Financing source: Executive Unit for Financing Higher Education, Research, Development and Innovation

Project code: PN-III-P4-ID-PCE-2016-0641.

Contract no.: 183 PCE / 2017.

Projects acronym: CEACAMMEL.

Projects Title:
Regression-based characterization of mechanisms involving CEACAM-1 in melanoma – an innovative approach for optimizing the management of patients with thin melanomas

Projects description – Abstract: Melanoma has overall rising incidence with increased incidence in young patients and high mortality rate in metastatic stage. The extreme variation in natural and therapeutic evolution and unexplained mortality of thin melanomas classified in the same TMN stage highlight the need of new biomarkers and ultrastaging.

The project brings in focus carcinoembyonic antigen cell adhesion molecule 1 (CEACAM1), key molecule involved in tumorigenesis, mainly in epithelial-mesenchymal transition (being responsible for gaining invasiveness and metastatic capacity) and immune escape (by inhibiting effector functions of tumor infiltrating lymphocytes). To decipher CEACAM1 role in modulating aggressive thin melanoma behavior, we chose to study it for the first time in regressing melanoma (RM) vs non-regressing melanoma (nRM), regression phenomenon being used as a valuable model for understanding antitumor immunity.

We will assess CEACAM1 isoforms expression using IHC, RT-PCR and WB in thin RM&nRM melanomas and ELISA for serologic tests. Retrospective studies [tissue microarrays from archived tumor tissue of 90 cases of RM, 90 nRM, 30 halo nevi and 30 common nevi (as benign control)] and prospective studies (plasma, frozen and paraffin-embedded tumor tissue from 30 cases of thin RM, 30 thin nRM, 10 halo nevi and 30 common nevi) will be performed. We assembled a multidisciplinary team including senior and junior researchers under the supervision of a highly experienced researcher with extensive expertise in multiple enzymatic and fluorescent IHC and complex morphometric analysis. We will analyze the correlation between CEACAM1 deregulation and prognosis, intratumor inflammatory infiltrate and consecrated progression-related tissue markers.

This approach will clarify the mechanisms through which CEACAM1 isoforms are involved in progression and regression of thin melanomas, as a starting point for new risk stratification of early melanomas and a better management of these patients.

Research team:
Coordonator (CO): University of Medicine and Pharmacy “Carol Davila” Bucharest
Projects Director: Prof. Dr. Nicolae Daniel Pirici

Projects objective:
Our main objective is to establish the role of CEACAM1 by its different isoforms in invasiveness and aggressiveness of thin melanomas. To accomplish this objective we will assess the differences of CEACAM1 isoforms expression in tumor and inflammatory cells in RM and nRM versus halo nevi and common nevi as control.

Considering the design of our study, several specific secondary objectives will also be fulfilled:

• elucidating the mechanisms involving CEACAM1 in inflammatory response in thin melanomas
• identifying the pathophysiological mechanisms through which CEACAM1 is involved in tumor regression/progression and evaluate its seric level as biomarker for thin melanomas progression.
• gaining new insights into the mechanisms involved in host’s immune response in RM and nRM vs control and subsequent correlation with other cell adhesion molecules and markers of EMT
• establishing the peculiarities of the relation between CEACAM1 expression and different types of immune responses in thin RM and nRM versus control.
• establishing a new criterion/ criteria for supplementary classification of thin melanomas

Estimated results:
In order to achieve all the foreseen results we structured our activity in six working packages. The estimated results are:

• Annual reports (M12, M24) and Final Report (M 30) – including scientific and financial report;
• Research report about CEACAM1 expression in RM and nRM melanomas versus control (M24);
• Research report: correlation between CEACAM1 expression and consecrated progression-related tissue markers in melanoma with and without regression versus control (M24);
• CEACAM1 isoform levels in plasma & tissue in relation to melanoma aggressiveness (M24);
• Scientific report: mechanisms involving CEACAM1 in melanoma (M30);
• three scientific papers (M12, 24, 30) and three presentations (M12, 24, 30)

Date projects starts: 12/07/2017.

Date projects ends: 31/12/2019.

