Vertical Growth Phase Research Articles

Melanoma in situ and low risk pT1a melanoma: Need for new diagnostic terminology

Melanoma incidence has risen rapidly, at least until recently, while mortality has changed only a little, a phenomenon suggestive of overdiagnosis, which can be defined as the diagnosis as “melanoma” of a lesion that would not have had the competence to cause death or symptoms even if it had not been excised. Overdiagnosis has been attributed to efforts at early diagnosis (“overdetection”), and to changes in criteria resulting in diagnosis as melanoma of lesions previously termed nevi (“overdefinition”). In terms of overdefinition, there is evidence that criteria for the histopathologic diagnosis of melanoma has changed over a period of approximately two decades. Specialization may play a role in overdefinition; research has shown that when pathologists interpret the same lesion, dermatopathologists are more likely to diagnose low stage (AJCC T1a) melanomas and general/surgical pathologists are more likely to diagnose atypical nevi. An important subset that contributes to overdiagnosis is those melanomas that lack the property of tumorigenic vertical growth phase, thus lacking metastatic competence, and perhaps not warranting diagnosis as overt melanomas. Studies have defined subsets of patients with very low stage lesions diagnosed as melanomas in which observed survival has been 100%. In the past, many of these lesions would have been diagnosed as nevi, constituting overdefinition. Other key characteristics for very low risk (or no risk) lesions that are currently termed invasive “melanomas” include low Breslow thickness, Clark's level II invasion, absence of mitoses, and clinically, lack of observed or experienced dynamic changes. We propose a provisional terminology for diagnosing extremely low risk subgroups as “Melanocytic neoplasms of low malignant potential”, aimed at reducing the negative personal and social effects of a cancer diagnosis for patients whose health and wellbeing are in reality not affected by an overdiagnosed “melanoma”. With additional confirmation and appropriate consensus, it is likely that some of these subgroups can be reclassified as atypical or dysplastic nevi.

ToF-SIMS imaging reveals changes in tumor cell lipids during metastatic progression of melanoma.

Most melanomas progress from radial to vertical growth phase before spreading locoregionally and distally. Much is still unknown about the metabolic changes in the tumor cells and their microenvironment during this metastatic progression. We aimed to gain new insight into the molecular characteristics of melanoma in regard to spatial lipidomics to deliver new knowledge regarding tumor metastatic progression. We included 10 fresh tumor samples from 10 patients including two insitu melanomas, two invasive primary melanomas, and six metastatic melanomas (four in-transit metastases and two distant metastases). In addition, we analyzed four healthy skin controls from the same patients. Time-of-flight imaging secondary ion mass spectrometry (ToF-SIMS) enabled detailed spatial-lipidomics that could be directly correlated with conventional histopathological analysis of consecutive H&E-stained tissue sections. Significant differences in the lipid profiles were found in primary compared to metastatic melanomas, notably an increase in phosphatidylethanolamine lipids relative to phosphatidylinositol lipids and an increase in GM3 gangliosides in the metastatic samples. Furthermore, analysis of the data from in transit versus distant metastases samples highlighted that specific phospholipids, and a difference in the long versus shorter chain GM3 gangliosides, discriminated the metastatic routes. Further studies are warranted to verify these preliminary findings. Lipidomic changes could serve as a novel biomarker for tumor progression and even serve as a target for novel treatments. Furthermore, analyzing the lipid profiles could help to differentiate between primary and metastatic melanomas in challenging cases.

CD44 and CD271 expression and chemoresistance in BRAF V600E/K mutation–positive melanoma.

