Cutaneous Surgical Wounds Have Distinct Microbiomes from Intact Skin.

Infections are relatively rare following cutaneous surgical procedures, despite the potential for wound exposure to pathogens both during surgery and throughout the healing process. Although gut commensals are believed to reduce the risk of intestinal infections, an analogous role for skin commensals has not been described. In fact, the microbiome of normally healing surgical skin wounds has not yet been profiled using culture-independent techniques. We characterized the wound microbiome in 53 patients who underwent skin cancer surgery and healed without signs or symptoms of infection. A week after surgery, several bacterial species displayed significant differences in relative abundance when compared to control, nonoperated skin from the same patient. The relative abundance of the most common bacterium found on intact skin, Cutibacterium acnes, was reduced in wounds 5-fold. Staphylococcus aureus, a frequent cause of postoperative skin infections, was enriched 6.4-fold in clinically noninfected wounds, suggesting active suppression of pathogenicity. Finally, members of the Corynebacterium genus were the dominant organism in postoperative wounds, making up 37% of the average wound microbiome. The enrichment of these bacteria in normally healing wounds suggests that they might be capable of providing colonization resistance. Future studies focused on the biological and clinical significance of the wound microbiome may shed light on normal wound healing and potential therapeutic opportunities to mitigate infection risk. IMPORTANCE Commensal bacteria on skin may limit the ability of pathogenic bacteria to cause clinically significant infections. The bacteria on healing acute wounds, which might provide such a protective effect, have not been described using culture-independent approaches in the absence of antibiotics. We compare the microbiome of wounds a week after skin cancer removal surgery with intact skin from the same patient. We find that the potentially pathogenic species S. aureus is common on these healing wounds despite the absence of symptoms or signs of infection. We report that bacteria often considered as potential skin probiotics, including Staphylococcus epidermidis, do not reach high relative abundance in wound microbiomes. In contrast, specific members of the Corynebacterium genus, rarely associated with infections, were significantly enriched in healing wounds compared to intact skin. Future work is needed to see if Corynebacterium species or derivatives thereof could be employed to lower the risk of wound infection.

Lethal Dermal Sarcoma in Immunosuppressed Patients.

Skin cancer is the leading malignancy in immunosuppressed patients, including organ transplant recipients (OTRs), which is increasing in incidence as OTRs live longer. We performed a single-center case series of 4 patients with scalp pleomorphic dermal sarcoma and a history of multiple keratinocyte carcinomas. Outcomes included incidence of dermal sarcoma, dermal sarcoma-related mortality, and histopathologic findings. Out of more than 200 patients followed over a 3-year period in Massachusetts General Hospital High Risk Skin Cancer Clinics, all skin cancer-related deaths (2/2) were due to metastatic dermal sarcoma. Three of 4 patients diagnosed with scalp dermal sarcoma were OTRs and had been on at least one immunosuppressive medication for a median of 9 years. For patients who died from dermal sarcoma, the median time between diagnosis and death was 6 months. Our findings suggest pleomorphic dermal sarcoma contributes to skin cancer-related morbidity and mortality in OTRs.

Brigham and Women's Hospital tumor classification system for basal cell carcinoma identifies patients with risk of metastasis and death.

BACKGROUND: Despite approximately 4400 locally advanced US cases annually, high-stage basal cell carcinoma (BCC) is ill-defined. OBJECTIVE: To develop a tumor (T) staging system for BCC that will predict metastasis/death and compare its performance with that of the American Joint Committee on Cancer 8th edition (AJCC8) T-staging system. METHODS: Brigham and Women's Hospital (BWH) T staging was developed from a previously published nested cohort of 488 primary BCCs. Tumors were staged via BWH and AJCC8 T-staging systems, and predictions of metastasis and/or death were compared. RESULTS: The BWH and AJCC8 T-staging systems both captured all metastases/deaths in high T stages (BWH, T2; AJCC8, T3/T4). BWH T2 included 54% fewer cases ≥2 cm than AJCC8 T3/T4. BWH had a higher specificity (0.92 vs 0.80; P < .001) and positive predictive value (24% vs 11%, P < .001) for identifying cases at risk for metastasis/death, and the C-statistic was superior for BWH (P < .001). The BWH T2 10-year cumulative incidence of metastasis/death was 37% (95% confidence interval, 21%-60%). LIMITATIONS: Two-center cohort. CONCLUSIONS: BWH and AJCC 8 BCC staging both capture all metastases and deaths in the upper stages. However, BWH staging does so in half the number of cases, thus minimizing inappropriate up-staging. The risk of metastasis or death in BWH T2 BCC is sufficient to warrant surveillance for recurrence and clinical trials of adjuvant therapy.

