Morphologische Charakterisierung der BRAF mutierten Melanome mittels Immunhistochemie, Dermatoskopie und konfokaler Laserscanmikroskopie

Background Since the advent of molecular targeted therapies, mutational profiling of malignant melanoma has become of outmost importance. Melanomas can be distinguished based on the presence of BRAF mutations. Estimated 40-60% of melanomas harbour a BRAF mutation, most of them V600E. Current guidelines recommend testing every advanced melanoma for BRAF mutation, since BRAF inhibitors are available for both adjuvant and therapeutic setting. BRAF Exon 15 PCR amplification and sequencing of genomic DNA now represents the gold standard for BRAF analysis in melanoma samples. Molecular genetic tests are however expensive and time consuming. Since different melanoma subtypes can be distinguished based on genetic mutations such as BRAFV600E, corresponding morphological patterns might be hypothesized. Aim of this study was to further characterize BRAF mutated melanomas compared to their wild type counterpart based on alternative methods such as in-vivo confocal microscopy (RCM), dermoscopy and immunohistochemistry (IHC). Methods Eight BRAFV600E mutated melanomas (six primary and two metastases), paired with age-, sex- and tumour thickness- matched wild-type controls were analysed with dermoscopy and in-vivo confocal microscopy. On the other side, eighteen melanomas with known BRAF mutational status (BRAFV600E, V600K, V600R and wild-type) were additionally evaluated through immunohistochemistry with anti-BRAF antibodies. Results Most common dermoscopic features in BRAFV600E mutated melanomas were irregularly distributed dots and globules and gray-blue blotches (62%), followed by irregular vessels, white regression, and peppering (50%). Most common RCM patterns were pleomorphic pagetoid cells, disarrangement at the dermoepidermal junction (DEJ), decohesed junctional nests, and bright particles at the dermal–epidermal junction (75%). Peppering in dermoscopy and plump bright cells in RCM were more frequently found in BRAFV600E mutated primary melanomas compared to wild-type ones (63% and 37%, respectively). In IHC, V600E-specific antibody stained all melanomas harbouring V600E and V600R mutation, but was not able to recognize BRAF V600K-mutated melanomas. Conclusions Dermoscopy and confocal microscopy are related to each other and might provide useful information in the non-invasive initial categorization of melanoma patients potentially harbouring a BRAFV600E mutation. At the same time, IHC might be a useful tool for the initial diagnosis of a BRAFV600E mutation in subjects with high risk or metastatic melanomas potentially benefiting from a systemic therapy with BRAF inhibitors. After the IHC screening, molecular techniques shall be used in V600E wild type melanomas, in the search for less frequent non-V600E BRAF mutations.Hintergrund Seit dem Aufkommen molekularer zielgerichteter Therapien ist die Mutationsprofilierung von malignen Melanomen von größter Bedeutung geworden. Melanome können anhand des Vorhandenseins von BRAF-Mutationen unterschieden werden. Schätzungsweise 40-60% der Melanome weisen eine BRAF-Mutation auf, die meisten davon V600E. Aktuelle Richtlinien empfehlen, jedes fortgeschrittene Melanom auf BRAF-Mutationen zu testen, da BRAF-Inhibitoren sowohl im adjuvanten als auch im therapeutischen Setting verfügbar sind. Molekularpathologische Techniken sind der Goldstandard für den Nachweis von BRAF-Mutationen, insbesondere die Ampflizierung und Sequenzierung von BRAF Exon 15 aus genomischer DNA mittels PCR. Solche Tests sind jedoch teuer und zeitaufwändig. Da verschiedene Melanom-Subtypen anhand genetischer Mutationen wie BRAFV600E unterschieden werden können, können entsprechende morphologische Muster angenommen werden. Ziel dieser Studie war es, BRAF-mutierte Melanome im Vergleich zu ihrem Wildtyp-Korrelat auf der Grundlage alternativer Methoden wie konfokaler in-vivo-Mikroskopie (RCM), Dermatoskopie und Immunhistochemie (IHC) weiter zu charakterisieren. Methoden Acht mutierte BRAFV600E-Melanome (sechs primäre und zwei Metastasen), gepaart mit alters-, geschlechts- und tumourdickenangepassten Wildtyp-Kontrollen, wurden mit Dermoskopie und konfokaler in-vivo-Mikroskopie analysiert. Auf der anderen Seite wurden 18 Melanome mit bekanntem BRAF-Mutationsstatus (BRAFV600E, V600K, V600R und Wildtyp) zusätzlich durch Immunhistochemie mit Anti-BRAF-Antikörpern bewertet. Ergebnisse Die häufigsten dermatoskopischen Merkmale bei mutierten BRAFV600E-Melanomen waren unregelmäßig verteilte Punkte und Schollen sowie grau-blaue homogene Areale (62%), gefolgt von unregelmäßigen Gefäßen, weißer Regression und Peppering (50%). Die häufigsten RCM-Muster waren pleomorphe pagetoide Zellen, Störungen der dermo-epidermalen Junktion (DEJ), atypische junktionale Melanozytennester und helle Partikel an der DEJ (75%). Peppering in der Dermatoskopie und plumpe helle Zellen in RCM wurden häufiger in mutierten primären BRAFV600E-Melanomen als in Wildtyp-Melanomen gefunden (63% vs. 37%). In der IHC färbte der V600E-spezifische Antikörper alle Melanome, die eine V600E- und V600R-Mutation enthielten, konnte jedoch keine BRAF V600K-mutierten Melanome erkennen. Schlussfolgerungen Dermatoskopie und konfokale Mikroskopie zeigen eine gute Korrelation zueinander und könnten nützliche Informationen für das nicht-invasive, preliminäre Screening und die Charakterisierung von BRAFV600E-mutierten Melanomen liefern. Gleichzeitig könnte die Immunhistochemie als erster Schritt zum Nachweis der BRAFV600E-Mutation bei der Auswahl von Patienten mit fortgeschrittenen Melanomen als Kandidaten für eine systemische Therapie mit BRAF-Inhibitoren wirksam eingesetzt werden. IHC sollte von molekularen Techniken bei p.V600E-negativen Melanomen gefolgt werden, um p.V600K und andere seltene Nicht-V600E-BRAF-Mutationen zu entdecken

