Open-top Bessel beam two-photon light sheet microscopy for three-dimensional pathology.

Nondestructive pathology based on three-dimensional (3D) optical microscopy holds promise as a complement to traditional destructive hematoxylin and eosin (H&E) stained slide-based pathology by providing cellular information in high throughput manner. However, conventional techniques provided superficial information only due to shallow imaging depths. Herein, we developed open-top two-photon light sheet microscopy (OT-TP-LSM) for intraoperative 3D pathology. An extended depth of field two-photon excitation light sheet was generated by scanning a nondiffractive Bessel beam, and selective planar imaging was conducted with cameras at 400 frames/s max during the lateral translation of tissue specimens. Intrinsic second harmonic generation was collected for additional extracellular matrix (ECM) visualization. OT-TP-LSM was tested in various human cancer specimens including skin, pancreas, and prostate. High imaging depths were achieved owing to long excitation wavelengths and long wavelength fluorophores. 3D visualization of both cells and ECM enhanced the ability of cancer detection. Furthermore, an unsupervised deep learning network was employed for the style transfer of OT-TP-LSM images to virtual H&E images. The virtual H&E images exhibited comparable histological characteristics to real ones. OT-TP-LSM may have the potential for histopathological examination in surgical and biopsy applications by rapidly providing 3D information.

Exploring Molecular Genetic Alterations and RAF Fusions in Melanoma: A Belvarafenib Expanded Access Program in Patients with RAS/RAF-Mutant Melanoma.

BACKGROUND: Melanoma incidence is on the rise in East Asia, yet studies of the molecular landscape are lacking in this population. We examined patients with melanoma who underwent next-generation sequencing (NGS) at a single tertiary center in South Korea, focusing on patients harboring NRAS or RAF alterations who received belvarafenib, a pan-RAF dimer inhibitor, through the Expanded Access Program (EAP). PATIENTS AND METHODS: Data were collected from 192 patients with melanoma who underwent NGS between November 2017 and May 2023. Variant call format data were obtained and annotated. Patients in the EAP received 450 mg twice daily doses of belvarafenib. RESULTS: Alterations in the RAS/RTK pathway were the most prevalent, with BRAF and NRAS alteration rates of 22.4% and 17.7%, respectively. NGS enabled additional detection of fusion mutations, including 6 BRAF and 1 RAF1 fusion. Sixteen patients with NRAS or RAF alterations received belvarafenib through the EAP, and disease control was observed in 50%, with 2 patients demonstrating remarkable responses. CONCLUSIONS: Our study highlights the value of NGS in detecting BRAF, NRAS mutations and RAF fusions, expanding possibilities for targeted therapies in malignant melanoma. Belvarafenib showed clinical benefit in patients harboring these alterations. Ongoing trials will provide further insights into the safety and efficacy of belvarafenib.

Comparison of full-thickness skin graft and punch grafting in the reconstruction of plantar defects.

Acral melanoma commonly occurs on weight-bearing areas of the sole. Reconstruction of such areas presents a surgical challenge due to limited availability and mobility of local skin. Thus, we aimed to compare the scar outcome of full-thickness skin graft (FTSG) and punch grafting in the reconstruction of plantar defects after melanoma surgery. We retrospectively reviewed six patients who underwent both FTSG for nonweight-bearing areas and punch grafting for weight-bearing areas. We compared results of FTSG and punch grafting within the same patient. Photos of completely healed scars were graded using the Stony Brook Scar Evaluation Scale (SBSES). The averages of the FTSG scores and the punch graft scores, as measured by the SBSES, were statistically compared. Punch grafting yielded a better outcome than FTSG, according to the SBSES. The average of the punch grafting scars was 4.67, which was significantly greater (p = 0.004) than that of FTSG scar scores at 1.83. For weight-bearing areas, punch grafting should be considered as the first option of reconstruction, with FTSG as a second option for nonweight-bearing areas. Overall, this combined approach provides an effective and safe method for reconstruction of extensive plantar wounds.