e21542 Background: Melanoma stem cells (MSC) are thought to play a role in cellular proliferation, migration, and chemotherapeutic resistance. We investigated native expression of several putative MSC biomarkers in human BRAF V600E/K mutation positive melanoma cell lines of different phases—radial growth phase (WM35), vertical growth phase (WM278)—and metastatic (LM-Mel-45, LM-Mel-71) cell lines. We examined cell viability of select cell subpopulations during in vitro treatment with BRAF and MEK inhibitor monotherapy and combination therapy. Methods: Using flow cytometry, we assessed surface expression of putative MSC biomarkers: CD44, CD271, CD133, CD186, and CD20. We sorted each cell line by CD44 and CD271 expression with a predetermined hierarchical gating strategy and compared 4 chemotherapy conditions: control, BRAF inhibition with 1.25 µM dabrafenib, MEK inhibition with 0.125 µM trametinib, and combination therapy. Cells (25,000) were collected directly into chemotherapy treated media and incubated for 72 hours. Cell viability was measured by MTT assay. Statistical analysis used mixed-effects ANOVA and Tukey HSD. Results: No significant expression of CD133, CD186, or CD20 was found in any cell line. Over 99% of live cells were CD44+ in all 4 cell lines. Only WM35 and LM-Mel-71 had both CD44+/CD271+ and CD44+/CD271- subpopulations. CD271 expression was restricted to CD44+ cells. Among live cells, the CD44+/CD271+ subpopulation was 31.2% for WM35 and 14.4% for LM-Mel-71. For WM35, cell viability in CD44+/CD271+ cells was greater than CD44+/CD271- cells for all treatments (p < 0.001). For LM-Mel-71 cells, CD271 expression did not affect cell viability within any treatment (p = 0.64). For chemotherapeutic treatments, cell viability in CD44+/CD271- subpopulation was higher in LM-Mel-45 cells than WM278 (p < 0.03) or LM-Mel-71 (p < 0.002), but not WM35 cells (p > 0.05). Trametinib monotherapy and combination therapy had similar cell viability in all 4 cell lines (p > 0.05). Conclusions: To our knowledge, our study is the first to show differences in phenotypic expression of CD44 and CD271 across nonmetastatic and metastatic melanoma cell lines and suggests co-expression of CD44 and CD271 confers increased chemotherapeutic resistance to in vitro BRAF and MEK inhibitor treatment in radial growth phase but not metastatic melanoma. More research is needed to better understand the role of CD44 and CD271 co-expression in melanoma initiation, progression, and chemotherapeutic resistance. CD271 remains a potential immunotherapeutic drug target in the treatment of BRAF V600E/K mutation positive melanoma.

Cell viability and chemotherapeutic resistance across melanoma lines from various stages of progression

Background: The melanoma stem cell (MSC) hypothesis holds that a subpopulation of cells with stem-cell like features are important for tumor progression and chemoresistance. We examined four V600E BRAF-mutated melanoma lines at various stages of melanoma progression. Within each cell line, we assessed the expression of putative MSC markers: CD20, CD44, CD133, CD186, and CD271. Subsequently, we assessed cell viability in the four lines during mono- or combination chemotherapy relative to surface marker expression. Methods: We examined two primary melanoma lines: WM35 (radial growth phase) and WM278 (vertical growth phase). Additionally, we analyzed two metastatic melanoma lines from brain metastases: LM-Mel-71 and LM-Mel-45, exhibiting epithelial and fibroblastic morphologies, respectively. MSC surface marker expression was determined using a Biorad S3e cell sorter to collect 25,000 cells each into one of 4 chemotherapeutic treatments on a 96-well plate. The treatments included: control, dabrafenib (1.25 μM, BRAF inhibitor), trametinib (0.125 μM, MEK inhibitor), and a combination of dabrafenib (1.25 μM) and trametinib (0.125 μM). Following 72 hours of incubation, we analyzed cell viability using an MTT assay. Statistical analysis was performed using ANOVA and Tukey posthoc testing. Results: All cell lines exhibited over 99% CD44+ cells. The only surface markers detected were CD44 and CD271; the remaining markers were undetectable across the four cell lines. LM-Mel-71 and WM35 cells presented both CD44+/CD271+ (14% and 31%, respectively) population and a larger CD44+/CD271- population. LM-Mel-45 and WM278 cells displayed a CD44+/CD271- population. LM-Mel-45 consistently exhibited higher viability than LM-Mel-71 (p<0.002) or WM271 (p<0.03) under both control and the three chemotherapeutic regimens. For LM-Mel-71, cell viability did not differ with CD271 expression in any treatment condition (p=0.64). For WM35 cells, viability was significantly higher in CD271+ compared to CD271- cells under all treatment conditions. Based on cell viability, monotherapy with trametinib or combination therapy appeared more effective than monotherapy with dabrafenib alone across all melanoma lines. Conclusions: The results suggest that there is not a simple relationship between CD271 expression and the origin of the cell line. While CD44+CD271+ expression may correlate with increased cell viability — as observed in WM35 radial growth phase melanoma — such an association is not universal, as CD44+/CD271+ and CD44+/CD271- cells demonstrated similar viability under each chemotherapeutic condition. Additional factors beyond the examined surface markers likely modulate melanoma progression and BRAF/MEK chemotherapy effcacy. Supported by A T Still Univeristy Student Research Fund Grant 560-950 to EH and MR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dermoscopy as a Tool for Identifying Potentially Metastatic Thin Melanoma: A Clinical-Dermoscopic and Histopathological Case-Control Study.

