. Based on these results, an algorithmic approach to challenging melanocytic neoplasms using CNV data is suggested, using as cutoff of >3 CNVs with some caveats, as the threshold for a positive result. Future clinical validation, using a larger cohort of relevant tumors, will be necessary.

p63 expression in Merkel cell carcinoma: comparative immunohistochemistry invokes TAp63 as the dominant isoform involved.

The literature suggests that p63 expression in Merkel cell carcinoma (MCC) is associated with a poor prognosis. p63 immunohistochemistry marks the 2 main isoforms of this transcriptional protein: TAp63 (tumor suppressor-like properties) and ∆Np63 (oncogenic properties). Little information about the isoform of relevance in MCC exists. p40 immunohistochemistry specifically marks ∆Np63, and using comparative, semiquantitative expression of p63 and p40, we sought to clarify the issue. Our cohort of 53 cases (28 men and 25 women, median age 79 years, interquartile range 71-88) was stratified by morphology and viral status. Immunohistochemistry (p63, p40, and cytokeratin 5/6) was performed, H-scores for nuclear expression of p63 and p40 were derived (2 observers; positivity ≥ 10), and interobserver agreement was evaluated. Clinical, pathological, and outcome data were documented. The results were analyzed statistically. Mortality amounted to 57% (median follow-up 686 days, interquartile range 292-1599). Positivity for Merkel cell polyomavirus was observed in 29 (55%) of cases. Expression of p63 and p40 was present in 36 (69%) and 4 (8%) of cases, respectively. Increased age (P = .0241), negative Merkel cell polyomavirus status (P = .0185), and p63 positivity (P = .0012) were significantly associated with mortality. The latter 2 variables were highly correlated (P = .004). The interclass correlation between the 2 sets of H-scores was 0.95. Our findings support an association between p63 expression and reduced overall survival in MCC and show consistency in scoring this prognostic parameter. TAp63 is the dominant isoform of the protein involved. The paradoxical tumor suppressor-like activity of this isoform in p63-positive MCCs with reduced overall survival requires further study.

Global PD-L1 Signals and Tumor-Infiltrating Lymphocytes: Markers of Immunogenicity in Different Subsets of Merkel Cell Carcinoma and Potential Therapeutic Implications.

We previously studied the genetic and immunohistochemical profiles of subsets of Merkel cell carcinoma (MCC) stratified by morphology and Merkel cell polyomavirus (MCPyV) status. Recent advances in the immunotherapy of this disease prompted us to examine markers of immunogenicity [PD-L1 expression and tumor-infiltrating lymphocytes (TILS) in these subsets]. The observed clinical responses to checkpoint inhibition of the PD-1/PD-L1 pathway have not correlated with PD-L1 expression by MCC cells, and recent evidence suggests that functions of this pathway within the immune tumor microenvironment may be relevant. We conducted a semiquantitative (high, moderate, and minimal) immunohistochemical evaluation of the global PD-L1 signal in 52 cases of MCC, segregated in 3 subsets [pure MCPyV-positive (n = 28), pure MCPyV-negative (n = 9), and combined MCPyV-negative (n = 15)]. TILS were categorized as brisk, nonbrisk, or absent. Intersubset comparisons revealed that high global PD-L1 signals were exclusively associated with pure MCPyV-positive MCCs contrasted with virus-negative cases (P = 0.0003). Moderate signals were seen across all 3 groups. Brisk TILS were significantly associated with MCPyV-positive MCCs compared with MCPyV-negative cases (P = 0.029). Neither parameter (PD-L1 or TILS) was significantly different between the MCPyV-negative groups. Of potential clinical relevance, MCPyV seems to convey greater immunogenicity to MCCs than the high mutational burden/greater neoantigen load of MCPyV-negative cases. Interesting too is the fact that subset-related profiles of these markers mirrored those noted at genetic and immunohistochemical levels, separating pure MCPyV-positive MCCs from the virus-negative subsets.

Inhibition of Pantothenate Synthetase by Analogs of ß-Alanine Precursor Ineffective as an Antibacterial Strategy.

BACKGROUND: Pantothenate, the fundamental precursor to coenzyme A, is required for optimal growth and virulence of microbial pathogens. It is synthesized by the enzyme-catalyzed condensation of ß-alanine and pantoate, which has shown susceptibility to inhibition by analogs of its molecular constituents. Accordingly, analogs of ß-alanine are gaining inquiry as potential antimicrobial chemotherapeutics. METHODS: We synthesized and evaluated 35 derivatives of ß-alanine, substituted at the α, ß, amine, and carboxyl sites, derived from in silico, dynamic molecular modeling to be potential competitive inhibitors of pantothenate synthetase. Employing the Clinical Laboratory Standards M7-A6 broth microdilution method, we tested these for inhibition of growth in Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. RESULTS: All compounds proved entirely ineffective in all species tested, with no inhibition of growth being observed up to 200 µM/mL. CONCLUSIONS: Upon revision of the literature, we conclude that high enzyme selectivity or external salvage mechanisms may render this strategy futile against most bacteria.

EWSR1-PBX3 gene fusion in cutaneous syncytial myoepithelioma.

