Telomere-Associated Changes in Nuclear Architecture of Cancer-Associated Macrophage-like Cells in Liquid Biopsies from Melanoma Patients.

During phagocytosis, tumor-associated macrophages (TAMs) can incorporate genetic material from tumor cells. The incorporation of extra genetic material may be responsible for advanced malignant behavior observed in some TAMs, making TAMs potentially important players in cancer progression. More recently, similar cells were described in the blood as cancer-associated macrophage-like cells (CAMLs). CAMLs may be equivalent to TAMs cells in the blood, and they express macrophage markers. However, their origin is still unclear. In a previous study, we showed for the first time the distinct telomere 3D structure of circulating tumor cells (CTCs) in melanoma and other cancers. In the present pilot study, we investigated, comparatively, the 3D telomere structure of CAMLs, CTCs and leucocytes from nine melanoma patients with metastatic cutaneous melanoma stage IV. CTC capture was performed by size-based filtration followed by cytological and immunocytological evaluation. Three-dimensional Quantitative Fluorescent in situ Hybridization was performed to measure differences in five 3D telomere parameters. Telomere parameters, such as number, length, telomere aggregates, nuclear volume, and a/c ratio, were compared among different cellular types (CTCs, CAMLs, and normal leucocytes). Three telomere parameters were significantly different between CAMLs and leucocytes. The combination of two telomere parameters (telomere length against the number of telomeres) resulted in the identification of two CAMLs subpopulations with different levels of genomic instability. Those populations were classified as profile 1 and 2. Profile 2, characterized by a high number of short telomeres, was observed in four of the nine melanoma patients. To our knowledge, this is the first pilot study to investigate 3D telomere parameters as hallmarks of nuclear architecture in CAMLs' population in comparison to leucocytes from the same patient. Further studies involving a larger patient sample size are necessary to validate these findings and explore their potential prognostic value.

Prevalence of T-cell antigen losses in mycosis fungoides and CD30-positive cutaneous T-cell lymphoproliferations in a series of 153 patients.

Mycosis fungoides (MF) and primary cutaneous CD30-positive T-cell lymphoproliferative disorders (CD30LPD) are the most frequent primary cutaneous T-cell lymphomas. Our objective was to study pan-T-cell antigens and PD-1 expression in a large cohort of MF and CD30LPD with a special interest in antigen losses as a diagnostic tool. We retrospectively reviewed 160 consecutive samples from 153 patients over a 3 year period, including 104 with MF and 49 with CD30LPD. As controls, benign inflammatory dermatoses (BID, n=19) were studied. A semi-quantitative evaluation of CD2, CD3, CD4, CD5, CD7, CD8 expression was performed. PD-1 and double stainings (CD3+CD7 and PD-1+CD7) were performed in a subset of MF cases. CD8+ MF was frequent (23%) and CD7 was the most frequently lost antigen in both MF (45%) and CD30LPD (86%), while no significant T-cell antigen loss was observed in BID. CD7 loss was less frequent in folliculotropic MF (p<0.001). PD-1 was variably expressed in MF with no differences with BID. The CD3+/CD7- and PD-1+/CD7- neoplastic lymphocytes were highlighted by the use of chromogenic double staining experiments in MF with a CD7 loss identified with single staining. Multiple pan T-cell antigen losses were mostly seen in CD30LPD with CD2 being the most frequently preserved marker (90%). While PD-1 does not discriminate between MF and BID, CD7 is frequently lost in MF infiltrates as well as other pan-T-cell antigens in CD30LPD, which can be used as routine markers for diagnosis. We recommend the use of CD7 in addition to CD3, CD4 and CD8 as a minimal immunohistochemical panel for MF assessment, and the use of double stainings for CD3 and CD7 in difficult cases.

Clinical and histological features of fixed drug eruption: a single-centre series of 73 cases with comparison between bullous and non-bullous forms.