Despite being early-stage tumors, thin cutaneous melanomas contribute significantly to mortality and have a rising incidence. A retrospective case-control study was performed to identify clinical-dermoscopic and histopathological variables linked to local and distant metastases in melanomas ≤0.8 mm. Data from 1 January 2000 to 22 June 2022 were analyzed from two Italian skin cancer referral centers. Sixteen patients with ≤0.8 mm melanomas developing metastases were studied compared to controls without metastases over 5 years. Statistical analysis involved Pearson's chi-squared test or Fisher's exact test. Of the 1396 cases, 1.1% progressed. The median diagnosis age was 49 (range 28-83), with 56.3% men and 43.7% women. The torso was the primary tumor site (43.7%). Clinically, lesions were pigmented (>10 mm diameter: 73.3%, ≥3 colors: 80%). Dermoscopically, the common features were white patches (73.3%), atypical vascular patterns (66.5%), blue-gray areas (60%) and absent pigment networks (60%). Histopathologically, all cases had adverse features like regression (87.4%), dermal mitoses (50%), a vertical growth phase (62.5%) and ulceration (12.5%). These findings were statistically significant compared to controls (p < 0.05). In ≤0.8 mm melanomas, specific clinical-dermoscopic traits might indicate higher metastatic potential when paired with adverse histopathological features.

The Keratinocyte in the Picture Cutaneous Melanoma Microenvironment.

Melanoma progression is a multistep evolution from a common melanocytic nevus through a radial superficial growth phase, the invasive vertical growth phase finally leading to metastatic dissemination into distant organs. Melanoma aggressiveness largely depends on the propensity to metastasize, which means the capacity to escape from the physiological microenvironment since tissue damage due to primary melanoma lesions is generally modest. Physiologically, epidermal melanocytes are attached to the basement membrane, and their adhesion/migration is under the control of surrounding keratinocytes. Thus, the epidermal compartment represents the first microenvironment responsible for melanoma spread. This complex process involves cell-cell contact and a broad range of secreted bioactive molecules. Invasion, or at the beginning of the microinvasion, implies the breakdown of the dermo-epidermal basement membrane followed by the migration of neoplastic melanocytic cells in the superficial papillary dermis. Correspondingly, several experimental evidences documented the structural and functional rearrangement of the entire tissue surrounding neoplasm that in some way reflects the atypia of tumor cells. Lastly, the microenvironment must support the proliferation and survival of melanocytes outside the normal epidermal-melanin units. This task presumably is mostly delegated to fibroblasts and ultimately to the self-autonomous capacity of melanoma cells. This review will discuss remodeling that occurs in the epidermis during melanoma formation as well as skin changes that occur independently of melanocytic hyperproliferation having possible pro-tumoral features.

Utilization of Imaging Modalities in the Diagnosis of Melanoma: A Scoping Review.

Melanomas arise de novo or in the context of a precursor lesion. Lesions typically grow radially and then undergo a vertical growth phase proceeding to local invasion and metastasis. This review describes the utility of different imaging modalities in diagnosis and melanocytic lesion monitoring. A literature search was performed in November 2023 utilizing EMBASE, Medline, and PubMed. The PRISMA diagram demonstrates the review process. Reflectance confocal microscopy (RCM) utilizes near-infrared light to help diagnose dermatologic lesions. RCMwas found to demonstrate nearly two times the positive predictive value compared to dermoscopy. The introduction of the Berlin Ultrasound (US) Morphology Criteria permitted a 65-80% improvement in diagnostic sensitivity. US with fine-needle aspiration cytology (FNAC) accurately predicts the necessity for sentinel lymph node biopsy and lymphadenectomy, sparing patients with metastasis and prompting biopsy for equivocal lesions. Single-photon emission computed tomography/computed tomography (SPECT/CT) is an adjunctive tool to anatomically and functionally assess lymphatic invasion. SPECT/CT improves the detection of sentinel nodes while decreasing operating time and improving cosmetic outcomes. 18F-fluorodeoxyglucose(18F-FDG) positron emission tomography/computed tomography (PET/CT) with small voxel reconstruction demonstrated increased specificity and sensitivity for detecting in-transit metastases of melanomas, specifically in the limbs. Dermoscopy allows providers to cost-effectively recognize common lesion patterns. Multiphoton microscopy assigns a weight-based score based on malignant features. Optical coherence angiography captures images of vessels to help diagnose equivocal lesions. Utilization of imaging techniques may increase diagnostic accuracy, reduce unnecessary procedures, and help guide treatment plans. Additional research is needed to further characterize the utility of these techniques in order to improve the diagnosis and treatment of melanomas.