Morphologische Charakterisierung der BRAF mutierten Melanome mittels Immunhistochemie, Dermatoskopie und konfokaler Laserscanmikroskopie

Background Since the advent of molecular targeted therapies, mutational profiling of malignant melanoma has become of outmost importance. Melanomas can be distinguished based on the presence of BRAF mutations. Estimated 40-60% of melanomas harbour a BRAF mutation, most of them V600E. Current guidelines recommend testing every advanced melanoma for BRAF mutation, since BRAF inhibitors are available for both adjuvant and therapeutic setting. BRAF Exon 15 PCR amplification and sequencing of genomic DNA now represents the gold standard for BRAF analysis in melanoma samples. Molecular genetic tests are however expensive and time consuming. Since different melanoma subtypes can be distinguished based on genetic mutations such as BRAFV600E, corresponding morphological patterns might be hypothesized. Aim of this study was to further characterize BRAF mutated melanomas compared to their wild type counterpart based on alternative methods such as in-vivo confocal microscopy (RCM), dermoscopy and immunohistochemistry (IHC). Methods Eight BRAFV600E mutated melanomas (six primary and two metastases), paired with age-, sex- and tumour thickness- matched wild-type controls were analysed with dermoscopy and in-vivo confocal microscopy. On the other side, eighteen melanomas with known BRAF mutational status (BRAFV600E, V600K, V600R and wild-type) were additionally evaluated through immunohistochemistry with anti-BRAF antibodies. Results Most common dermoscopic features in BRAFV600E mutated melanomas were irregularly distributed dots and globules and gray-blue blotches (62%), followed by irregular vessels, white regression, and peppering (50%). Most common RCM patterns were pleomorphic pagetoid cells, disarrangement at the dermoepidermal junction (DEJ), decohesed junctional nests, and bright particles at the dermal–epidermal junction (75%). Peppering in dermoscopy and plump bright cells in RCM were more frequently found in BRAFV600E mutated primary melanomas compared to wild-type ones (63% and 37%, respectively). In IHC, V600E-specific antibody stained all melanomas harbouring V600E and V600R mutation, but was not able to recognize BRAF V600K-mutated melanomas. Conclusions Dermoscopy and confocal microscopy are related to each other and might provide useful information in the non-invasive initial categorization of melanoma patients potentially harbouring a BRAFV600E mutation. At the same time, IHC might be a useful tool for the initial diagnosis of a BRAFV600E mutation in subjects with high risk or metastatic melanomas potentially benefiting from a systemic therapy with BRAF inhibitors. After the IHC screening, molecular techniques shall be used in V600E wild type melanomas, in the search for less frequent non-V600E BRAF mutations.Hintergrund Seit dem Aufkommen molekularer zielgerichteter Therapien ist die Mutationsprofilierung von malignen Melanomen von