Looking Beyond the Hutchinson Sign: A Retrospective Study of Clinical Factors Indicating the Presence and Invasiveness of Nail Unit Melanoma in Patients With Longitudinal Melanonychia.

BACKGROUND: The data underlying this article are available in the article.Longitudinal melanonychia (LM) presents a challenge because nail unit melanoma (NUM) must be considered as a differential diagnosis. Because nail matrix biopsy may result in nail dystrophy, it is important to distinguish NUM from LM. OBJECTIVE: To provide evidence of previously reported clinical factors indicative of NUM in patients with LM. METHODS: This was a retrospective study of patients who presented with LM and had biopsy-confirmed NUM from 2005 to 2021. Benign LM was either confirmed by biopsy or considered benign if followed without the need for biopsy. Clinical factors associated with LM and NUM were compared by multivariate regression. RESULTS: A total of 177 patients (97 LM and 80 NUM) were included. Multivariate regression showed that high band color intensity (p = .0031), variegation (p = .0005), nail plate splitting (p = .0017), Hutchinson sign (p = .0027), and band change (p = .001) correlated with malignancy. Nail plate splitting was associated with Breslow thickness. CONCLUSION: Malignancy should be suspected and biopsy performed in patients with LM and high band color intensity, variegation, nail plate splitting, Hutchinson sign, and band change.

Successful Treatment of Recurrent Earlobe Keloid Through Removal of Concealed Underlying Epidermoid Cysts: A Case Report.

In modern clinical practice, earlobe keloids demonstrate a high cure rate through surgical intervention and suitable adjuvant therapies. Furthermore, the concurrence of keloids and epidermoid cysts is uncommon, potentially attributed to the lack of skin appendages within keloid tissue. This case report presents the successful treatment of a recurrent earlobe keloid through the removal of concealed underlying epidermoid cysts. The lesion recurred even after the second excision and proper adjuvant treatments. It was finally stabilized following the removal of epidermoid cysts within the earlobe at the third surgical procedure. These findings emphasize the importance of identifying underlying conditions associated with keloids and addressing inflammation, as these factors significantly influence treatment outcomes and resistance.

Risk of recurrence of nail unit melanoma after functional surgery versus amputation.

BACKGROUND: Minimally invasive nail unit melanoma (NUM) can be treated with functional surgery (FS) instead of amputation. OBJECTIVE: To determine risk factors associated with recurrence in NUM. METHODS: We retrospectively reviewed patients with NUM between 2008 and 2022 at a tertiary referral center. Multivariable Cox regression models adjusted for male sex and Breslow thickness (BT) were generated. Receiver operating characteristic analysis was performed to determine optimal cut-off points of the BT for stratifying recurrence risk. RESULTS: We evaluated 140 NUM cases (33 amputation and 107 FS). The mean BT values were 3.14 ± 2.62 mm (amputation) and 0.70 ± 1.36 mm (FS). Recurrence occurred in 10 (30.30%) patients with amputation and 23 (21.5%) with FS. Distant disease occurred in 10 (30.30%) patients with amputation and 8 (7.48%) with FS. Male sex, greater BT, amelanotic color, ulcers, and nodules were associated with greater risk for recurrence or distant disease. A BT of 0.8 mm was deemed the optimal cut-off for stratifying recurrence risk after surgery (odds ratio, 5.32; 95% CI, 2.04-13.85). LIMITATIONS: Small sample. CONCLUSION: FS can be considered for NUM with a BT < 0.8 mm, providing an amputation-sparing benefit. However, NUM with risk factors for recurrence requires patient counselling and close follow-ups.

High-contrast visualization of human skin cancers with combined reflectance confocal and moxifloxacin-based two-photon microscopy: An ex vivo study.

