Tumoral PD-L1 expression in desmoplastic melanoma is associated with depth of invasion, tumor-infiltrating CD8 cytotoxic lymphocytes and the mixed cytomorphological variant.

Recently, patients with metastatic desmoplastic melanoma (DM) have been shown to respond more favorably to anti-PD1/PD-L1 therapy than other melanoma subtypes. Given this, we evaluated PD-L1/2 expression in primary DM samples and correlated these with subtype, CD8+ lymphocyte status, histopathological prognosticators, and select genetic alterations. Eighty-six (36 mixed DM, 50 pure DM) archival annotated samples met inclusion criteria and were immunohistochemically semiquantitatively evaluated. Per established criteria, for PD-L1/L2, cases with ⩾5% tumoral expression, and for CD8, cases with a predominantly peri/intratumoral CD8+ infiltrate were scored positive. Univariate analysis (chi-square and Wilcoxon) identified potential confounders and a nested case-control study was accomplished using multiple logistic regression. For PD-L1, 49% of cases were positive and 71% of cases with thickness >4 mm were positive; PD-L1 expression differed by median depth (3.29 mm, interquartile range=3.58 mm for PD-L1 positives vs 1.75 mm, interquartile range=2.04 mm for PD-L1 negatives, P=0.0002) and was linearly associated with increasing depth of invasion (P=0.0003). PD-L1-positive cases were more likely to display CD8+ lymphocytes (60 vs 28% P=0.0047).The presence of CD8+ lymphocytes correlated significantly with depth of invasion >1 mm (P=0.022). On multivariate analysis, PD-L1 was 6.14 × more likely to be expressed in mixed DM than pure DM (P=0.0131), CD8+ staining was 6.22 × more likely in PD-L1 positive cases than in PD-L1 negative (P=0.0118), and tumor depth was associated with greater odds of PD-L1 expression (OR=1.61, P=0.0181). PD-L2 expression was observed in 48% of cases but did not correlate with any variables. Correlation of tumoral PD-L1 with increased depth and CD8+ lymphocytes implicates the tumoral immune microenvironment with advancing disease in DM. Enhanced tumoral PD-L1 expression in the mixed cytomorphological variant provides an insight into the differential pathogenesis of the subtypes and suggests that these patients are likely better candidates for anti-PD/PD-L1 therapy.

Frequency of telomerase reverse transcripter promoter mutations in desmoplastic melanoma subtypes: analyses of 76 cases.

Estimates of the frequency of telomerase reverse transcripter (TERT) mutations in desmoplastic melanoma (DM) are limited. DM is categorized into subtypes, pure and mixed, differing in prognosis, suggesting genetic heterogeneity. Given this, our aims were to determine the incidence of TERT promoter mutations in DM subtypes and to evaluate its relationship with established histopathologic prognosticators, BRAF and RETp status, and neurofibromin protein expression. Of the archival annotated samples retrieved, 76 cases of DM (48 pure and 28 mixed) fulfilled the criteria for inclusion. PCR amplification of the TERT promoter region was performed on DNA extracted from formalin-fixed paraffin-embedded tissue using primers5'-GCCGATTCGACCTCTCTCC-3' (forward) and 5'-CAGCGCTGCCTGAAACTC-3' (reverse). For each case, appropriate C>T mutations were identified on the electropherograms. Univariate analysis using χ-test was carried out to identify potential confounders; a nested case-control study of demographic, clinical, histopathological, and genetic determinants was carried out using multiple logistic regression. Significant differences in TERT promoter mutation frequencies were noted in the subtypes (mixed vs. pure; 15/28, 54% vs. 11/48, 23%, respectively, P=0.0066). After adjusting for potential confounding, multivariate analyses indicated a three-fold increase in the odds of the TERT mutation for those with the mixed subtype compared with the pure subtype (P=0.04, adjusted odds ratio =3.32). No other significant associations were noted (sex/junctional component/Breslow depth/ulceration/mitoses/host response/RETp, BRAF status, and neurofibromin protein expression). Our findings, the largest to date investigating TERT promoter mutations in DM, support the hypothesis that the subtypes have distinct genetic drivers and underscore the relevance of telomere integrity in the etiopathogenesis of the mixed variant.

Neurofibromin protein loss in desmoplastic melanoma subtypes: implicating NF1 allelic loss as a distinct genetic driver?