Measuring Melanoma Nanomechanical Properties in Relation to Metastatic Ability and Anti-Cancer Drug Treatment Using Scanning Ion Conductance Microscopy.

A cell's mechanical properties have been linked to cancer development, motility and metastasis and are therefore an attractive target as a universal, reliable cancer marker. For example, it has been widely published that cancer cells show a lower Young's modulus than their non-cancerous counterparts. Furthermore, the effect of anti-cancer drugs on cellular mechanics may offer a new insight into secondary mechanisms of action and drug efficiency. Scanning ion conductance microscopy (SICM) offers a nanoscale resolution, non-contact method of nanomechanical data acquisition. In this study, we used SICM to measure the nanomechanical properties of melanoma cell lines from different stages with increasing metastatic ability. Young's modulus changes following treatment with the anti-cancer drugs paclitaxel, cisplatin and dacarbazine were also measured, offering a novel perspective through the use of continuous scan mode SICM. We found that Young's modulus was inversely correlated to metastatic ability in melanoma cell lines from radial growth, vertical growth and metastatic phases. However, Young's modulus was found to be highly variable between cells and cell lines. For example, the highly metastatic cell line A375M was found to have a significantly higher Young's modulus, and this was attributed to a higher level of F-actin. Furthermore, our data following nanomechanical changes after 24 hour anti-cancer drug treatment showed that paclitaxel and cisplatin treatment significantly increased Young's modulus, attributed to an increase in microtubules. Treatment with dacarbazine saw a decrease in Young's modulus with a significantly lower F-actin corrected total cell fluorescence. Our data offer a new perspective on nanomechanical changes following drug treatment, which may be an overlooked effect. This work also highlights variations in cell nanomechanical properties between previous studies, cancer cell lines and cancer types and questions the usefulness of using nanomechanics as a diagnostic or prognostic tool.

Clinicopathological Profile of a Cohort of Patients With Malignant Melanoma in the United Kingdom.

Introduction Malignant melanoma (MM) is potentially a fatal type of skin cancer and a major health concern for the Caucasian population. It is a heterogeneous disease with a wide spectrum of manifestations. Therefore, in this study, we evaluated the clinicopathological characteristics of MM. Methods We retrospectively studied the clinicopathological characteristics of MM in 167 biopsy-proven cases of MM reported between January 2020 and December 2021 at Kings Mill Hospital, Sutton-in-Ashfield, United Kingdom. Clinical data such as the age, sex, and anatomical site of the lesion were obtained from the clinical referral forms. Biopsies of the lesions were performed, and the specimens collected were sent to the laboratory for histopathological study and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation evaluation. Formalin-fixed paraffin-embedded blocks (FFPE) were prepared, sectioned, and stained with hematoxylin and eosin for histological examination. Results A total of 167 cases of MM were included in the study. The age range was 23-96 years, with the median age at diagnosis found to be 66 years; males were more commonly affected (52.1%). The median Breslow thickness was 1.20 mm. The median mitotic activity was 1.0/mm2. The lower limb was the most common site of involvement (27.5%), followed by the thorax (25.1%). The most common histological subtype was superficial spreading melanoma (SSM) (77.8%), followed by nodular melanoma (14.4%). The in situ component was present in 95.8% of cases; a majority (92.2%) of the cases showed vertical growth phase, 71.9% of cases were at Clark's level IV of invasion, regression was noted in 70.7% of cases, ulceration was present in 21.6% of cases, and microsatellites were present in 3% of cases. Perineural invasion was present in 3% of cases, and lymphovascular invasion (LVI) was present in 4.2% of cases. BRAF mutation testing was performed on 36 cases, out of which 20 cases (55.6%) showed BRAF mutation. Acral lentiginous melanoma and nodular melanoma were most likely to show ulceration (66.7% and 37.5%, respectively). SSM and lentigo maligna melanoma were more likely to be associated with regression. Conclusion The study demonstrated that MM is prevalent among the elderly population with male predominance; SSM was found to be the most common subtype. The study further demonstrated various clinicopathological features of MM and its association with histological subtypes.