größter Bedeutung geworden. Melanome können anhand des Vorhandenseins von BRAF-Mutationen unterschieden werden. Schätzungsweise 40-60% der Melanome weisen eine BRAF-Mutation auf, die meisten davon V600E. Aktuelle Richtlinien empfehlen, jedes fortgeschrittene Melanom auf BRAF-Mutationen zu testen, da BRAF-Inhibitoren sowohl im adjuvanten als auch im therapeutischen Setting verfügbar sind. Molekularpathologische Techniken sind der Goldstandard für den Nachweis von BRAF-Mutationen, insbesondere die Ampflizierung und Sequenzierung von BRAF Exon 15 aus genomischer DNA mittels PCR. Solche Tests sind jedoch teuer und zeitaufwändig. Da verschiedene Melanom-Subtypen anhand genetischer Mutationen wie BRAFV600E unterschieden werden können, können entsprechende morphologische Muster angenommen werden. Ziel dieser Studie war es, BRAF-mutierte Melanome im Vergleich zu ihrem Wildtyp-Korrelat auf der Grundlage alternativer Methoden wie konfokaler in-vivo-Mikroskopie (RCM), Dermatoskopie und Immunhistochemie (IHC) weiter zu charakterisieren. Methoden Acht mutierte BRAFV600E-Melanome (sechs primäre und zwei Metastasen), gepaart mit alters-, geschlechts- und tumourdickenangepassten Wildtyp-Kontrollen, wurden mit Dermoskopie und konfokaler in-vivo-Mikroskopie analysiert. Auf der anderen Seite wurden 18 Melanome mit bekanntem BRAF-Mutationsstatus (BRAFV600E, V600K, V600R und Wildtyp) zusätzlich durch Immunhistochemie mit Anti-BRAF-Antikörpern bewertet. Ergebnisse Die häufigsten dermatoskopischen Merkmale bei mutierten BRAFV600E-Melanomen waren unregelmäßig verteilte Punkte und Schollen sowie grau-blaue homogene Areale (62%), gefolgt von unregelmäßigen Gefäßen, weißer Regression und Peppering (50%). Die häufigsten RCM-Muster waren pleomorphe pagetoide Zellen, Störungen der dermo-epidermalen Junktion (DEJ), atypische junktionale Melanozytennester und helle Partikel an der DEJ (75%). Peppering in der Dermatoskopie und plumpe helle Zellen in RCM wurden häufiger in mutierten primären BRAFV600E-Melanomen als in Wildtyp-Melanomen gefunden (63% vs. 37%). In der IHC färbte der V600E-spezifische Antikörper alle Melanome, die eine V600E- und V600R-Mutation enthielten, konnte jedoch keine BRAF V600K-mutierten Melanome erkennen. Schlussfolgerungen Dermatoskopie und konfokale Mikroskopie zeigen eine gute Korrelation zueinander und könnten nützliche Informationen für das nicht-invasive, preliminäre Screening und die Charakterisierung von BRAFV600E-mutierten Melanomen liefern. Gleichzeitig könnte die Immunhistochemie als erster Schritt zum Nachweis der BRAFV600E-Mutation bei der Auswahl von Patienten mit fortgeschrittenen Melanomen als Kandidaten für eine systemische Therapie mit BRAF-Inhibitoren wirksam eingesetzt werden. IHC sollte von molekularen Techniken bei p.V600E-negativen Melanomen gefolgt werden, um p.V600K und andere seltene Nicht-V600E-BRAF-Mutationen zu entdecken

Line-field confocal optical coherence tomography-Practical applications in dermatology and comparison with established imaging methods