BACKGROUND AND OBJECTIVES: Precise determination of cancer margin during skin cancer surgery is crucial for complete resection and further clinical prognosis. Although reflection confocal microscopy (RCM) has been used for perioperative guiding, its reflection contrast has limitations in detecting cancer cells in the dermis. We previously developed combined reflection confocal (RC) and moxifloxacin-based two-photon (MB-TP) microscopy for sensitive cancer detection by using multiple contrast mechanisms. In this study, the performance of combined microscopy was characterized in various skin cancer specimens and compared with standard methods. MATERIALS AND METHODS: Seven human skin specimens in total including two normal ones, three basal cell carcinomas (BCCs), and two squamous cell carcinomas (SCCs) were collected and imaged in fresh condition. Moxifloxacin ophthalmic solution was topically instilled for cell labeling for 3-5 minutes, then mosaic imaging with the combined microscopy was conducted. The imaged specimens were imaged again after exogenous nuclear labeling for comparison and then processed for standard hematoxylin and eosin histology. RESULTS: Combined RC and MB-TP microscopy visualized both cell and extracellular matrix structures of the skin specimens with multiple contrasts of reflection, moxifloxacin fluorescence, autofluorescence, and second harmonic generation. It distinguished normal cell structures in the skin dermis such as hair follicles, sebaceous and eccrine glands from BCC nests, and SCCs based on cell organization. Normal cell structures had organized cell arrangements for their functions, while cancer cell structures had dense and disorganized cell arrangements. Cellular features found by combined microscopy images were confirmed by both TP microscopy with nuclear labeling and histological examination. CONCLUSIONS: The imaging results showed the potential of combined microscopy for sensitive cancer detection and in vivo guiding of skin cancer surgery.

Discrimination of Invasive Human Skin Tumor Using an Ultrafast ATP-Proton AND-Gate Probe.

Cancer cells undergo unscheduled proliferation resulting from dysregulation of the cell cycle, and hence, evaluation in tumor is of keen interest to examine the invasiveness and recurrence of cancer in the lesion. Molecular probes capable of discriminating actively growing tumor from resting ones remain unexplored despite their vast importance. Here, we describe a novel strategy to visualize invasive areas in tumor with a fluorescence probe that implements synergistic fluorescence response toward the slightly acidic environment of tumor and an ATP-abundant nature of actively growing cells. The probe has been designed for ultrafast detection of ATP with high specificity. We demonstrate its utility in visualizing invasive areas in tumor by distinguishing basal cell carcinomas and squamous cell carcinomas at their early stages by two-photon microscopy.

Prediction of Immune-Checkpoint Blockade Monotherapy Response in Patients With Melanoma Based on Easily Accessible Clinical Indicators.

BACKGROUND: Immune checkpoint blocker (ICB) has shown significant clinical activity in melanoma. However, there are no clinically approved biomarkers to aid patient selection. We aimed to identify patients with advanced or metastatic melanoma who are likely to benefit from ICB monotherapy using easily accessible clinical indicators. MATERIALS AND METHODS: We retrospectively reviewed the records of 134 patients with advanced or metastatic melanoma who received ICB monotherapy between 2014 and 2018. Prognostic factors of overall survival (OS) and progression-free survival (PFS) were determined using Cox regression analysis. RESULTS: During the median follow-up of 13.7 months, the median OS and PFS were 18.4 and 3.4 months, respectively. Visceral/central nervous system (CNS) metastasis (OS: adjusted hazards ratio [HR], 1.82; p=.014; PFS: HR, 1.59; p=.024), lymphopenia (<1000 cells/µL) within 3 months (OS: HR, 1.89, p=.006; PFS: HR, 1.70; p=.010), and elevated baseline lactate dehydrogenase (LDH) level (OS: HR, 2.61; p<.001; PFS: HR, 2.66; p<.001) were independent prognostic factors for both poor OS and PFS. Development of immune-related adverse events (irAE; e.g., hypothyroidism or vitiligo) within 6 months showed a trend toward better OS in multivariable analysis (HR, 0.37; p=.058). Patients with normal LDH levels and no visceral/CNS metastasis had a substantially better OS than the others (median, 40.4 vs. 13.6 months; p<.001). Among others, patients who developed irAE within 6 months achieved long-term OS (median, 43.6 vs. 13.1 months; p=.008). A decision tree was suggested using four risk factors, and the risk stratification provided significant distinction between the survival curves. CONCLUSION: The four easily accessible clinical indicators associated with better treatment outcomes after ICB monotherapy in patients with advanced or metastatic melanoma were LDH level, the extent of disease, lymphopenia, and irAE. The combined use of these indicators can be clinically useful in improving risk stratification of patients treated with ICB monotherapy.