Dual-Wavelength Optical Polarization Imaging for Detecting Skin Cancer Margins.

Treatment of keratinocyte carcinomas requires an assessment of the extent of tumor spread. Visual delineation of tumor margins is error-prone owing to the limited contrast between cancerous and normal skin. In this contribution, we introduce spectrally-encoded optical polarization imaging and evaluate its performance for preoperative demarcation of keratinocyte carcinomas. Subjects with basal or squamous cell carcinoma, scheduled for Mohs surgery, were enrolled. The surgeon outlined the clinical boundary of each lesion preoperatively. Optical images of the lesions were then acquired at 440 and 640 nm. Spectral encoding of the experimental images minimized the impact of background pigmentation and vascularization. The surgeon was blinded to the imaging results. Margin assessments by imaging and by the surgeon were recorded and compared with the intraoperative histopathology. In total, 53 lesions were imaged in vivo. Thirteen cases required more than one Mohs stage. In all these cases, images accurately visualized the tumor. For cases negative following the first Mohs stage, margin assessments correlated with histopathology in 39 out of 40 cases. Imaging demonstrated 100% sensitivity and 98% specificity. Spectrally-encoded optical polarization imaging may prove valuable for real-time noninvasive preoperative delineation of skin cancer.

Factors predictive of recurrence, metastasis, and death from primary basal cell carcinoma 2 cm or larger in diameter.

BACKGROUND: Basal cell carcinoma (BCC) recurrence and metastatic rates are known to be very low. The risk factors for these rare outcomes are subsequently not well studied. OBJECTIVE: To identify risk factors independently associated with local recurrence (LR) and metastasis and/or death (M/D) in large (≥2 cm) BCC. METHODS: BCCs histologically confirmed between 2000 and 2009 were retrospectively screened for tumor diameter at 2 academic centers. Medical records of all large BCCs and an equal number of randomly selected small BCCs were reviewed for LR and M/D. RESULTS: Included were 248 large BCC and 248 small BCC tumors. Large BCCs had a significantly higher risk of LR and M/D than small BCCs (LR: 8.9% vs 0.8%, P < .001; M/D: 6.5% vs. 0%, P < .001). Because the risks were so low in small BCCs, they were excluded from further analysis. On multivariable logistic regression, head/neck location (odds ratio [OR], 9.7; 95% confidence interval [CI], 3.0-31.3) and depth beyond fat (OR, 3.1; 95% CI, 1.0-9.6) were associated with LR in large BCCs. Risk of LR was lower with Mohs micrographic surgery (OR, 0.14; 95% CI, 0.04-0.5). Head/neck location (OR, 5.3; 95% CI, 1.2-23.2), tumor diameter ≥4 cm (OR, 11.9; 95% CI, 2.4-59.4), and depth beyond fat (OR, 28.6; 95% CI, 6.7-121) were significant predictors of M/D in large BCCs. LIMITATIONS: Retrospective cohort design. CONCLUSIONS: Large BCCs, particularly those with additional risk factors, have a high enough risk of recurrence and metastasis to warrant further investigation to optimize management.

Immunity to commensal papillomaviruses protects against skin cancer.