The clinical and pathological aspects of fixed drug eruption (FDE) have been described based on a few case series. To compare bullous FDE (BFDE) and non-bullous FDE (NBFDE) and to determine whether BFDE can be histologically distinguished from other dermatoses presenting with an apoptotic pan-epidermolysis. In this retrospective monocentre study (2005-2016), FDE was classified as BFDE or NBFDE and localized (one anatomical site) or generalized (≥ two sites; GBFDE). Clinical data were extracted from charts, and images were reviewed. Skin biopsies were analysed and compared to the clinical presentation. Three dermatopathologists, blinded to the final clinical diagnosis, evaluated a subset of BFDE cases (n = 8) and 25 biopsies of other bullous diseases known to have an epidermal necrolysis (EN)-like pattern. In total, 73 patients were included in the study. Patients with BFDE (n = 58; GBFDE n = 48) were significantly older (p < 0.001). All patients with GBFDE were hospitalized; 25 had a complication (infectious; n = 19), and eight died (median age: 80). Histology revealed spongiotic (6.7%), interface dermatitis (48.3%) and EN-like (66.3%) patterns. The EN-like pattern was more frequent in BFDE than NBFDE (74% vs 27%; p = 0.008). Melanophages (100% vs 66%; p = 0.02) and massive dermal melanosis (40% vs 4%; p = 0.0005) were more prominent in NBFDE than BFDE. BFDE could not be reliably distinguished from other bullous diseases with EN-like patterns. BFDE belongs to the spectrum of skin conditions with an EN pattern, for which the concept of acute syndrome of apoptotic pan-epidermolysis (ASAP) was previously introduced. Clinical-pathological correlation is mandatory for a diagnosis of BFDE.

[Humoral immune diseases: Cutaneous vasculitis and auto-immune bullous dermatoses]. / Immunité à médiation humorale : vascularites cutanées et maladies bulleuses auto-immunes.

Humoral immunity is the cause of multiple diseases related to antibodies (IgA, IgG, IgM) produced by the patient. Two groups of diseases are identified. The first group is related to circulating antigen-antibody complexes. The antigens are various. They are often unknown. These immune complexes cause a vascular inflammation due to the complement fixation. Consequently, this group is dominated by inflammatory vasculitis. In the second group, the pathology is due to the fixation in situ of antibodies to a target antigen of the skin that is no more recognized by the patient. This group is represented by the auto-immune bullous dermatoses.

Droplet digital PCR of circulating tumor cells from colorectal cancer patients can predict KRAS mutations before surgery.

In colorectal cancer (CRC), KRAS mutations are a strong negative predictor for treatment with the EGFR-targeted antibodies cetuximab and panitumumab. Since it can be difficult to obtain appropriate tumor tissues for KRAS genotyping, alternative methods are required. Circulating tumor cells (CTCs) are believed to be representative of the tumor in real time. In this study we explored the capacity of a size-based device for capturing CTCs coupled with a multiplex KRAS screening assay using droplet digital PCR (ddPCR). We showed that it is possible to detect a mutant ratio of 0.05% and less than one KRAS mutant cell per mL total blood with ddPCR compared to about 0.5% and 50-75 cells for TaqMeltPCR and HRM. Next, CTCs were isolated from the blood of 35 patients with CRC at various stage of the disease. KRAS genotyping was successful for 86% (30/35) of samples with a KRAS codon 12/13 mutant ratio of 57% (17/30). In contrast, only one patient was identified as KRAS mutant when size-based isolation was combined with HRM or TaqMeltPCR. KRAS status was then determined for the 26 available formalin-fixed paraffin-embedded tumors using standard procedures. The concordance between the CTCs and the corresponding tumor tissues was 77% with a sensitivity of 83%. Taken together, the data presented here suggest that is feasible to detect KRAS mutations in CTCs from blood samples of CRC patients which are predictive for those found in the tumor. The minimal invasive nature of this procedure in combination with the high sensitivity of ddPCR might provide in the future an opportunity to monitor patients throughout the course of disease on multiple levels including early detection, prognosis, treatment and relapse as well as to obtain mechanistic insight with respect to tumor invasion and metastasis.