Loss of the NF1 allele, coding for the protein neurofibromin, and polymorphism in the proto-oncogene RET (RETp) are purportedly common in desmoplastic melanoma (DM). DM is categorized into pure (PDM) and mixed (MDM) subtypes, which differ in prognosis. Most NF1 mutations result in a truncated/absent protein, making immunohistochemical screening for neurofibromin an ideal surrogate for NF1 allelic loss. Using antineurofibromin, our aims were to ascertain the incidence of neurofibromin loss in DM subtypes and to evaluate the relationship with RET, perineural invasion (PNI) and established histopathologic prognosticators. A total of 78 archival samples of DM met criteria for inclusion (54 cases of non-DM serving as controls). Immunohistochemistry was performed for neurofibromin, whereas direct DNA sequencing was used for RETp and BRAF mutation status. Statistical analyses included χ(2) test as well as Fisher exact test. Neurofibromin loss was more common in DM than non-DM (69% versus 54%; P=.02). In DM, significant differences in neurofibromin loss were noted in the following: non-head and neck versus head and neck biopsy site (88% versus 55%) and PDM versus MDM variants (80% versus 56%). No significant associations were noted with sex, presence of a junctional component, Breslow depth, ulceration, mitoses, host response, RETp, BRAF status, or PNI. RETp was marginally associated with PNI-positive DM versus PNI-negative DM (36 versus 18%; P=.08). Our findings, the largest to date investigating neurofibromin in DM, validate the incidence of NF1 mutations/allelic loss in DM and suggest that the DM subtypes have distinct genetic drivers.

Melan-A positive dermal cells in malignant melanoma in situ.

The presence of Melan-A positive dermal cells in excisions for melanoma in situ represents a frequent conundrum for pathologists. These cells may represent superficially invasive melanoma, benign, incidental, dermal nevi or non-specific staining of dermal melanophages. Occasionally, rare, Melan-A positive dermal cells are present which do not clearly correspond to the above three categories. Our objective was to further characterize these Melan-A positive dermal cells. To do this, immunoperoxidase staining for Melan-A and SOX-10 was performed on 188-cutaneous excisions, including examples of melanoma in situ, atypical junctional melanocytic hyperplasia and non-melanocytic tumors. These were evaluated for the presence of Melan-A and SOX-10 positive dermal cells. Dermal cells, positive for both markers, were identified in 17% of the excisions. The cells were present in 10% of cases from the melanocytic group and 31% of the cases from the non-melanocytic group. These cells did not exhibit cytologic atypia and resembled neither the co-existing neoplasm nor melanophages. We conclude that positivity of these rare Melan-A positive cells for SOX-10 argues that they represent true melanocytes and not non-specific staining. The absence of cytologic atypia in these cells and their presence in excisions of non-melanocytic neoplasms argues that they are benign, reactive, dermal melanocytes.

Microvessel density, lymphovascular density, and lymphovascular invasion in primary cutaneous melanoma-correlation with histopathologic prognosticators and BRAF status.

The relationship between microvessel density (MVD), lymphovascular density (LVD), and lymphovascular invasion (LVI) in primary cutaneous melanoma (PCM) remains unclear. Given this, a total of 102 PCMs were assessed for MVD (vascular endothelial growth factor receptor 2 and Endocan), LVD (D2-40), and LVI (immunostaining with D2-40/S-100 and hematoxylin and eosin); tumoral S-100A13, vascular endothelial growth factor receptor 2, and Endocan; and BRAF status. LVD was associated with MVD (P = .01). MVD was higher in PCMs with depth greater than or equal to 2 mm and ulceration (P = .04, .05), whereas LVD was higher in PCMs with depth greater than or equal to 2 mm and mitoses (P = .03, .02). After adjusting for MVD and LVD, only ulceration was associated with LVI (P < .02). A BRAF mutation was seen in 30.4% cases, and when present, both LVD and host response (P = .0008 and .04, respectively) were significantly associated with MVD. Immunostaining with S-100A13 was noted in 99% of cases and a significant association noted only with ulceration (P = .05). Immunostaining increased LVI positivity (46.5% versus 4.9% by hematoxylin and eosin, P < .0001). MVD and LVD are not associated with LVI, appear to be closely related with each other, and are associated with select markers of poor prognosticative value. The association between a host response and LVD and MVD in PCMs with a BRAF mutation suggests that they exhibit potential for strategizing immunotherapies.

Reactive granular histiocytosis secondary to arthroplasty prosthesis: a novel reaction pattern.

A reactive histiocytic infiltrate can be seen as an incidental finding in a lymph node biopsy from a patient with a history of joint arthroplasty. We report the case of a 74-year-old female who underwent surgical revision of a polyethylene-based right total knee prosthesis due to chronic wear. At the time of surgery, a soft tissue mass adjacent to the tibial prosthetic insert was noted and excised. Histopathologic examination revealed a sheet-like proliferation of large, histiocytoid cells within the subcutis and superficial fascia. The cells showed abundant eosinophilic, granular cytoplasm and small round bland nuclei. Immunohistochemical evaluation revealed the cells to be positive only for CD68. In addition, abundant PAS-positive cytoplasmic granules were found, and minute particles of polarizable material were noted intracellularly and scattered throughout the interstitium of the infiltrate. These findings were interpreted as consistent with a reactive, non-Langerhans cell histiocytosis secondary to the patient's polyethylene knee prosthesis. This finding appears to be a local correlate of the process previously described in regional lymph nodes as reactive granular histiocytosis. Dermatopathologists should be cognizant of this uncommon reaction pattern to avoid mistaking it for a neoplastic process.