Immunosuppression increases the risk of cancers that are associated with viral infection1. In particular, the risk of squamous cell carcinoma of the skin-which has been associated with beta human papillomavirus (ß-HPV) infection-is increased by more than 100-fold in immunosuppressed patients2-4. Previous studies have not established a causative role for HPVs in driving the development of skin cancer. Here we show that T cell immunity against commensal papillomaviruses suppresses skin cancer in immunocompetent hosts, and the loss of this immunity-rather than the oncogenic effect of HPVs-causes the markedly increased risk of skin cancer in immunosuppressed patients. To investigate the effects of papillomavirus on carcinogen-driven skin cancer, we colonized several strains of immunocompetent mice with mouse papillomavirus type 1 (MmuPV1)5. Mice with natural immunity against MmuPV1 after colonization and acquired immunity through the transfer of T cells from immune mice or by MmuPV1 vaccination were protected against skin carcinogenesis induced by chemicals or by ultraviolet radiation in a manner dependent on CD8+ T cells. RNA and DNA in situ hybridization probes for 25 commensal ß-HPVs revealed a significant reduction in viral activity and load in human skin cancer compared with the adjacent healthy skin, suggesting a strong immune selection against virus-positive malignant cells. Consistently, E7 peptides from ß-HPVs activated CD8+ T cells from unaffected human skin. Our findings reveal a beneficial role for commensal viruses and establish a foundation for immune-based approaches that could block the development of skin cancer by boosting immunity against the commensal HPVs present in all of our skin.

Collagen disruption as a marker for basal cell carcinoma in presurgical margin detection.

INTRODUCTION AND OBJECTIVE: Nonmelanoma skin cancers (NMSCs) are the most common malignancies in the United States. Surgery is the most common treatment for these tumors, but pre-operative identification of surgical margins is challenging. The objective in this study was to determine whether optical polarization imaging (OPI) could be used prior to surgery to detect the extent of subclinical tumor spread by monitoring disruption in collagen. MATERIALS AND METHODS: OPI is a non-invasive and rapid imaging modality that highlights the structure of dermal collagen. OPI was used preoperatively at wavelengths of 440 and 640 nm to perform imaging of NMSCs on six patients scheduled to undergo Mohs surgery for biopsy-proven basal cell carcinoma. This pilot study did not alter the course of routine MMS for any of the patients. The surgeon was blinded from the preoperative imaging results and completed the entire procedure without relying on the new technology. The study was conducted in an outpatient surgical setting. Patients over 18 years of age with biopsy-proven basal cell carcinoma participated. RESULTS AND CONCLUSION: OPI accurately predicted the presence or absence of tumor at the surgical margin in six out of six cases, as confirmed on histology. OPI may allow efficient surgical planning by identifying tumor extension beyond visibly involved skin. Lasers Surg. Med. 50:902-907, 2018. © 2018 Wiley Periodicals, Inc.

The RNA-binding protein YBX1 regulates epidermal progenitors at a posttranscriptional level.

The integrity of stratified epithelia depends on the ability of progenitor cells to maintain a balance between proliferation and differentiation. While much is known about the transcriptional pathways underlying progenitor cells' behavior in the epidermis, the role of posttranscriptional regulation by mRNA binding proteins-a rate-limiting step in sculpting the proteome-remains poorly understood. Here we report that the RNA binding protein YBX1 (Y-box binding protein-1) is a critical effector of progenitors' function in the epidermis. YBX1 expression is restricted to the cycling keratinocyte progenitors in vivo and its genetic ablation leads to defects in the architecture of the skin. We further demonstrate that YBX1 negatively controls epidermal progenitor senescence by regulating the translation of a senescence-associated subset of cytokine mRNAs via their 3' untranslated regions. Our study establishes YBX1 as a posttranscriptional effector required for maintenance of epidermal homeostasis.

Optical mapping of nonmelanoma skin Cancers-A pilot clinical study.