Did circulating tumor cells tell us all they could? The missed circulating tumor cell message in breast cancer.

PURPOSE: To compare circulating tumor cell (CTC) detection rates in patients with early (M0) and metastatic (M+) breast cancer using 2 positive-selection methods or size-based unbiased enrichment. METHODS: Blood collected at baseline and at different times during treatment from M0 patients undergoing neoadjuvant therapy and from M+ women starting a new line of treatment was processed in parallel using AdnaTest EMT-1/ and EMT-2/Stem CellSelect/Detect kits or ScreenCell Cyto devices. CTC positivity was defined according to the suggested cutoffs and cytological parameters, respectively. RESULTS: Higher CTC detection rates were obtained with the AdnaTest approach when using for CTC-enrichment antibodies against ERBB2 and EGFR in addition to MUC1 and the classical epithelial surface marker EPCAM (13% vs. 48%). In M0 patients mainly, CTC positivity rates further increased when EMT- and stemness-related marker expression (PIK3CA, AKT2 and ALDH1) was evaluated in addition to EPCAM, MUC1 and ERBB2. When the physical properties of tumor cells were exploited, CTCs were detected at higher percentages than with positive-selection-based methods, without any difference between clinical stages (78% in M0 vs. 72% in M+ cases at baseline). Circulating tumor microemboli (CTMs) were detected in addition to single CTCs with significantly higher frequency in M0 than M+ samples (78% vs. 27%, p = 0.0002). CONCLUSIONS: Different approaches for CTC detection probably identify distinct tumor cell subpopulations, but need technical standardization before their clinical validity and biological specificity may be adequately investigated. The distinct role of CTMs compared with CTCs as prognostic and predictive biomarkers represents a further challenge.

[Cutaneous lymphoproliferations: proposal for the use of diagnostic algorithms based on 2760 cases of cutaneous lymphoproliferations taken from the INCa networks (LYMPHOPATH and GFELC) over a two-year period]. / Lymphoproliférations cutanées : proposition d'algorithmes diagnostiques à partir de l'expérience sur 2ans des réseaux INCa (LYMPHOPATH et GFELC) sur 2760 lymphoproliférations cutanées.

INTRODUCTION: Taking as a base our retrospective study of 2760 cases of cutaneous lymphoproliferations from the LYMPHOPATH and GFELC networks, we analyzed the doubtful and discordant cases between non-expert and expert pathologists, and the interest of clinicopathological confrontation. MATERIAL AND METHODS: We defined the main diagnostic difficulties presented by cutaneous lymphoproliferations. We then designed and tested the algorithms on 20 random cases with 20 pathologists, in order to be used by any pathologist (not necessarily specialised in dermatopathology). RESULTS: The problematic differential diagnoses most frequently encountered are the following: MF or reactive dermatose; lymphoma without any other precision or reactive infiltrate; small B cell lymphoproliferation: lymphoma or reactive infiltrate; phenotyping of large B cell lymphoproliferation. We also analyzed less common problematic differential diagnoses, on the grounds that they are over- or under- diagnosed. Our test had a 72% success rate among the 20 randomly tested cases. The use of several algorithms for the same case is possible. DISCUSSION: Our study shows that an expert second-opinion is of interest in the area of cutaneous lymphoproliferations. A second opinion is useful for distinguishing a small B cell lymphoma from a HLR, and for defining a final diagnosis when the first pathologist doubts between lymphoma and reactive infiltrate. However, we demonstrate that for the problem MF or reactive dermatose, an initial clinicopathological confrontation produces more results than a second-opinion pathology review. CONCLUSION: This is the first study of cutaneous lymphoproliferations that, without excluding reactionary infiltrates, concentrates on doubtful and discordant diagnoses between non expert and expert pathologists, and which has produced tested diagnostic algorithms.