Intracellular mechanics and TBX3 expression jointly dictate the spreading mode of melanoma cells in 3D environments

Cell stiffness and T-box transcription factor 3 (TBX3) expression have been identified as biomarkers of melanoma metastasis in 2D environments. This study aimed to determine how mechanical and biochemical properties of melanoma cells change during cluster formation in 3D environments. Vertical growth phase (VGP) and metastatic (MET) melanoma cells were embedded in 3D collagen matrices of 2 and 4 mg/ml collagen concentrations, representing low and high matrix stiffness. Mitochondrial fluctuation, intracellular stiffness, and TBX3 expression were quantified before and during cluster formation. In isolated cells, mitochondrial fluctuation decreased and intracellular stiffness increased with increase in disease stage from VGP to MET and increased matrix stiffness. TBX3 was highly expressed in soft matrices but diminished in stiff matrices for VGP and MET cells. Cluster formation of VGP cells was excessive in soft matrices but limited in stiff matrices, whereas for MET cells it was limited in soft and stiff matrices. In soft matrices, VGP cells did not change the intracellular properties, whereas MET cells exhibited increased mitochondrial fluctuation and decreased TBX3 expression. In stiff matrices, mitochondrial fluctuation and TBX3 expression increased in VGP and MET, and intracellular stiffness increased in VGP but decreased in MET cells. The findings suggest that soft extracellular environments are more favourable for tumour growth, and high TBX3 levels mediate collective cell migration and tumour growth in the earlier VGP disease stage but play a lesser role in the later metastatic stage of melanoma.

Prognosis of polypoid melanoma: a comparative study with non-polypoid melanomas.

Polypoid melanoma is considered an exophytic and frequently non-pigmented variant of nodular melanoma with an adverse prognosis; however, very few studies have been published about it with contradictory results. Therefore, our objective was to determine the prognostic value of this configuration in melanomas. A transversal retrospective study of 724 cases was analyzed according to the main configuration (polypoid vs. non-polypoid) regarding their clinicopathologic characteristics and survival analysis. Of the 724 cases, 35 (4.8%) met the definition of polypoid melanoma; such cases, compared with non-polypoid melanomas, were associated with a high Breslow thickness (7 mm vs. 3 mm), 68.6% had a Breslow >4 mm; showed different clinical stages of presentation, and presented more ulceration (77.1 vs. 51.4%). In the 5-year overall survival (OS) analysis, polypoid melanoma is associated with a lower 5-year OS, together with lymph node metastasis, Breslow thickness, clinical stage, mitoses per mm 2 , vertical growth phase, ulceration, and state of the surgical margins; however, in the multivariate analysis, the factors that remained independent predictors of death were the Breslow thickness groups, the clinical stage, the presence of ulceration, and the state of the surgical margins. Polypoid melanoma was not an independent predictor of OS. We found a prevalence of 4.8% of polypoid melanomas, which showed a worse prognosis than non-polypoid melanomas, explained by a higher proportion of ulcerated cases, greater Breslow thickness, and ulceration. However, polypoid melanoma was not an independent predictor of death.

TERT Promoter Mutational Analysis as an Ancillary Diagnostic Tool for Diagnostically Challenging Melanocytic Neoplasms.

Telomerase reverse transcriptase promoter mutations (TPMs) have been shown to be common in melanoma and uncommon in benign nevi. To assess the use of TPMs as an ancillary diagnostic tool, we report the concordance of the TPM status with the final diagnosis in clinical cases with distinct differential diagnostic scenarios: dysplastic nevus versus melanoma, atypical Spitz nevus versus melanoma, atypical deep penetrating nevus (DPN) versus melanoma, and atypical blue nevus versus malignant blue nevus. In a control cohort, we found a positive TPM in 51/70 (73%) of the total melanomas with the highest frequency in vertical growth phase melanoma cases. Conversely, only 2/35 (6%) dysplastic nevi in our control cases were TPM-positive and b were severely atypical dysplastic nevi. Our clinical cohort of 257 cases had a positive TPM in 24% of cases diagnosed as melanoma and in 1% of cases with a benign diagnosis. The overall concordance of the TPM status with the final diagnosis was 86%. The TPM status had the greatest concordance (95%) with the final diagnosis in the atypical DPN versus melanoma group, with the rest of the groups ranging between 50% and 88%. Overall, our results suggest that TPMs are most useful in the differential diagnosis of atypical DPN versus melanoma. It also has some value in the differential diagnosis of atypical Spitz tumor versus melanoma and dysplastic nevus versus melanoma, whereas in our cohort, it did not contribute meaningfully to differentiating malignant blue nevus and atypical blue nevus.