Initial topical monotherapy may increase the risk of recurrence in patients with extramammary Paget's disease.

Although topical therapies are used to treat extramammary Paget's disease (EMPD), reliable treatment outcomes and the effects of these therapies on subsequent surgical treatments are unclear. To assess the clinical outcomes of topical treatment of EMPD and establish the treatment guidelines the medical records of 166 patients diagnosed with EMPD were retrospectively reviewed. The recurrence rate was evaluated according to the previous use of topical agents. Thirty-four patients (34/166, 20.5%) were initially treated with topical agents such as imiquimod, 5-fluorouracil, and ingenol mebutate. Three patients (3/34, 8.8%) showed clinical response, while 31 patients required subsequent therapy owing to treatment failure. Analysis of the prognostic factors of recurrence in 166 patients revealed that initial topical treatment increased the hazard ratio (HR) in both univariate and multiple Cox proportional hazards models (HR = 3.770, 95% confidence interval [CI] = 1.768-8.037, P = 0.001, and adjusted HR = 3.628, 95% CI = 1.558-8.450, P = 0.003). Patients treated with topical agents showed significantly poorer 3-year recurrence-free survival than the nontreated group (66.3% vs 88.6%, P < 0.001). Topical treatment may be deleterious for some EMPD patients, thus increasing the recurrence risk.

Hypopigmentation in Extramammary Paget Disease Is an Important Prognostic Factor for High Recurrence Rate and Poor Surgical Outcome.

BACKGROUND: Several studies have reported the presence of hypopigmentation in extramammary Paget disease (EMPD). However, an in-depth analysis regarding its clinical implication is lacking. OBJECTIVE: To evaluate the clinical characteristics of EMPD in the Korean population and to determine the implication of hypopigmentation on clinical outcomes. METHODS: We retrospectively reviewed 124 cases of EMPD who underwent surgical treatment from a single tertiary hospital from December 2005 to March 2019. Baseline characteristics of the patients and hypopigmentation patterns were analyzed. Moreover, the number of stages of Mohs micrographic surgery (MMS) and recurrence rate were evaluated in relation to the hypopigmentation. RESULTS: A total of 67.7% (n = 84) of the patients showed hypopigmentation. The adjusted odds ratio for recurrence in the hypopigmented group was 5.980, which was statistically significant (95% confidence interval = 1.347-26.553, p-value = 0.019). Furthermore, the average number of MMS stages was 2.92 in the hypopigmentation group, compared with 1.82 in the nonhypopigmentation group (p-value = .0016). CONCLUSION: Hypopigmented lesions may disguise the tumor margin, thus raising the recurrence rate after surgery and the number of stages of MMS. The hypopigmentation status must be considered when deciding the surgical margin.

Checkmark rotation flap for nasal tip reconstruction in Asian patients.

Background: Nasal tip reconstruction (NTR) of the Mohs defect is challenging, especially in Asians who have a relatively low nasal bridge and thin cartilages.Objective: To evaluate the usefulness of a new flap design, the checkmark rotation flap (CRF), for NTR in AsiansMethods and materials: We retrospectively reviewed 31 cases of NTR. Among these, 15 cases underwent CRF, which uses skin from the columellar or infra-tip area with a checkmark v-shaped incision (CRF group). The remaining 16 cases underwent reconstruction with other flaps such as the bilobed or island pedicle flap (Others group).Results: The average defect area was larger in the CRF than in the Others group (1.85 ± 1.01 cm2 vs. 1.10 ± 0.72 cm2, p = .0499), but the visual analog scale score was significantly higher in the CRF group (7.97 ± 1.36 vs. 6.25 ± 1.62, p = .0059). For complications, a depressed scar was observed in four cases (26.7%) in the CRF group; nine cases (56.3%) in the Others group presented with a depressed scar, hypertrophic scar, or scarring similar to a trap door deformity.Conclusion: CRF may be a favorable option for the reconstruction of intermediate-sized nasal tip defects, providing good cosmetic outcomes.