Projects duration (months): 30.

Total financed value: 850.000,00 lei.

Total co-financed value: 0 lei.

Phase 1/2017

Update Phase 1

The First Phase of this project includes management and dissemination activities, as well as tasks 1 and 2 of WP2 and task 1 of WP4.

The members of the research team have agreed to communicate online at regular intervals (one week), as well as to document the meetings through written memos and verbal processes.

For a better supervision of the project, Gantt graphics are used.  A detailed plan of activities involving the assignment of activities, responsibilities, intermediate and final deadlines as well as the efficient and equitable division of the tasks between the members of the research team was made.

1. WP1: Management

1.1The creation of the electronic support of the patient database; the initiation of the registration of patients in the database

The database has been built in compliance with recent literature, with similar studies as well as with the informational requirements of the project, so that it could allow for rapid access to complete info on the patient as well as to clinical, dermatoscopic and hystopathological images.

1.2 The creation and implementation of the research project’s web page

All information regarding the actual state of the project (actualized data regarding the project’s objectives, the research team’s members as well as the activity plan for 2018) is available on the project’s web page.

1.3 The study of the bibliography (in physical, electronic and online format); the creation of the database for the bibliography

The access to national and international databases (Medline, Pubmed, ScienceDirect, Thomson ISI) allowed for the selection and analysis of peer-reviewed publications regarding the following themes: cell adhesion molecules, CEACAM1, melanoma regression, the role of dendritic cells in melanoma regression, with the prioritization of the articles published in the last 5 years in prestigious publications (British Journal of Dermatology, Journal of the American Academy of Dermatology, Archives of Dermatological Research, Nature Biotechnology, Skin Research and Technology).

The bibliography was encompassed in a database containing approximately 100 full text articles together with abstracts and documents of the main international conferences from the last three years treating the following topics: dermatooncology, dermatopathology, molecular biology, immunohistochemistry, dermatoscopy.

In order to identify the latest international tendences in the scientific research and medical practice, a comprehensive scientometric study was made.

The operationalised scientometric methodology involved the combined use of two semantic analysis software instruments (Tropes Zoom) and network analysis (Gephi). The semantic analysis instrument integrates two work models: the sentence analysis of the speech and the predictive sentence analysis.

The analysed text body was composed of the abstracts of 19.024 scientific articles recorded in PubMed (883 articles containing the keyword ,,CEACAM1’’, published in the timeframe 1981-2017 as well as 18.141 articles containing the keyword ,,melanoma’’ published in the last three years).

The operationalizing of the semantic analysis involved the creation of an analysis dictionary (analysis scenario) with 18 semantic classes, 61subclasses and 231 keywords. The semantic classes represent functional categories of the tissue markers (referenced in the analysed article body and of interest to this project), analytical techniques (ICH, immunofingerprint and ELISA) as well as other generic terms of interest for the analysis (such as melanoma and other types of cancer); the semantic subclasses represent the tissue markers (which are individualised in the semantic structure in order to stress the particular correlations as they are reflected in the analysed scientific articles).

The semantic analysis software instrument allows for the export of semantic ontology in a Gephi-compatible format, allowing for mathematical and statistical supplementary processings which are relevant for the scientometric analysis.

2. WP2: The expression of CEACAM1 isoforms and the correlation with the inflammatory response in the regression melanoma (RM) and non-regression melanoma (nRM), respectively

2.1. The assembly of the multitisular paraffin blocks (WP2, task 1)

45 cases belonging to patients who had been histopathologically diagnosed with thin malignant melanoma (≤ 2mm, corresponding to stages pTis, pT1, pT2) were selected from the archive of the Pathological Anatomy Service of the Clinical Hospital Colentina. Using the consultation sheets, the patients’ clinical and follow-up data (sex, age, localization, dimensions) have been identified.

From a histopathological point of view, the presence of consecrated or about to be validated prognosis factors was analysed: the Breslow index, the Clark level, the invasion of different normal adjacent structures, the presence of ulceration, of vascular invasion, of perineural invasion, of intratumoral inflammatory infiltrate, the mitotic index, the presence of regression areas, the presence of satellite nodules/metastases of the lymphatic ganglia, the presence of regression areas, of satellite nodules/ transitory metastases, the presence of lymphatic ganglion metastases, of visceral metastases and of nevic associated lesions.