Aggressive epidermotropic cutaneous CD8+ lymphoma: a cutaneous lymphoma with distinct clinical and pathological features. Report of an EORTC Cutaneous Lymphoma Task Force Workshop.

AIMS: Aggressive epidermotropic cutaneous CD8(+) lymphoma is currently afforded provisional status in the WHO classification of lymphomas. An EORTC Workshop was convened to describe in detail the features of this putative neoplasm and evaluate its nosological status with respect to other cutaneous CD8(+) lymphomas. METHODS AND RESULTS: Sixty-one CD8(+) cases were analysed at the workshop; clinical details, often with photographs, histological sections, immunohistochemical results, treatment and patient outcome were discussed and recorded. Eighteen cases had distinct features and conformed to the diagnosis of aggressive epidermotropic cutaneous CD8(+) lymphoma. The patients typically present with widespread plaques and tumours, often ulcerated and haemorrhagic, and histologically have striking pagetoid epidermotrophism. A CD8(+) /CD45RA(+) /CD45RO(-) /CD2(-) /CD5(-) /CD56(-) phenotype, with one or more cytotoxic markers, was found in seven of 18 patients, with a very similar phenotype in the remainder. The tumours seldom involve lymph nodes, but mucosal and central nervous system involvement are not uncommon. The prognosis is poor, with a median survival of 12 months. Examples of CD8(+) mycosis fungoides, lymphomatoid papulosis and Woringer-Kolopp disease presented the typical features well documented in the CD4(+) forms of those diseases. CONCLUSIONS: Aggressive epidermotropic cutaneous CD8(+) lymphoma is a distinct lymphoma that warrants inclusion as a distinct entity in future revisions of lymphoma classifications.

Three-Dimensional Telomeric Analysis of Isolated Circulating Tumor Cells (CTCs) Defines CTC Subpopulations.

Circulating tumor cells (CTCs) have been identified with the potential to serve as suitable biomarkers for tumor stage and progression, but the availability of effective isolation technique(s) coupled with detailed molecular characterization have been the challenges encountered in making CTCs clinically relevant. For the first time, we combined isolation of CTCs using the ScreenCell filtration technique with quantitative analysis of CTC telomeres by TeloView. This resulted in the identification and molecular characterization of different subpopulations of CTCs in the same patient. Three-dimensional (3D) telomeric analysis was carried out on isolated CTCs of 19 patients that consisted of four different tumor types, namely, prostate, colon, breast, melanoma, and one lung cancer cell line. With telomeric analysis of the filter-isolated CTCs, the level of chromosomal instability (CIN) of the CTCs can be determined. Our study shows that subpopulations of CTCs can be identified on the basis of their 3D telomeric properties.

Prognostic value of histologic features of toxic epidermal necrolysis.

BACKGROUND: The prognosis of toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), and SJS/TEN overlap syndrome has been assessed using a disease-specific severity score (SCORTEN) based on clinical and laboratory data. Histologic data may improve outcome prediction. OBJECTIVE: We sought to evaluate whether dermal mononuclear infiltration and epidermal necrosis predict survival of patients with TEN, SJS, or SJS/TEN. METHODS: We conducted a retrospective review of clinical records and skin biopsy specimens read without knowledge of clinical data. RESULTS: We identified 108 patients (SJS, n = 42; SJS/TEN, n = 36; TEN, n = 30). Overall mortality was 21.3%. Dermal infiltration and epidermal necrosis were not associated with time from disease onset to biopsy. Extensive dermal infiltrates were seen in 19 (18.5%) patients and full-thickness epidermal necrosis in 56 (52%) patients. Dermal infiltrate severity was not associated with day-1 (D1) SCORTEN or hospital death. Epidermal necrosis severity showed trends toward associations with D1 SCORTEN (P = .11) and hospital death (P = .06). In univariate analyses, full-thickness epidermal necrosis was significantly associated with hospital death (32.1% vs 11.4%, P = .017) and worse D1 SCORTEN values (1.98 ± 1.29 vs 1.55 ± 1.21; P = .04). In the bivariate analysis, however, D1 SCORTEN remained significantly associated with hospital death (odds ratio = 3.07, 95% confidence interval 1.83-5.16) but the association with full-thickness epidermal necrosis was no longer significant (odds ratio = 2.02, 95% confidence interval 0.65-7.12). LIMITATIONS: Retrospective study design and indirect assessment of progression are limitations. CONCLUSION: Full-thickness epidermal necrosis was associated with mortality but did not independently predict hospital death after adjustment based on the SCORTEN value. Dermal infiltrate severity was not associated with hospital death.