Application of secondary intention for the restoration of the apical triangle after Mohs micrographic surgery.

BACKGROUND: Restoring the apical triangle (AT) to maintain the symmetry of the face after Mohs micrographic surgery can be challenging. We have applied secondary intention (SI) after partial closure in cases with large defects. OBJECTIVE: To compare the cosmetic results between immediate closure (IC) and SI. METHODS & MATERIALS: We retrospectively reviewed 24 patients (IC group: n = 15, SI group: n = 9). To evaluate the symmetry, the comparative ratio (CR; area of the AT of the involved side/area of the normal contralateral side) was calculated. RESULTS: The defects were significantly larger in the SI group than in the IC group (588.89 ± 346.53 mm2 vs. 252.87 ± 196.52 mm2, p < .01). While there was no statistically significant difference in average CR, the standard deviation was higher in the SI group (0.95 ± 0.08 vs. 0.93 ± 0.31, p = .3359), indicating the difficulty in predicting the results of SI healing. The average visual analog scale score evaluated by two dermatologists was higher in the IC group, albeit without a significant difference (8.23 ± 0.96 vs. 7.78 ± 1.52, p = .5267). CONCLUSION: SI after partial closure can be an option for large defects in the AT area.

Artificial Intelligence in Cutaneous Oncology.

Skin cancer, previously known to be a common disease in Western countries, is becoming more common in Asian countries. Skin cancer differs from other carcinomas in that it is visible to our eyes. Although skin biopsy is essential for the diagnosis of skin cancer, decisions regarding whether or not to conduct a biopsy are made by an experienced dermatologist. From this perspective, it is easy to obtain and store photos using a smartphone, and artificial intelligence technologies developed to analyze these photos can represent a useful tool to complement the dermatologist's knowledge. In addition, the universal use of dermoscopy, which allows for non-invasive inspection of the upper dermal level of skin lesions with a usual 10-fold magnification, adds to the image storage and analysis techniques, foreshadowing breakthroughs in skin cancer diagnosis. Current problems include the inaccuracy of the available technology and resulting legal liabilities. This paper presents a comprehensive review of the clinical applications of artificial intelligence and a discussion on how it can be implemented in the field of cutaneous oncology.

High-speed combined reflectance confocal and moxifloxacin based two-photon microscopy.

Reflectance confocal microscopy (RCM) is a non-invasive high-resolution optical imaging technique used in clinical settings as a diagnostic method. However, RCM has limited diagnostic ability by providing non-specific morphological information only based on reflection contrast. Various multimodal imaging techniques have been developed to compensate the limitations of RCM, but multimodal techniques are often slow in imaging speed compared to RCM alone. In this report, we combined RCM with moxifloxacin based two-photon microscopy (TPM) for high-speed multimodal imaging. Moxifloxacin based TPM used clinically compatible moxifloxacin for cell labeling and could do non-invasive cellular imaging at 30 frames/s together with RCM. Performance of the combined microscopy was characterized in the imaging of mouse skin and cornea, in vivo. Detail tissue microstructures including cells, extra-cellular matrix (ECM), and vasculature were visualized. The combined microscopy was applied to human skin cancer specimens, and both cells and ECM in the skin cancer and normal skin regions were visualized at high imaging speeds. The combined microscopy can be useful in the clinical applications of RCM by providing multiple contrasts.

Clinical Outcomes of Immune Checkpoint Blocker Therapy for Malignant Melanoma in Korean Patients: Potential Clinical Implications for a Combination Strategy Involving Radiotherapy.