Cutaneous syncytial myoepithelioma (CSM) is a recently recognized, histopathological variant of myoepithelial (ME) tumors of the skin. It is characterized by a syncytial arrangement of spindled, ovoid, and/or epithelioid cells forming a well-circumscribed, unencapsulated dermal nodule. There is a paucity of intervening stroma, and absent duct or gland formation. Strong immunohistochemical staining for S100 and epithelial membrane antigen (EMA) has been described, while cytokeratin expression has been uncommon. The majority of CSMs harbor a rearrangement involving the EWSR1 gene. Although various fusion partner genes have been discovered in ME tumors at other anatomic sites, none has yet been described in CSM. We present a case of CSM represented clinically by a papule on the mid-upper back of a healthy 44-year-old female. It exhibited morphological and immunohistochemical features of a CSM with strong, diffuse S100 and alpha-actin expression, and focal positivity for EMA and cytokeratin AE1/AE3. Fluorescence in-situ hybridization showed an EWSR1 gene rearrangement. Massively parallel next-generation RNA sequencing revealed PBX3 as the fusion partner. The EWSR1-PBX3 gene fusion has been previously identified in three cases of ME tumors of bone and soft tissue, and in a case of retroperitoneal leiomyoma. This is the first report of an EWSR1-PBX3 fusion in CSM.

Molecular testing of borderline cutaneous melanocytic lesions: SNP array is more sensitive and specific than FISH.

Melanocytic lesions with borderline features are diagnostically challenging. Single-nucleotide polymorphism (SNP) arrays, which detect genomic copy number alterations (CNAs), can be helpful in distinguishing between nevi and melanoma. Fluorescence in situ hybridization (FISH) has been used as a more rapid, less expensive alternative to SNP array, using a panel of probes that are often gained or lost in melanoma. We used SNP array data from 63 borderline cutaneous melanocytic lesions and 44 definitive melanomas to predict the performance of FISH testing. Lesions were considered positive by "virtual FISH" if 1 or more of the 5 FISH-probed loci demonstrated appropriate CNAs by SNP array. Cases were classified as positive by SNP array if ≥3 CNAs were present, based on internal validation studies, or if FISH criteria were met. Conventional FISH was performed in 33 cases (17 borderline lesions, 16 melanomas). Of the 63 borderline cases, 44 (70%) were positive by SNP array and 30 (48%) were positive by virtual FISH. A higher proportion of melanomas were positive by SNP array (41/44, 93% sensitivity) and virtual FISH (36/44, 82% sensitivity). Virtual FISH had 61% sensitivity in the borderline group using SNP array as the gold standard, whereas specificity was 84%. There was good correlation between conventional and virtual FISH, with agreement in 30 of 33 (91%) cases. Although FISH is highly effective in distinguishing between nevi and melanoma in cases where the histological diagnosis is straightforward, it is not nearly as sensitive or specific as SNP array when applied to borderline lesions.

Immunohistochemical profiles of different subsets of Merkel cell carcinoma.

The literature records many examples of Merkel cell carcinoma (MCC) exhibiting aberrant immunohistochemical profiles. These can lead to diagnostic difficulty. The objectives of the current study were (1) to examine the immunohistochemical profile of different subsets of MCC to determine whether predictable subset-specific patterns exist and (2) to establish whether shared immunophenotypic patterns might reveal links between individual subsets, as demonstrated previously at a genetic level. In 52 cases of MCC, stratified by viral status and morphology, we studied 5 markers commonly used in the diagnostic evaluation of these tumors (CK20, CK7, chromogranin, neurofilament and TTF-1). Expression of these proteins was recorded as quantitative (H-scores) and absolute (positive vs negative) variables. In general, our data indicate that the "classical" or expected panel (CK20+, NF+, Chromo+, TTF-1, CK7-) is observed significantly more often in pure Merkel cell polyomavirus (MCPyV)-positive than in MCPyV-negative cases (78% vs 25%; P = .002). Neurofilament was less frequently encountered in MCPyV-negative than in MCPyV-positive tumors (66.7% vs 100%; P = .001) and expression of TTF-1 (37.5% vs 3.6%; P = .003) and CK7 (45.8 vs 14.3; P = .02) was more frequent. No significant immonophenotypic differences were observed between pure and combined MCPyV-negative tumors. Recognition of the more aberrant immunohistochemical profile of MCPyV-negative MCC should inform the diagnostic approach to this tumor. Moreover, the shared aberrant immunophenotype in pure and combined MCPyV-negative tumors supports a link between these entities and serves to separate them from MCPyV-positive tumors.

Florid Granuloma Annulare-Like Reaction in Regional Lymph Nodes After "Regression" of Red Pigment in Tattoos.

A healthy 50-year-old woman had a tattoo performed on the posterior aspect of her neck and another on the dorsum of her left foot. Several weeks later, she noted redness, tenderness, and intense pruritis at both tattoo sites. Treatment with cephalexin and hydrocortisone cream was instituted, without success. Within a few months, the red, but not black, pigment had disappeared from both tattoos and was replaced by pale areas of scarring. Persistently enlarged left supraclavicular and suboccipital lymph nodes were excised 7 and 10 months after receipt of the tattoos, respectively. The nodes were pigmented on gross examination, and on microscopy, a granuloma annulare-like reaction was observed. Normal lymphoid tissue was seen to be replaced by large palisading granulomas with central degenerative change, abundant stromal mucin, and scattered deposits of tattoo pigment. Histochemical stains, tissue culture, and serological studies revealed no evidence of infection. There are rare reports of granuloma annulare-like reactions in tattoos, and these are believed to represent delayed-type hypersensitivity reactions. Our case is unique in the observation of this reaction pattern in regional lymph nodes, and it expands the spectrum of complications known to be associated with tattoos.