Breeding of New Strains of Gracilariopsis lemaneiformis with High Agar Content by ARTP Mutagenesis and High Osmotic Pressure Screening.

ARTP (atmospheric and room temperature plasma mutagenesis) mutagenesis was tried on G. lemaneiformis, and mutagenesis conditions were confirmed. An osmotic pressure screening program was established. Mutants were identified and characterized of relevant physiological traits. The aim of the study is to try to use ARTP mutagenesis and osmotic pressure screening for the breeding of high-agar G. lemaneiformis. Treatment time of 46s was found to be an optimal mutagenesis time. The mutagenized spores were initially screened with 58‰salinity artificial seawater, and then, the surviving spores were screened twice with 60‰ salinity artificial seawater in their vertical growth phase and branch growth phase, respectively. Four fast-growing and hypertonic resistance gametophytes were selected. The actual photosynthetic efficiency [Y(PSII)], photochemical quenching (qL), and non-photochemical quenching (NPQ) of four mutants were measured. The values of Y(PSII) and qL of HAGL-X3 and HAGL-X5 were higher than those of the control in the early stage of salt stress. NPQs of HAGL-X3 and HAGL-X5 were higher than control in most of the times. The growth rates of the four mutants were higher than that of the control. HAGL-X4 was the highest. The agar content was measured; HAGL-X5 displayed the highest agar content among the tested strains. HAGL-X5 was more in line with expectations, because of its high agar content and good hypertonic resistance. In this study, the mutant of G. lemaneiformis with high agar content was obtained by the procedure, which provided a certain reference for the selection of G. lemaneiformis strains with high agar content.

Abstract A001: Shared gene expression and immune pathway changes associated with progression from nevi to melanoma

Abstract There is a need to identify biomarkers of melanoma progression to assist the development of chemoprevention strategies to lower melanoma incidence. In this study, we assessed the feasibility of creating a molecular signature for melanomagenesis using three publicly available RNA sequencing and microarray expression datasets. We performed differential expression and regularized regression analyses across nevi and melanoma samples to identify consistent genes associated with melanomagenesis. The regularized regression models demonstrated that a small number of genes could successfully distinguish between nevi and melanoma, providing evidence for the feasibility of creating a molecular signature. Differential expression analysis identified consistent upregulation of C1QB, CXCL9, CXCL10, DFNA5 (GSDME), FCGR1B, and PRAME in melanoma and consistent downregulation of SCGB1D2 in melanoma compared to nevi. Additionally, each of these genes demonstrated a linear association with the progression from benign nevi to dysplastic nevi, to radial growth phase melanoma to vertical growth phase melanoma, providing additional evidence for their role in melanomagenesis. Subsequent pathway analysis demonstrated significant enrichment of immune-related pathways among the differentially expressed genes. Overall, this study 1) demonstrates the feasibility of creating a gene signature for melanomagenesis and 2) highlights genes and pathways of interest for melanoma progression. We are in the process of generating a new dataset with benign nevi, dysplastic nevi, and melanoma with which to build and validate a molecular signature of melanoma. Citation Format: Elizabeth S. Borden, Anngela C. Adams, Kenneth H. Buetow, Melissa A. Wilson, Julie E. Bauman, Clara Curiel-Lewandrowski, H.-H. Sherry Chow, Bonnie J. LaFleur, Karen Taraszka Hastings. Shared gene expression and immune pathway changes associated with progression from nevi to melanoma [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A001.

Abstract A006: Shared gene expression and immune pathway changes associated with progression from nevi to melanoma

Abstract There is a need to identify biomarkers of melanoma progression to assist the development of chemoprevention strategies to lower melanoma incidence. In this study, we assessed the feasibility of creating a molecular signature for melanomagenesis using three publicly available RNA sequencing and microarray expression datasets. We performed differential expression and regularized regression analyses across nevi and melanoma samples to identify consistent genes associated with melanomagenesis. The regularized regression models demonstrated that a small number of genes could successfully distinguish between nevi and melanoma, providing evidence for the feasibility of creating a molecular signature. Differential expression analysis identified consistent upregulation of C1QB, CXCL9, CXCL10, DFNA5 (GSDME), FCGR1B, and PRAME in melanoma and consistent downregulation of SCGB1D2 in melanoma compared to nevi. Additionally, each of these genes demonstrated a linear association with the progression from benign nevi to dysplastic nevi, to radial growth phase melanoma to vertical growth phase melanoma, providing additional evidence for their role in melanomagenesis. Subsequent pathway analysis demonstrated significant enrichment of immune-related pathways among the differentially expressed genes. Overall, this study 1) demonstrates the feasibility of creating a gene signature for melanomagenesis and 2) highlights genes and pathways of interest for melanoma progression. We are in the process of generating a new dataset with benign nevi, dysplastic nevi, and melanoma with which to build and validate a molecular signature of melanoma. Citation Format: Elizabeth S. Borden, Anngela C. Adams, Kenneth H. Buetow, Melissa A. Wilson, Julie E. Bauman, Clara Curiel-Lewandrowski, H.-H. Sherry Chow, Bonnie J. LaFleur, Karen Taraszka Hastings. Shared gene expression and immune pathway changes associated with progression from nevi to melanoma [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A006.