BACKGROUND AND OBJECTIVE: Nonmelanoma skin cancer (NMSC) is the most common human cancer. Because these tumors often affect the face, there is a strong need for both accurate removal of these neoplasms to prevent recurrence and maximal tissue preservation to prevent cosmetic or functional deformity. Polarization-enhanced reflectance and fluorescence imaging (PERFI) is a new bedside method that uses fluorescent chromophores to image NMSC. While the feasibility of the technique has been successfully demonstrated in ex vivo studies, this is the first pilot study to extend the use of PERFI to in vivo intraoperative imaging of NMSC. MATERIALS AND METHODS: Subjects were recruited from a population of patients with biopsy-confirmed NMSC, scheduled for Mohs micrographic surgery. Eight cases were studied. Sterile methylene blue (MB) was diluted in anaesthetic solution and infused into the peritumoral space. Digital photographs of the lesion were taken and Mohs surgery was performed. Then, the surgical bed was re-imaged. Each excision was also imaged ex vivo and processed for routine histopathology. Optical images were processed and compared with histopathology. RESULTS AND CONCLUSIONS: The injection of MB was well tolerated. We observed a transient blue staining of the treated area, which disappeared completely within 1 week in all of the patients. In all subjects, the contrast agent, MB, was preferentially retained in the tumor. The ex vivo images correlated well with histopathology. In vivo images qualitatively delineated the tumor margins. The results of our pilot trial indicate that PERFI may be useful for accurate and rapid delineation of NMSC during surgery. Lasers Surg. Med. 49:803-809, 2017. © 2017 Wiley Periodicals, Inc.

Multimodal imaging for nonmelanoma skin cancer margin delineation.

BACKGROUND AND OBJECTIVE: Nonmelanoma skin cancer (NMSC) is the most common form of cancer worldwide. The most effective form of treating this cancer is its surgical removal. As NMSC rarely metastasize, its complete excision is often curative. We investigated the potential of combining Terahertz Pulsed Imaging (TPI) with polarization enhanced reflectance optical imaging for the accurate intraoperative delineation of NMSC. MATERIALS AND METHODS: Fresh thick samples with residual cancer were obtained from surgeries. The samples were imaged within two hours using polarization optical and TPI systems without remounting. Analysis of the TPI results was performed in the frequency domain. Co- and cross-polarized optical images were acquired at 410 nm. Superficial optical images were obtained by subtracting cross-polarized from the respective co-polarized images. Terahertz, optical, and histological images were overlaid and compared. RESULTS AND CONCLUSIONS: Our results show that the frequency powers of diseased and normal skin tissues differ significantly at 0.47 THz. While TPI has demonstrated contrast between diseased and normal tissue, it can also highlight normal structures. As TPI alone lacks the resolution necessary to distinguish between tissue types morphologically, polarization optical imaging was used for the inspection of the suspicious areas highlighted by TPI. Combined TPI and optical imaging has the potential for quick intraoperative delineation of cancers. Lasers Surg. Med. 49:319-326, 2017. © 2016 Wiley Periodicals, Inc.

Sustained Akt Activity Is Required to Maintain Cell Viability in Seborrheic Keratosis, a Benign Epithelial Tumor.

Seborrheic keratoses (SKs) are common benign skin tumors that share many morphological features with their malignant counterpart, squamous cell carcinoma. SKs frequently have acquired oncogenic mutations in the receptor tyrosine kinase/phosphatidylinositol 3-kinase/Akt signaling cascade. We developed a reliable culture system to study SKs in vitro and screened these cells using a library of selective kinase inhibitors to evaluate effects on cell survival. These benign tumors are sensitive to inhibition by ATP-competitive Akt inhibitors, including A-443654 and GSK690693. RNA interference-mediated Akt suppression mimicked the effects of enzyme inhibition in cultured cells. Akt inhibition suppressed phosphorylation of downstream targets of Akt kinase that are critical for cell survival, including MDM2 and FOXO3a, and induced apoptosis. Cell death was also dependent on p53, mutations in which, although common in cutaneous squamous cell carcinoma, have not been identified in SKs. Intact explants of SKs were also sensitive to Akt inhibition. In addition to the obvious therapeutic implications of these findings, identifying the signaling characteristics that differentiate benign and malignant tumors may inform our understanding of the malignant state.

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy.

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.

Imaging of ex vivo nonmelanoma skin cancers in the optical and terahertz spectral regions optical and terahertz skin cancers imaging.