Background Non-invasive diagnostic techniques in dermatology gained increasing popularity in the last decade. Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) are meanwhile established in research and clinical routine. While OCT is mainly indicated for detecting non-melanoma skin cancer, RCM has proven its usefulness additionally in distinguishing melanocytic lesions. Line-field confocal optical coherence tomography (LC-OCT) is an emerging tool combining the principles of both above-mentioned methods. Methods Healthy skin at different body sites and exemplary skin lesions (basal cell carcinoma, malignant melanoma, actinic keratosis) were examined using dermoscopy, RCM, OCT and LC-OCT. Standard features for RCM and OCT and comparable features for LC-OCT were analysed. Results LC-OCT has a lower penetration depth but superior resolution compared to OCT. In comparison with RCM, which provides only horizontal sections, LC-OCT creates both vertical and horizontal images in real time and has nearly the same cellular resolution. Discussion Our preliminary experiences suggest that LC-OCT combines the advantages of RCM and OCT, with optimal resolution and penetration depth to diagnose all types of skin cancer. Larger systematic studies are needed to further characterize the field of use of this device and its sensitivity and specificity compared to histology

Giuseppe Moscati: a man, a physician and a scientist

The life of Giuseppe Moscati (1880-1927) as a man, as a physician and as a scientist may be framed within the cultural climate of Positivism, which spread over the last years of the 19th century and the beginning of the 20th Century. His activity contributed to patients' care improvement; in addition to meticulous drug regimens, he also prescribed a methodology of spiritual care, involving meditation and self-control as part of an holistic approach to healthcare. Our review deals with his published researches, highlighting the innovative findings on the juvenile diabetes treatment and extensive clinical changes consequent upon nephritis. This extraordinary man put considerable emphasis on primary care and holistic health in Italy, pioneering a new patient-centred, and holistic approach to medicine

GIUSEPPE MOSCATI: ČOVJEK, LIJEČNIK I ZNANSTVENIK

The life of Giuseppe Moscati (1880-1927) as a man, as a physician and as a scientist may be framed within the cultural climate of Positivism, which spread over the last years of the 19th century and the beginning of the 20th Century. His activity contributed to patients’ care improvement; in addition to meticulous drug regimens, he also prescribed a methodology of spiritual care, involving meditation and self-control as part of an holistic approach to healthcare. Our review deals with his published researches, highlighting the innovative findings on the juvenile diabetes treatment and extensive clinical changes consequent upon nephritis. This extraordinary man put considerable emphasis on primary care and holistic health in Italy, pioneering a new patient-centred, and holistic approach to medicine.Život Giuseppea Moscatija (1880.–1927.) kao čovjeka, liječnika i znanstvenika može biti smješten unutar kulturne klime pozitivizma, koji se proširio posljednjih godina 19. i početkom 20. stoljeća. Njegova aktivnost doprinijela je unaprjeđenju njege pacijenata; uz pedantan režim lijekova, on je također propisao metodologiju duhovne skrbi, uključujući meditaciju i samokontrolu kao dio holističkog pristupa zdravstvenoj skrbi. Naš pregled bavi se njegovim objavljenim istraživanjima, naglašavajući inovativna otkrića o liječenju juvenilnog dijabetesa i opsežnih kliničkih promjena koje slijede nakon nefritisa. Ovaj izuzetan čovjek stavljao je znatan naglasak na primarnu skrb i holističko zdravlje u Italiji, utirući put novom pacijentu orijentiranom i holističkom pristupu medicini

BRAFV600E mutated and wild type melanomas: dermoscopy and reflectance confocal microscopy characterization

The advent of modern molecular approaches was of crucial importance for the identification of melanoma genetic signatures, opening new horizons in the treatment of metastatic disease with molecular targeted therapies. Similarly the melanoma diagnosis is aided by reflectance confocal microscopy (RCM): a promising technique that allows non-invasive imaging from the skin surface to the upper dermis with quasi-histologic resolution. The most common melanoma mutation involves the gene BRAF and it is represented by the BRAFV600E, however, V600K, V600R and V600D mutations are also known. Because different genetic aberrations categorize melanoma subtypes with distinct clinical characteristics, it is reasonable to hypothesize that a distinctive molecular signature corresponds to specific morphologic patterns. A comparison between the dermoscopic patterns of BRAF p.V600E, BRAF p.V600K and wild-type BRAF primary melanomas was assessed from a collection of 12 lesions (4 primary melanomas per each BRAFV600 mutated status and 4 wt). In 9 cases the RCM images were available and the frequency of the RCM descriptors was examined. The RCM analysis showed that the presence of plump bright cells, collagen bundles and inflammatory cells in the dermis were frequently observed even when dermoscopy showed no regression features. Our study showed that regression phenomena and the associated dermoscopic and RCM descriptors could help the clinician to discriminate between the different BRAF mutated status, providing key information for patient screening, management and follow-up