DOUBLE V-Y FLAP IN EYEBROW AFTER WIDE EXCISION IN MELANOMA

Melanoma is a malignant tumor with a high metastatic capacity, hence the importance of early diagnosis. Suspected melanoma lesions must be excised at 2 stages. First, a biopsy is performed to obtain histological conrmation by a punch or a narrow margin excisional biopsy, such as an elliptical incision or double M-plasty technique. Then, a re-excision with wider surgical margins, depending on the Breslow thickness, is done. We present a 33 year old woman with an atypical pigmented lesion at her right eyebrow. A narrow margin excision was performed through double M- plasty, obtaining a diagnosis of supercial extensive melanoma in vertical growth phase, Breslow 0.5 mm. After a wide excision with 1 cm margin, a reconstruction with a double V-Y advancement ap was performed with satisfactory aesthetic results and without tumor recurrence 5 years after diagnosis.

Abstract LB056: The spatial landscape of progression and immunoediting in primary melanoma at single cell resolution

Abstract Cutaneous melanoma is a highly immunogenic malignancy, surgically curable at early stages, but life-threatening when metastatic. The spatial organization of the tumor ecosystem during early-stage melanoma is not well understood. Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially-resolved micro-region transcriptomics to study immune evasion and immunoediting in primary melanoma. We collected highly multiplexed single-cell data from 70 distinct histological regions from 13 specimens (patients) selected to have multiple progression-associated histologies within a single resection. These histologies range from pre-malignant fields in which melanocytic atypia represents the first steps in cancer initiation to non-invasive (radial growth phase) and invasive (vertical growth phase) primary melanoma that eventually gives rise to disseminated disease. We find that recurrent cellular neighborhoods involving tumor, immune, and stromal cells change significantly along a progression axis involving precursor states, melanoma in situ, and primary invasive tumor. Hallmarks of immunosuppression were detectable as early as the melanoma precursor stage, and when tumors become locally invasive, a consolidated and spatially restricted environment with multiple overlapping immunosuppressive mechanisms forms along the tumor-stromal boundary. This environment is established by cytokine gradients that promote expression of MHC-II and IDO1 and by PDL1-expressing macrophages and dendritic cells engaging activated T cells. However, only a few millimeters away, T cells synapse with melanoma cells in fields of tumor regression. Thus, invasion and immunoediting can co-exist within a few millimeters of each other in a single specimen. Multiplexed single-cell imaging and micro-region mRNA profiling link morphological and molecular features of tumor evolution within and across primary cancer specimens, revealing highly localized programs of immune and tumor cell communication via paracrine cytokine signaling and direct cell-cell contact. Citation Format: Ajit Johnson Nirmal, Zoltan Maliga, Tuulia Vallius, Brian Quattrochi, Alyce Chen, Connor Jacobson, Roxanne Pelletier, Clarence Yapp, Raquel Arias-Camison, Yu-An Chen, Christine Lian, George F. Murphy, Sandro Santagata, Peter K. Sorger. The spatial landscape of progression and immunoediting in primary melanoma at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB056.

Clinical and pathological predictors of micrometastasis in marginal thickness melanoma.