The hematoxylin-eosin stained slides have been re-examined and the areas presenting interest from a histopathological point of view have been identified, sectioned and manually included in multitisular blocks.

Sections with a thickness of 3 microns have been made from each multitissue block and collected on polysine slides, in order to be analysed using immunohistochemistry for CEACAM1, N-cadherin, E-cadherin, β-catenin, TGF-β, MMP, TIMP, p53, p16, p21, Ki67, fas and VEGF.

Also, in order to prepare the second Phase of the Project’s Implementation, articles regarding the tissue expression and CEACAM isoforms have been consulted.

Multitisular paraffin blocks as well as their corresponding hematoxylin-eosin stained slides have been obtained.

The processing of the data obtained after consulting the archive and examining the slides has given the following data:

• the actual lot includes 19 men and 26 women with ages between 26 and 84 years (mean age 55 years);
• the minimal Breslow index (which is also an important pronostic factor in the thin melanoma subgroup [2]) was 0,20mm (the maximum one having been chosen of 2mm), 14 lesions (31% of the total) with the Breslow index between 0,20 and 1mm, the rest being thinner than 1mm.
• 14 lesions presented tumour regression, 14 ulceration, and 4 vascular invasion. The neural invasion was absent on all the examined lesions.

2.2. The expression of the CEACAM1 isoforms in the RM and nRM compared to the control group (WP2, Task 2)

An ample documentation regarding the expression of the CEACAM1 isoforms in regression melanomas (RM) and non-regression melanomas (nRM), respectively, has been made.

Following this documentation it has been decided that, in order to analyse the expression of CEACAM1 as a key element of the immunological mechanisms involved in the progression/regression of thin melanomas, immunohistochemical tests as well as a serological evaluation of cell adhesion molecules will be performed. The expression of CEACAM1 will be correlated with tissue markers which are already consecrated in the progression of thin melanoma, using benign naevi as control group.

3. The collection of biological samples (fresh tissue and serum) (WP4, task 1)

The consents and approvals were prepared in accordance to the research ethics and medical practice guidelines, to the international WHO and EU guidelines as well as to the national legislation.

After having been explained the procedures and having, subsequently, signed the consent, a case has been enroled in the prospective study. For sampling of the biological products destined to be analysed at the Virology Institute, a preestablished protocol has been followed. Blood has been collected and transported to the Virology Institute, where the samples were centrifuged, aliquoted and afterwards stored at -80°C.

In order to obtain tumour tissue samples, after the surgical excision, the tumour was transported to the pathological anatomy laboratory, where a fragment of about 10 mg was cut and afterwards stored at -80°C.

In order to realise a large database so that the obtained results could have statistical significance, collaborations with family doctors and other medical specialists from the Elias Universitary Hospital and Colentina Clinical Hospital as well as other medical units have been established. The patients have been counselled for free clinical, dermatoscopical and optical tomography evaluation of premalignant and malignant cutaneous lesions. The patients were informed about the study’s objectives to compare the performances of different imagistic methods for early detection of cutaneous cancers as well as about the fact that the obtained information will be available for them in order to establish the consequent treatment plan.

Phase 1/2017

4. Dissemination of the study’s results

The objectives set in this Phase were completely accomplished, with the achievement of the results expected in the Activity Plan.

The preliminary results of the deployed research activity were included in three papers presented at international conferences as well as four papers presented at the National Dermatology Congress (with international participation). After consulting the studies in this domain from the current literature, two articles have been elaborated, among which one has already been published in a BDI-indexed magasine, and the second has been accepted for publishment in the first trimester of 2018.

The annual congress of the European Academy of Dermatology and Venerology represents a prestigious event for European dermatologists, which has the objectives the promotion of excellence in research, the clinical care and the continuous medical education. The papers presented at Geneva were elaborated by the members of the research team, starting from the cases evaluated during the phase of enrolement of the patients in the study.