PURPOSE: We investigated the clinical efficacy of immune checkpoint blocker (ICB) therapy for metastatic or advanced melanoma in Korean patients. As well, we assessed whether the effects of ICBs can be enhanced by combination therapy with palliative radiotherapy (RT). MATERIALS AND METHODS: We retrospectively reviewed the records of 127 patients with metastatic melanoma who received ICB with or without palliative RT between 2014 and 2018. The melanoma subtypes were classified as follows: chronic sun-damaged (CSD), acral, mucosal, and uveal. The primary endpoint was the objective response rate (ORR). RESULTS: The overall ORR was 15%, with 11 complete and eight partial responses. ORRs for CSD, acral/mucosal, and uveal melanomas were 50%, 16.5%, and 0%, respectively (p=0.009). In addition to the subtype, stage at treatment, total tumor burden at treatment, and ICB type were significantly associated with ORR (all p < 0.05). Palliative RT was administered in 44% of patients during the treatment, and it did not affect ORR. Clinical responders to ICB therapy exhibited significantly higher 1-year progression-free and overall survival rates than nonresponders. CONCLUSION: ORR for ICB monotherapy in Korean patients with melanoma is relatively modest compared with that in Western patients because the non-CSD subtypes are predominant in the Korean population. Our findings regarding combination therapy with ICB provided a rationale for the initiation of our phase II study (NCT04017897).

Advantages of A Hairline Incision for the Excision of Forehead Lipomas.

BACKGROUND: Direct incision (DI) for forehead lipoma removal has a considerable risk of damaging the vertical vessels and leaving a visible scar. OBJECTIVE: To evaluate the usefulness of the hairline incision (HI) in minimizing scars and neurovascular damage. PATIENTS AND METHODS: Retrospective analysis was done for 30 patients with forehead lipomas who underwent excision between 2011 and 2019 at the Severance Hospital of the Yonsei University Health System, Seoul, Korea. Fourteen patients underwent DI, and 16 underwent HI. Comparison of the cosmetic outcomes, complications, and patient's subjective satisfaction was performed. RESULTS: In the HI group, superior cosmetic outcomes, including patients' subjective satisfaction and photographic assessment findings, were observed. In the DI group, there were 2 cases of skin necrosis with scarring change and 3 cases of recurrence. Periorbital edema was the most common complication in the HI group, which spontaneously resolved within 1 week. CONCLUSION: Hairline incision using a loupe should be considered as a first-line treatment in the removal of forehead lipomas, because it enables complete removal of lipoma with few complications and minimal scarring. Validation of our treatment algorithm requires further exploration.

Male sex and Breslow thickness are important risk factors for recurrence of localized melanoma in Korean populations.

BACKGROUND: Predicting the recurrence of localized melanoma is important; however, studies investigating risk factors for recurrence of localized melanoma are lacking in Asian populations. OBJECTIVE: To identify risk factors for recurrence of localized melanoma in Korean patients. METHODS: We retrospectively reviewed patients with cutaneous melanoma without evidence of metastasis from 2000 to 2017. Logistic and Cox regression analyses were conducted for recurrence. The average follow-up time was 46.2 months. RESULTS: We reviewed the data of 340 patients diagnosed with cutaneous melanoma and staged as melanoma in situ, stages I and II. Acral melanoma (70.3%, 239/340) was the predominant subtype. Ninety-two patients (27.1%) had a recurrence after primary melanoma removal (29 local recurrences, 49 regional metastases, and 28 distant metastases). Some patients had multiple types of recurrence at the same time. Male sex (P = .030) and Breslow thickness greater than 1 mm (P = .008) correlated with an increased risk of recurrence. Breslow thickness greater than 2.5 mm in males and greater than 4 mm in females showed a higher predictive value for recurrence than traditional stages IIB and IIC (hazard ratio 3.743 vs 2.972). LIMITATIONS: This was a single-center retrospective study. CONCLUSION: In patients with localized cutaneous melanoma, male sex and Breslow thickness are the most important prognostic factors for recurrence in Korean populations. Different cutoff values of Breslow thickness may better predict recurrence according to sex.