e21585 Background: Sentinel lymph node biopsy (SLNB) remains controversial in patients with localized marginal thickness melanoma (MTM). Nonetheless, micrometastasis (miLN) i.e. the presence of clinically occult lymph node (LN) metastasis, upstages localized tumors to Stage III, indicating the need for more extensive surgical resection and escalated systemic therapy. We aim to identify disease predictors associated with miLN in MTM. Methods: This National Cancer Database (2004-2018) analysis includes patients with T1a/T1b, N0, and M0 (AJCC 8th edition) melanoma (Breslow’s depth: 0.76-1.00 mm). We selected 2 cohorts based on the pathological status of LN metastasis, as per SLNB or elective lymphadenectomy: negative LN versus miLN. Bivariate association of various clinical characteristics and LN status was assessed using Chi-square and Fisher’s exact tests. Significant covariates were integrated into a multivariable logistic regression (MLR) with a backward elimination model to identify the risk factors associated with the presence of miLN. A multivariable Cox regression model with backward elimination compared overall survivor (OS) between both cohorts, adjusting for significantly unbalanced factors. Results: N = 72,945 patients with clinical N0 MTM were identified. N = 68,912 (94.5%) had negative LN and N = 4,033 (5.5%) had miLN on pathology. Ethnicity (p = 0.260), Charlson/Deyo comorbidity score (p = 0.0894) and LDH levels (p = 0.2278) did not significantly vary across cohorts. On MLR analysis, patients with miLN were more likely to be younger and have tumors located outside the head &amp; neck. Tumors with miLN were more likely to show lymphovascular invasion, vertical growth, and a mitotic rate of ≥ 1 but less likely to show positive tumor regression. Ulceration was not an independent risk factor of miLN (p = 0.9305). Multivariable analysis showed that patients with miLN had lower OS rates than those with negative LN (HR = 1.66; 95% CI: 1.45-1.90; p &lt; 0.0001). Conclusions: miLN is associated with a worse prognosis, warranting early recognition and prompt treatment initiation. Despite NCCN guidelines incorporating tumor ulceration into decision-making regarding the need for SLNB in clinical N0 MTM, we show that tumor ulceration was not significantly associated with miLN in MTM. However, patient age, tumor location, tumor mitotic rate, lymphovascular invasion, and vertical growth phase were associated with miLN and thus need to be assessed when considering SLNB.[Table: see text]

Disruption of Redox Homeostasis by Alterations in Nitric Oxide Synthase Activity and Tetrahydrobiopterin along with Melanoma Progression.

Cutaneous melanoma emerges from the malignant transformation of melanocytes and is the most aggressive type of skin cancer. The progression can occur in different stages: radial growth phase (RGP), vertical growth phase (VGP), and metastasis. Reactive oxygen species contribute to all phases of melanomagenesis through the modulation of oncogenic signaling pathways. Tetrahydrobiopterin (BH4) is an important cofactor for NOS coupling, and an uncoupled enzyme is a source of superoxide anion (O2•−) rather than nitric oxide (NO), altering the redox homeostasis and contributing to melanoma progression. In the present work, we showed that the BH4 amount varies between different cell lines corresponding to distinct stages of melanoma progression; however, they all presented higher O2•− levels and lower NO levels compared to melanocytes. Our results showed increased NOS expression in melanoma cells, contributing to NOS uncoupling. BH4 supplementation of RGP cells, and the DAHP treatment of metastatic melanoma cells reduced cell growth. Finally, Western blot analysis indicated that both treatments act on the PI3K/AKT and MAPK pathways of these melanoma cells in different ways. Disruption of cellular redox homeostasis by the altered BH4 concentration can be explored as a therapeutic strategy according to the stage of melanoma.

Current Management and Treatment of Extramammary Paget's Disease.

Extramammary Paget's disease (EMPD) is a rare neoplastic disease affecting areas rich in apocrine glands in the elderly. EMPD clinically resembles a benign inflammatory skin disease, and ill-defined tumor borders can lead to misdiagnosis and incomplete excision. Several prognostic factors have been reported, including nodule formation, tumor thickness, tumor invasion, lymphovascular invasion, and a perianal location, which are characteristic of primary tumors. EMPD typically presents as an in situ tumor spreading horizontally within the epidermis and then invading into the dermis as it transitions to a vertical growth phase. For this reason, tumor thickness, rather than tumor size, is correlated with patient prognosis. The best treatment for resectable EMPD is complete surgical removal of the tumor. EMPD sometimes has unclear tumor borders, and it can unexpectedly spread beyond its clinical boundaries. Surgical resection in such cases is often associated with tumor-positive margins, which can result in recurrence. However, surgical excision with wide margins may deteriorate patients' organ functions and quality of life. Mohs micrographic surgery may be ideal for controlling the surgical margins and minimizing the sacrifice of normal tissue, but this technique is not always feasible because of constraints associated with the medical environment. No standard treatment for unresectable or metastatic EMPD has been established. Although conventional chemotherapy has been used as the first-line treatment, it frequently causes adverse events, and consequently, targeted therapy will become more valuable in the near future.