Cutaneous metastasis of prostatic adenocarcinoma: a cautionary tale.

With the exception of skin cancer, prostatic adenocarcinoma represents the most common cancer among men in the United States and the second most common cause of cancer mortality. Mortality is often associated with metastatic disease, which in the case of prostatic adenocarcinoma typically involves bones and only rarely affects the skin. Although clinical history and examination, laboratory tests and routine pathology can suggest the prostate as a source of metastatic disease, immunohistochemistry - specifically, for prostate-specific antigen (PSA) - is often used to help establish the diagnosis. We report a case of cutaneous metastatic prostatic adenocarcinoma presenting in the inguinal region of a 78-year-old man 5 years after his initial diagnosis. The case is unusual in that the clinical appearance mimicked a vascular proliferation and in that the metastatic prostatic adenocarcinoma failed to express PSA. Rather, expression of prostatic acid phosphatase was observed.

Stem cell markers (cytokeratin 15, cytokeratin 19 and p63) in in situ and invasive cutaneous epithelial lesions.

The inherent longetivity of stem cells causes them to be susceptible to multiple genetic hits. Thus, it is not surprising that stem cells are implicated in the etiopathogenesis of select cutaneous neoplasms. However, most studies to date are restricted to the use of a single marker (p63, cytokeratin-15 or cytokeratin-19) and do not appear to compare distribution of stem cell markers in a spectrum of cutaneous in situ versus invasive epithelial malignancies. In this study, we evaluate expression of cytokeratin-15, cytokeratin-19, and p63 in a series of primary cutaneous epithelial lesions that include actinic keratosis (n=29), squamous cell carcinoma in situ (n=30), bowenoid papulosis (n=15) and squamous cell carcinoma, well differentiated (n=29) in order to evaluate the role of stem cell marker expression in the grading and development of in situ and invasive malignancies. For cytokeratin-15, expression was retained in actinic keratosis (38%), squamous cell carcinoma in situ (53%) and bowenoid papulosis (60%) but appeared to be lost in squamous cell carcinoma (3%) with statistically significant differences observed between groups that retained versus those that did not (P<0.05 for all three); for cytokeratin-19, patchy yet basal expression was noted in actinic keratosis (21%), patchy and suprabasal expression was noted in squamous cell carcinoma in situ (37%), bowenoid papulosis (13%) and squamous cell carcinoma (24%) with no statistically significant differences between groups; for p63, expression was retained in actinic keratosis (90%), squamous cell carcinoma in situ (87%), bowenoid papulosis (60%) and squamous cell carcinoma (100%) with no statistically significant differences between groups. In summary, our findings expand the neoplasms which involve stem cells to include cutaneous epithelial malignancies. Differential localization of each of these markers argues in favor of stem cell heterogeneity.

Perineural involvement: what does it mean?

Perineural invasion is an important mechanism for local spread in certain malignant cutaneous neoplasms and is associated with aggressive tumor growth, increased frequency of recurrence, and increased morbidity and mortality. Thus, perineural invasion is often used both as a marker of malignancy and an indicator of aggressive behavior. There exists, however, a limited number of cutaneous and noncutaneous benign neoplasms in addition to reactive lesions that either demonstrates perineural involvement or mimics it. Given the association of the term "invasion" with malignant neoplasms, we use the term "perineural involvement" to describe neoplastic cells of any type infiltrating within nerves. Despite the presence of perineural involvement in these benign lesions, there is no evidence of aggressive behavior compared with similar examples which do not demonstrate perineural involvement. The aim of this article is to review cutaneous and noncutaneous benign neoplasms and reactive conditions that may demonstrate or mimic perineural involvement. Recognition of the spectrum of benign processes that may resemble perineural involvement may help prevent diagnostic confusion, misdiagnosis, and overly aggressive treatment.

When dead cells tell tales-cutaneous involvement by precursor T-cell acute lymphoblastic lymphoma with an uncommon phenotype.