At the National Dermatology Congress which took place in Brasov, in October, the team members have presented their colleagues a series of relevant cases for this type of tumour pathology.

The scientometric analysis will be integrated in one of the scientific articles which have been planned for the next Phase of the project’s implementation.

Articles accepted for publication in journal indexed in IDB:

Stîngă Patricia-Irina, Stoica Alina, Cioplea Mirela Daniela, Zurac Sabina, Nichita Luciana, Popp Cristiana, Cioroianu Alexandra Ioana, Bastian Alexandra Eugenia. SPITZOID MELANOMA-CASE REPORT; POTENTIAL ROLE OF ALK EXPRESSION IN THE DIAGNOSIS OF SPITZOID MELANOCYTIC LESIONS - Romanian Journal of Clinical and Experimental Dermatology

Conferences:

Balmes G., Todorovic T., Zurac S., Brinzea A., Nedelcu R., Turcu G. Nodulul sister Mary Joseph - Congresul National de Dermatologie 2017, Brasov, Romania, De la 04/10/2017 Pâna la 07/10/2017

Balaban M., Brinzea A., Turcu G., Ion D.A., Zurac S., Cioplea M.D., Stoica A., Popescu R., Popescu C., Antohe M., Nedelcu R. Epiteliomul calcificat Malherbe la nivel facial - Congresul National de Dermatologie 2017, Brasov, Romania, De la 04/10/2017 Pâna la 07/10/2017

Balaban M., Turcu G., Brinzea A., Ion D.A., Zurac S., Stoica A., Cioplea M.D., Popescu R., Popescu C., Antohe M., Nedelcu R. Dermatofibrosarcoma protuberans Darier-Ferrand: raportare de caz - Congresul National de Dermatologie 2017, Brasov, Romania, De la 04/10/2017 Pâna la 07/10/2017

Brinzea A., Popescu R., Popescu C., Hodorogea A., Balaban M., Calinescu A., Nedelcu R., Turcu G., Ion D.A., Antohe M., Ionescu M., Teodorescu C., Vacarelu C., Gosa R., Balaban M. Aspecte dermatoscopice în epiteliomul bazocelular pigmentat vs. nepigmentat - Congresul National de Dermatologie 2017, Brasov, Romania, De la 04/10/2017 Pâna la 07/10/2017

Update Phase 2

This report concerns the second phase of implementation of the project, namely  the interval between January and December 2018.

The materials assigned for this phase of the scientific project (month 18) are a management report, at least 3 articles submitted for publishing and at least one presentation at a scientific manifestation. Although the scientific reports for WP2, WP3, WP4 were planned for July 2019, in order to ensure the clarity of the report, short reports with the partial results of these WPs have been made.

Management (WP1): The members have established a weekly communication protocol using the internet. The activity plan at the level of subactivities  was detailed; the Gantt graphic was used to supervise the assigned activities.

National and international databases such have been used, with the selection of peer-reviewed recent journals with a high impact factor. The bibliographical database has been updated. The informatic support for the database of the patients has been used.

The expression of CEACAM1 and the correlation with the inflammatory response in regression and non-regression melanomas (WP2):

53 consecutive cases of thin melanomas (thickness of maximum 2 mm, diagnosed immunohistochemicaly and histopathological in the last two years), among which 21 with regression and 32 without regression, were analysed.  The presence of consecrated or about to be validated prognosis factors  - tumor subtype, tumor thickness, presence or absence of ulceration, mitotic index, Breslow and Clark indexes, CEACAM1 expression was analysed. Sections with a thickness of 3 mm have been obtained and stained using hematoxylin-eosine. The corresponding cases have been further analysed using immunohistochemistry techniques. For all the cases, the final diagnosis was of melanoma with a Breslow index smaller than 2 mm.

The seriate sections have been analysed for the detection of CEACAM1. Three different isoforms of this marker have been analysed.