GIUSEPPE MOSCATI: ČOVJEK, LIJEČNIK I ZNANSTVENIK

The life of Giuseppe Moscati (1880-1927) as a man, as a physician and as a scientist may be framed within the cultural climate of Positivism, which spread over the last years of the 19th century and the beginning of the 20th Century. His activity contributed to patients’ care improvement; in addition to meticulous drug regimens, he also prescribed a methodology of spiritual care, involving meditation and self-control as part of an holistic approach to healthcare. Our review deals with his published researches, highlighting the innovative findings on the juvenile diabetes treatment and extensive clinical changes consequent upon nephritis. This extraordinary man put considerable emphasis on primary care and holistic health in Italy, pioneering a new patient-centred, and holistic approach to medicine.Život Giuseppea Moscatija (1880.–1927.) kao čovjeka, liječnika i znanstvenika može biti smješten unutar kulturne klime pozitivizma, koji se proširio posljednjih godina 19. i početkom 20. stoljeća. Njegova aktivnost doprinijela je unaprjeđenju njege pacijenata; uz pedantan režim lijekova, on je također propisao metodologiju duhovne skrbi, uključujući meditaciju i samokontrolu kao dio holističkog pristupa zdravstvenoj skrbi. Naš pregled bavi se njegovim objavljenim istraživanjima, naglašavajući inovativna otkrića o liječenju juvenilnog dijabetesa i opsežnih kliničkih promjena koje slijede nakon nefritisa. Ovaj izuzetan čovjek stavljao je znatan naglasak na primarnu skrb i holističko zdravlje u Italiji, utirući put novom pacijentu orijentiranom i holističkom pristupu medicini

Line-field confocal optical coherence tomography: a new tool for the differentiation between nevi and melanomas?

SIMPLE SUMMARY: Typical benign nevi and advanced melanomas can be easily discriminated, but there are still some melanocytic lesions where even experts are not sure about the correct diagnosis and degree of malignity. The high penetration depth of optical coherence tomography (OCT) allows an assessment of tumor thickness of the lesion precisely, but without cellular resolution the differentiation of melanocytic lesions remains difficult. On the other hand, reflectance confocal microscopy (RCM) allows for very good morphological identification of either a nevus or a melanoma, but cannot show the infiltration depth of the lesion because of its low penetration depth. Since the new device of line-field confocal optical coherence tomography (LC-OCT) technically closes the gap between these other two devices, in this study, we wanted to examine if it is possible to differentiate between nevi and melanomas with LC-OCT, and which criteria are the most important for it. ABSTRACT: Until now, the clinical differentiation between a nevus and a melanoma is still challenging in some cases. Line-field confocal optical coherence tomography (LC-OCT) is a new tool with the aim to change that. The aim of the study was to evaluate LC-OCT for the discrimination between nevi and melanomas. A total of 84 melanocytic lesions were examined with LC-OCT and 36 were also imaged with RCM. The observers recorded the diagnoses, and the presence or absence of the 18 most common imaging parameters for melanocytic lesions, nevi, and melanomas in the LC-OCT images. Their confidence in diagnosis and the image quality of LC-OCT and RCM were evaluated. The most useful criteria, the sensitivity and specificity of LC-OCT vs. RCM vs. histology, to differentiate a (dysplastic) nevus from a melanoma were analyzed. Good image quality correlated with better diagnostic performance (Spearman correlation: 0.4). LC-OCT had a 93% sensitivity and 100% specificity compared to RCM (93% sensitivity, 95% specificity) for diagnosing a melanoma (vs. all types of nevi). No difference in performance between RCM and LC-OCT was observed (McNemar’s p value = 1). Both devices falsely diagnosed dysplastic nevi as non-dysplastic (43% sensitivity for dysplastic nevus diagnosis). The most significant criteria for diagnosing a melanoma with LC-OCT were irregular honeycombed patterns (92% occurrence rate; 31.7 odds ratio (OR)), the presence of pagetoid spread (89% occurrence rate; 23.6 OR) and the absence of dermal nests (23% occurrence rate, 0.02 OR). In conclusion LC-OCT is useful for the discrimination between melanomas and nevi