The thymic type of precursor T-cell acute lymphoblastic lymphoma (pre-T ALL), an uncommon T-cell malignancy, typically presents as a thymic mass and expresses terminal deoxonucleotidyl transferase, CD7, and cytoplasmic CD3, with variable expression of other markers. Cutaneous presentation in pre-T ALL is highly unusual. We describe a case of pre-T ALL presenting as 2 papulonodular lesions on the face of an otherwise asymptomatic 27-year-old man. Microscopic examination of both lesions revealed a moderate to dense pandermal infiltrate of medium-sized lymphocytes with extensive "crush" artifact, whereas immunohistochemistry revealed positive staining of lesional cells for CD45, CD3, Bcl-2, Ki-67, CD5, CD7, and CD34 but negative staining for CD4, CD8, CD30, CD56, CD10, CD117, anaplastic lymphoma kinase protein, TdT, myeloperoxidase, CD79a, and CD20. Gene rearrangement studies performed on both biopsies identified a clonal population of T lymphocytes. A subsequent computed tomography scan revealed a 9-cm mediastinal mass encasing all major mediastinal vessels, whereas a bone marrow biopsy revealed blasts with an immunophenotype similar to that of the cutaneous lesions. Features unique to this case include the cutaneous presentation and the immunophenotype-absence of CD4, CD8, and TdT with expression of CD34-both highly unusual features for pre-T ALL.

Regional recurrence after negative sentinel lymph node biopsy for melanoma.

OBJECTIVE: Sentinel lymph node (SLN) biopsy has shown great utility in the management of melanoma. An analysis of regional recurrence in previously mapped negative SLN basins as the first site of relapse is performed. METHODS: A retrospective query of a prospective melanoma database from 1994 to 2006 identified 1287 patients who underwent successful SLN biopsy. One thousand sixty patients (82.4%) were SLN negative and 227 (17.6%) patients SLN positive. Clinical variables were examined for the impact on regional recurrence by multivariate analysis. RESULTS: Mean follow-up was 44.3 months (range 3-155 months). Thirty-five patients (3.3%) presented with false-negative (FN) SLN biopsy. Pathologic review of the SLNs harvested from these basins found 7 (20.0%) samples positive for metastatic melanoma. Multivariate analysis found head and neck site [hazard ratio 3.67; 95% confidence interval (CI), 1.77-7.60, P < 0.001] and tumor thickness (hazard ratio 1.16; 95% CI, 1.04-1.30, P = 0.01) to be predictive of FN SLN biopsy. The 5-year melanoma specific survival calculated from the date of the SLN biopsy was 57.6% (95%CI, 35.7-41.9) in the FN group, which was not statistically different than the SLN positive group 60.0% (95% CI, 29.6-40.1; P = 0.14). CONCLUSIONS: Head and neck tumor site and tumor thickness are predictors of a FN SLN biopsy. Mechanisms other than pathologic SLN sampling error may contribute to the failure of the SLN biopsy in some patients. Patients with regional recurrence after negative SLN biopsy have a similar 5-year survival compared with patients with positive SLNs.

Activity of the A3 adenosine receptor gene promoter in transgenic mice: characterization of previously unidentified sites of expression.

Sites of A3 adenosine receptor gene expression have not been fully explored nor has this gene's promoter activity been confirmed in vivo. Transgenic mice were generated in which 2.3 kb upstream of the transcriptional start site of the mouse A3 adenosine receptor was coupled to a beta-galactosidase reporter gene. Selective transgene expression was detected in testis and brain as well as at other sites in which A3 adenosine receptor message has not been previously reported, including retinal ganglion cells and smooth muscle cells of the cerebrospinal vasculature. Our study suggests that this promoter may be useful in the selective targeting of gene expression to specific tissues.

Overexpression of A3 adenosine receptors in smooth, cardiac, and skeletal muscle is lethal to embryos.

The profile of expression of the A3 adenosine receptor (A3AR) and its importance during embryo development were explored. To this end, different ages of mouse embryos (8.5 days and older) were subjected to in situ hybridization with an A3AR riboprobe. No expression was found in any embryonic tissue except for the aorta and heart of 15.5-day embryos. To investigate further the role of the A3AR gene in development, we overexpressed this gene in A3AR knockout and wild-type mice by using the SM22 alpha promoter. This promoter is active in smooth, cardiac, and skeletal muscle lineages during early embryogenesis (at 8.5 days or earlier), becoming restricted to vascular and visceral smooth muscle cells in late fetal development and adult mice. We observed that moderate copy number incorporation (four copies) of the A3AR gene driven by the SM22 alpha promoter is sufficient to induce lethality at an early stage of embryo development. Remains of 8.5-day transgenic embryos were collected, including fragmented DNA. Hence, we speculate that A3AR homeostasis is critical for embryo viability and proper development. This finding is intriguing in view of the reported effects of sustained activation of the A3AR on induction of DNA fragmentation and apoptosis in cultured myocytes and other cell types.