The seriate sections have been stained using immunohistochemical techniques and a comparison between the expression of CEACAM1 in regression and non-regression areas of regression melanoma, respectively, as well as in non-regression melanomas, has been made. The positivity of the tumor cell membranes in the non-regression melanomas and in the areas without regression of the regresion melanomas has been analysed. At the same time, we have analysed the intensity of the expression in different areas of the tumors (junctional, dermal, corresponding to the invasion) and we have quantified it using a semiquantitative scale: 0 - absent, 1 – weak positivity, 2 – moderate positivity and 3 – intense positivity.

All data has been introduced into a database and statistically analysed using Microsoft Excel and SPSS. The t test and Pearson correlation test, with a value p <0,05, have been used.

52 of the cases were superficial melanomas and only one case (presenting regression) was an acral-lentiginous melanoma. Also, only one case (with regression) presented ulcerations, all the other ones lacking this characteristic. The medium Breslow index was 0,688 mm (with values between 0,16 and 1,75 mm), higher in non-regression melanomas (0,770 mm) compared to the regression ones (0,660 mm).

The mitotic index was evaluated between 0 and 9 mitotic divisions/ mm², with a mean value of 1 division/mm².

All the three CEACAM1 isoforms had similar reactivity. CEACAM1 was positive in the epithelial cells of the sudoral gland ducts and of the sebaceous glands (used as a positive internal control).

Out of the 32 cases of non-regression melanoma, only in 3 cases CEACAM1 was negative in the tumoral cells, in all the other cases the positivity of the membrane being identified (as weak – 13 cases, mild – 14 cases and intense – 2 cases).

A significant difference between the tumor cells remaining from the regressed areas and the tumor cells from the non-regressed areas has been observed in the group of regressed melanomas. In the regressed areas, the tumor cells were negative for CEACAM1 in 16 cases and weakly positive in just 5 cases. In the non-regressed areas, the tumor cells were positive for CEACAM1 in 19 cases and negative in just 2 cases.

The statistical correlation have shown that the difference in the reactivity of CEACAM1 between the tumor regressed and non-regressed region is highly significant (t-test, p <0,0001).

The correlation between the Breslow index and the CEACAM1 reactivity was significant only in the regressed melanomas (Pearson correlation test, p = 0,22589). The thicker tumors had a more intense global positivity for CEACAM1 in all the studied tumors, but in the non-regression melanomas the correlation was not statistically significant.

In 34 cases – approximately 64% of all the cases, we have observed a more intense CEACAM1 expression in the tumor cells in the deep area of invasion. This characteristic has been observed in the regression melanomas (11 cases out of 21) as well as in the non-regression melanomas (23 cases out of 32).

Thus, in the areas without regression, a more intense signal of the CEACAM1 molecule has been identified in the profound area of invasion. In the thin melanomas, the increased expression of CEACAM1 was correlated with the invasivity, suggesting the fact that the melanomas positive for CEACAM-1 are more aggressive.

The correlation of the CEACAM-1 expression with the expression of the tisular progression of consecrated markers in the regression and non-regression melanomas (WP3):

We have selected all the cases of melanoma at the patients under 31 years old, diagnosed between 01.01.2013 - 31.08.2017, from the archive of the Pathology Service of the Colentina Clinical Hospital. The youngest patient was 15 years old. Using clinical and histological reports, more data, including the sex, age, localization, tumor diameter, histological subtype, Breslow index, Clark index, mitotic rate, ulceration, vascular invasion, metastases and presence of the associated nevus were identified.

All the tissue samples were processed, and multiple sections of 2,5 mm resulted. The sections were stained using hematoxylin and eosin and analysed independently by two pathologists with experience in skin tumors. A dermatopathology expert’s input has been used, as well. The statistical interpretation of the data was made using the t test.

The studies included 28 patients with the histopathological diagnosis of melanoma and ages between 15 and 30 years old, the median age being 24,25. 25% of the patiens were between 15 and 20 years old, 25% between 21 and 25 years old and 50% between 26 and 30 years old..

19 patients were women and 9 men, this being in accordance to the literature data which indicates the increased incidence of melanoma in young women.