Skin tumors induced by sorafenib; paradoxic RAS-RAF pathway activation and oncogenic mutations of HRAS, TP53, and TGFBR1.

PURPOSE: The emergence of skin tumors in patients treated with sorafenib or with more recent BRAF inhibitors is an intriguing and potentially serious event. We carried out a clinical, pathologic, and molecular study of skin lesions occurring in patients receiving sorafenib. EXPERIMENTAL DESIGN: Thirty-one skin lesions from patients receiving sorafenib were characterized clinically and pathologically. DNA extracted from the lesions was screened for mutation hot spots of HRAS, NRAS, KiRAS, TP53, EGFR, BRAF, AKT1, PI3KCA, TGFBR1, and PTEN. Biological effect of sorafenib was studied in vivo in normal skin specimen and in vitro on cultured keratinocytes. RESULTS: We observed a continuous spectrum of lesions: from benign to more inflammatory and proliferative lesions, all seemingly initiated in the hair follicles. Eight oncogenic HRAS, TGFBR1, and TP53 mutations were found in 2 benign lesions, 3 keratoacanthomas (KA) and 3 KA-like squamous cell carcinoma (SCC). Six of them correspond to the typical UV signature. Treatment with sorafenib led to an increased keratinocyte proliferation and a tendency toward increased mitogen-activated protein kinase (MAPK) pathway activation in normal skin. Sorafenib induced BRAF-CRAF dimerization in cultured keratinocytes and activated CRAF with a dose-dependent effect on MAP-kinase pathway activation and on keratinocyte proliferation. CONCLUSION: Sorafenib induces keratinocyte proliferation in vivo and a time- and dose-dependent activation of the MAP kinase pathway in vitro. It is associated with a spectrum of lesions ranging from benign follicular cystic lesions to KA-like SCC. Additional and potentially preexisting somatic genetic events, like UV-induced mutations, might influence the evolution of benign lesions to more proliferative and malignant tumors.

S100B and neurofibromin immunostaining and X-inactivation patterns of laser-microdissected cells indicate a multicellular origin of some NF1-associated neurofibromas.

Neurofibromatosis 1 (NF1) is an autosomal dominant disease that predisposes individuals to developing benign neurofibromas. Some features and consequences of NF1 appear to result from partial deficiency of neurofibromin (Nfn), the NF1 gene protein product, as a result of haploinsufficiency for the NF1 gene. Other features and consequences of NF1 appear to involve total deficiency of Nfn, which arises as a result of either loss of function of the second NF1 allele or excess degradation of Nfn produced by the second allele in a particular clone of cells. We used immunofluorescence to assess the presence of Nfn in putative Schwann cells (S100B(+) ) and non-Schwann cells (S100B(-) ) in 36 NF1-derived benign neurofibromas classified histologically as diffuse or encapsulated. The S100B(+) /Nfn(-) cell population made up only 18% ± 10% (mean ± standard deviation) of the neurofibroma cells in both the diffuse and encapsulated neurofibromas. The proportion of S100B(+) /Nfn(+) cells was significantly higher and the proportion of S100B(-) /Nfn(-) cells was significantly lower in diffuse neurofibromas than in encapsulated neurofibromas. We isolated S100B(+) /Nfn(+) , S100B(+) /Nfn(-) , and S100B(-) /Nfn(+) cells by laser microdissection and, using X-chromosome inactivation profiles, assessed clonality for each cell type. We showed that, although some neurofibromas include a subpopulation of S100B(+) /Nfn(-) cells consistent with clonal expansion of a Schwann cell progenitor that has lost function of both NF1 alleles, other neurofibromas do not show evidence of monoclonal proliferation of Schwann cells. Our findings suggest that, although clonal loss of neurofibromin function is probably involved in the development of some NF1-associated neurofibromas, other pathogenic processes also occur.