We tested the hypothesis that polarization sensitive optical and terahertz imaging may be combined for accurate nonmelanoma skin cancer (NMSC) delineation. Nine NMSC specimens were imaged. 513 µm and 440 nm wavelengths were used for terahertz and optical imaging, respectively. Histopathology was processed for evaluation. Terahertz reflectance of NMSC was quantified. Our results demonstrate that cross-polarized terahertz images correctly identified location of the tumours, whereas cross-polarized and polarization difference optical images accurately presented morphological features. Cross-polarized terahertz images exhibited lower reflectivity values in cancer as compared to normal tissue. Combination of optical and terahertz imaging shows promise for intraoperative delineation of NMSC.

Continuous wave terahertz transmission imaging of nonmelanoma skin cancers.

BACKGROUND AND OBJECTIVE: Continuous wave terahertz imaging has the potential to offer a safe, noninvasive medical imaging modality for delineating human skin cancers. Terahertz pulse imaging (TPI) has already shown that there is contrast between basal cell carcinoma and normal skin. Continuous-wave imaging offers a simpler, lower cost alternative to TPI. The goal of this study was to investigate the feasibility of continuous wave terahertz imaging for delineating skin cancers by demonstrating contrast between cancerous and normal tissue in transmission mode. MATERIALS AND METHODS: Two CO(2) optically pumped far-infrared molecular gas lasers were used for illuminating the tissue at two frequencies, 1.39 and 1.63 THz. The transmitted signals were detected using a liquid Helium cooled Silicon bolometer. Fresh skin cancer specimens were obtained from Mohs surgeries. The samples were processed and imaged within 24 hours after surgery. During the imaging experiment the samples were kept in pH-balanced saline to prevent tissue dehydration. At both THz frequencies two-dimensional THz transmission images of nonmelanoma skin cancers were acquired with spatial resolution of 0.39 mm at 1.4 THz and 0.49 mm at 1.6 THz. For evaluation purposes, hematoxylin and eosin (H&E) histology was processed from the imaged tissue. RESULTS: A total of 10 specimens were imaged and it was determined that for both frequencies, the areas of decreased transmission in the THz image correlated well with cancerous areas in the histopathology. Two negative controls were also imaged. The difference in transmission between normal and cancerous tissue was found to be approximately 60% at both frequencies, which suggests that contrast between normal and cancerous tissue at these frequencies is dominated by differences in water content. CONCLUSIONS: Our results suggest that intraoperative delineation of nonmelanoma skin cancers using continuous-wave terahertz imaging is feasible.

A new American Joint Committee on Cancer staging system for cutaneous squamous cell carcinoma: creation and rationale for inclusion of tumor (T) characteristics.

BACKGROUND: The incidence of cutaneous squamous cell carcinoma (cSCC) is increasing. Although most patients achieve complete remission with surgical treatment, those with advanced disease have a poor prognosis. The American Joint Committee on Cancer (AJCC) is responsible for the staging criteria for all cancers. For the past 20 years, the AJCC cancer staging manual has grouped all nonmelanoma skin cancers, including cSCC, together for the purposes of staging. However, based on new evidence, the AJCC has determined that cSCC should have a separate staging system in the 7th edition AJCC staging manual. OBJECTIVE: We sought to present the rationale for and characteristics of the new AJCC staging system specific to cSCC tumor characteristics (T). METHODS: The Nonmelanoma Skin Cancer Task Force of AJCC reviewed relevant data and reached expert consensus in creating the 7th edition AJCC staging system for cSCC. Emphasis was placed on prospectively accumulated data and multivariate analyses. Concordance with head and neck cancer staging system was also achieved. RESULTS: A new AJCC cSCC T classification is presented. The T classification is determined by tumor diameter, invasion into cranial bone, and high-risk features, including anatomic location, tumor thickness and level, differentiation, and perineural invasion. LIMITATIONS: The data available for analysis are still suboptimal, with limited prospective outcomes trials and few multivariate analyses. CONCLUSIONS: The new AJCC staging system for cSCC incorporates tumor-specific (T) staging features and will encourage coordinated, consistent collection of data that will be the basis of improved prognostic systems in the future.