Most of the lesions (15) were localised on sun-exposed areas. We have microscopically diagnosed 18 superficial melanomas, 4 nodular melanomas and 6 in situ melanomas.

The Breslow index has varied between 0,25 mm and 13 mm, with a median of 2,32 mm. In this group, the thin melanomas (under 1 mm in diameter) were the most frequent (15 cases). Only 7 lesions were thicker than 2 mm. The youngest patient in our group (15 years old) had the largest value of the Breslow index (13 mm).

The mitotic index varied a lot between 0 and 30 de mitoses/mm².In almost half of the patients (n= 13), there are no tumor identified mitoses. Three of them had in situ melanoma. The Breslow index also correlates with the mitotic index.

Overall, the study identified a higher risk for an increased tumoral width in young patients.

The evaluation of CEACAM1 level in tumor and plasma (WP4):

According to the already established protocol for the prelevation of biological samples for the tests made at the Virology Institute, in 2018 more patients were enrolled in the prospective study.

Thus, the blood samples were prelevated, processed and the CEACAM1 expression level in the serum of patients with dermatological conditions was analysed using the kit „Human CEACAM1 ELISA Kit (ABCAM, SUA; ab215540), according to the manufacturer’s indications.

After the surgical resection, the tissue samples were cut into fragments of minimum 10 mg and deposited at -80°C.

In order to evaluate the expression of the CEACAM1 isoforms, total RNA was extracted from the bioptic fragments and, after determining its concentration and purity it was reverse-transcribed starting with 2 µg RNA.

In the qPCR reaction were introduced 50 ng cDNA for each sample, the used primers being CEACAM1 - Hs00266109_m1 for the isoforms 2 si 5; CEACAM1 - Hs00989786_m1 for the isoforms 1,3 si 4; CEACAM1 - Hs00992687_mH for the X1 variant; as well as TIMP3 - Hs00165949_m1. The quantification of the expression level was made using Plus Real-Time PCR System (Thermo Fisher Scientific, SUA), each experiment being made in triplicate. The results were analysed with StepOne Software v2.3 (Thermo Fisher Scientific, SUA), the comparison of the relative expression levels being made using the ΔC_T method_. The level of expression of the GAPDH gene was used as endogen control.

The results were analysed using the simple standardization (endogen control) and the double standardization (considering NEV control group).

The blood levels of CEACAM1 in the first 28 patients included in the study have already been determined. The rest of the blood samples are still being analysed. According to the histopathological result, the 28 patients have been divided into 5 diagnosis cathegories, in order for the statistical analysis to be performed (nevus, dysplasic nevus, melanoma, dermatofibroma, basocellular epithelioma), as follows: Nevus - 5 cases, Dysplasic Nevus - 5 cases, Melanoma - 14 cases, Dermatofibroma - 2 cases, Basocellular epithelioma (abbr.EPI) 2 cases. During the intermediate statistical analysis, no significant differences between MEL and NEV, on one side, and NVD, on the other side, were found.

In the analysed tissue samples, the expression level of RNA of the isoforms 2 and 5 was increased. Regarding the CEACAM, as well as the TIMP3 isoforms, the expression increased proportionally to the severity of the malignancy.

Scientific dissemination (WP6):

The results of the scientific activity of the project’s research team for 2018 are: 28 presentations at international scientific events and 19 presentation and workshops at national with international participation scientific events. There have been elaborated: one book chapter; 4 articles published in BDI-indexed journals; 5 articles published/accepted for publishing in ISI-indexed journals, among which one is published in an ISI-indexed journal (IF=3,298) in 2018, one has been published in an ISI-indexed journal in 2017 without after the report corresponding to the respective phase and 3 articles have been accepted for publishing in 2019.

The team members have presented numerous papers regarding the cases which were evaluated during the patient enrollement phase at the annual congress of the Dermatology and Venerology European Academy.

The team members have presented at the National Dermatology Congress (Brasov, October 2018), many relevant cases for this type of tumor pathology.

During the ,,Interdisciplinary congruences in imunodermatology’’ manifestation organised by the Imuno-Dermatology Romanian Association, the team members have held three oral presentations centered around the CEACAM-1 molecule, immune system and cutaneous cancers, the main issues studied in this project.