Different patterns of mast cells distinguish diffuse from encapsulated neurofibromas in patients with neurofibromatosis 1.

Multiple neurofibromas are cardinal features of neurofibromatosis 1 (NF1). Several different types of NF1-associated neurofibromas occur, each distinct in terms of pathological details, clinical presentation, and natural history. Mast cells are present in most neurofibromas and have been shown to be critical to the origin and progression of neurofibromas in both human NF1 and relevant mouse models. In this investigation, the authors determined whether mast cell involvement is the same for all types of NF1-associated neurofibromas. They examined the density and distribution of mast cells within 49 NF1-associated neurofibromas classified histopathologically as diffuse or encapsulated on the basis of the presence or absence of the perineurium or its constituent cells. They made two observations: (1) Diffuse neurofibromas had significantly higher densities of mast cells than did encapsulated neurofibromas, and (2) mast cells were evenly distributed throughout diffuse neurofibromas but were primarily restricted to the periphery of encapsulated neurofibromas. The differences in mast cell density and distribution differentiate the two basic types of NF1-associated neurofibromas, suggesting that the pathogenesis of diffuse and encapsulated neurofibromas may be significantly different.

A new device for rapid isolation by size and characterization of rare circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) likely derive from clones in the primary tumor, suggesting that they can be used for all biological tests applying to the primary cells. MATERIALS AND METHODS: The ScreenCell® devices are single-use and low-cost innovative devices that use a filter to isolate and sort tumor cells by size. RESULTS: The ScreenCell® Cyto device is able to isolate rare, fixed, tumor cells, with a high recovery rate. Cells are well preserved morphologically. Immunocytochemistry and FISH assays can be performed directly on the filter. The ScreenCell® CC device allows isolation of live cells able to grow in culture. High quality genetic materials can be obtained directly from tumor cells isolated on the ScreenCell® MB device filter. CONCLUSION: Due to their reduced size, versatility, and capacity to isolate CTCs within minutes, the ScreenCell® devices may be able to simplify and improve non-invasive access to tumor cells.

Influence of evaluation of clinical pictures on the histopathologic diagnosis of inflammatory skin disorders.

BACKGROUND: Clinical information on histologic referral sheets is usually very limited, and particularly for inflammatory skin disorders, dermatopathologists often ask referring physicians for clinical correlation. OBJECTIVE: In this study we tested the value of clinicopathologic correlation in the histopathologic diagnosis of inflammatory skin disorders. METHODS: One-hundred biopsy specimens were digitalized and stored on 3 DVDs along with the clinical images. All cases were evaluated by 9 independent full-time dermatopathologists, initially without looking at the clinical pictures and subsequently after checking them. All diagnoses were finally compared with the "reference" diagnosis established in Graz, Austria, and the results were statistically analyzed. RESULTS: After evaluation of the clinical images, the number of dermatopathologists making a correct diagnosis was increased in 70 cases, unchanged in 25 cases, and decreased in 5 cases. The total number of correct diagnoses increased from 332 (diagnoses before evaluation of clinical pictures) to 481 (diagnoses after evaluation of clinical pictures), with a 16.6% increase in the total. LIMITATIONS: The computerized setting is different from real-life dermatopathology and physical examination of patients. CONCLUSION: Our study clearly shows that clinical pictures should be added to biopsy request slips of inflammatory skin disorders whenever possible, as they allow a better interpretation of histopathologic findings.