The objectives set for this phase have been successfully accomplished and all the conditions to pass to the next phase of the project have thus been created.

Book chapter

A Bastian, L Nichita, S Zurac. Matrix Metalloproteinases in Melanoma with and without Regression. The Role of Matrix Metalloproteinase in Human Body Pathologies, IntechOpen, London, UK, 2018. ISBN 978-953-51-3717-7

ISI articles

1. A Călinescu, G Turcu, RI Nedelcu, A Brînzea, A Hodorogea, M Antohe, C Diaconu, C Bleotu, D Pirici, LB Jilăveanu, DA Ion, IA Bădărau. On the Dual Role of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 (CEACAM1) in Human Malignancies. Journal of Immunology Research, 2018, IF=3.29
2. C Dumitru, A Bastian, C Popp, M Neagu, C Constantin, M Cioplea, L Nichita, R Andrei, T Vassu, S Zurac. Immunohistochemical features of partial regression in cutaneous melanoma – E-cadherin key molecule. Human & Veterinary Medicine International Journal of the Bioflux Society, 2017
3. M Antohe, RI Nedelcu, L Nichita, C Popp, M Cioplea, A Brînzea, A Hodorogea, A Călinescu, DA Ion, C Diaconu, C Bleotu, ND Pirici, SA Zurac, G Turcu. Tumor infiltrating lymphocytes - the regulator of melanoma evolution. Oncology Letters, accepted for publication, IF=1.66

4. R Nedelcu, M Balaban, G Turcu, A Brînzea, D Ion, M Antohe, A Hodorogea, A Călinescu, C Popp, M Cioplea, L Nichita, S Popescu, C Diaconu, C Bleotu, D Pirici, R Popescu, C Popescu, S Zurac. Efficacy of methotrexate as anti-inflammatory and anti-proliferative drug in dermatology. Experimental and Therapeutic Medicine, accepted for publication, IF=1.41
5. L Nichita, S Zurac, A Bastian, P Stinga, R Nedelcu, A Brînzea, G Turcu, D Ion, L Sticlaru, C Popp, M Cioplea.^.Comparative analysis of CEACAM-1 expression in thin melanomas with and without regression Oncology Letters, accepted for publication, IF=1.66

Articles published in BDI journals

1. M Cioplea, S Zurac, C Popp, P Stinga, A Cioroianu, R Nedelcu, A Brînzea, G Turcu, DA Ion, A Bastian. Malignant melanoma in young patients: histopathologic particularities. ROJCED-Romanian Journal of Clinical and Experimental Dermatology, vol 5, no 1:12-17, 2018
2. G Balmeș, TA Todorovic, I Nedelcu, RI Nedelcu, A Brînzea, G Turcu. Autoimmune progesterone dermatitis: a thorough clinical history may lead to the diagnosis of a rare disorder – EMJ Dermatology 1 2018
3. M Balaban, G Turcu, A Brînzea, A Hodorogea, A Călinescu, M Antohe, D Ion, R Nedelcu. An atypical case of acrokeratosis paraneoplastica Bazex. – . EMJ Dermatology 1 2018
4. A Călinescu, R Popescu, C M Popescu, A Hodorogea, A Brînzea, L Zeiler, RI Nedelcu, G Turcu, M Antohe. Polymorphic eruption of pregnancy with postpartum onset-case report, EMJ Dermatology  1 2018

Workshops

1. RI Nedelcu, A Brînzea, G Turcu, M Antohe, M Balaban, A Hodorogea, A Călinescu, E Balasescu, R Popescu, C Popescu, DA Ion. Workshop-De ce dermatoscopia?. ZEM-Zilele Educatiei Medicale, Bucuresti, 2018
2. A Brînzea, RI Nedelcu, G Turcu, M Antohe, M Balaban, A Hodorogea, A Călinescu, E Bălășescu, DA Ion, R Popescu, C Popescu. Workshop : Veștile din spatele dermatoscopului. ZEM-Zilele Educatiei Medicale, Bucuresti, 2018