Gerald Saldanha - Academia.edu (original) (raw)

Papers by Gerald Saldanha

Journal of Cutaneous Pathology, 2022

BackgroundInvasive width, the distance between the most peripheral invasive melanoma cells on the... more BackgroundInvasive width, the distance between the most peripheral invasive melanoma cells on the section where Breslow thickness (BT) was measured, was recently identified as a prognostic feature. It is unclear whether a routine measurement is justified, given that macroscopic width is already included in many melanoma histopathology reports and may itself be a prognostic feature. This study sought to investigate this.MethodsA retrospective cohort of 718 melanoma patients in which macroscopic width had been stated in the original histopathology report was used. Survival analysis was performed.ResultsMacroscopic and invasive widths were positively correlated (p < 0.001). Invasive width was typically smaller than the paired macroscopic width (median difference 3.7 mm, p < 0.001), a difference seen across all T groups. Both macroscopic and invasive widths were significantly associated with melanoma survival in Kaplan–Meier analysis, including overall survival, but invasive width...

Social Science Research Network, 2019

The Hedgehog signalling pathway and its role in cancer Gerald Saldanha Mutations in Hedgehog (Hh)... more The Hedgehog signalling pathway and its role in cancer Gerald Saldanha Mutations in Hedgehog (Hh) pathway genes such as Patched1 are of established importance in Basal cell carcinoma (BCC). However, BCC is unusual because it has indolent behaviour and rarely metastasises. Hh pathway gene mutations are rare in cancers with more typical malignant behaviour, leading to the hypothesis that the pathway plays an important role in BCC but has limited importance in cancers with typical malignant behaviour. This has been addressed in three ways. First, evidence that superficial BCC is a clonal proliferation driven by alterations at the Patched1 locus was sought. Second, evidence of Wnt pathway activation in BCC was sought by looking for nuclear accumulation of p-catenin, because Wnt is a putative Hedgehog pathway target gene. Third, to determine whether the Hh pathway was important in a cancer with typical malignant behaviour, breast cancer was studied. In superficial BCC, microdissection of...

JCO Precision Oncology, 2019

Successful treatment of primary cutaneous diffuse large B cell lymphoma leg type with single agen... more Successful treatment of primary cutaneous diffuse large B cell lymphoma leg type with single agent venetoclax

Orbit (Amsterdam, Netherlands), Jan 20, 2018

Microcystic adnexal carcinoma (MAC) is a very rare and locally aggressive sweat gland tumour. MAC... more Microcystic adnexal carcinoma (MAC) is a very rare and locally aggressive sweat gland tumour. MAC has been well reported presenting as a periocular cutaneous lesion, rarely with subsequent direct orbital invasion and only once as a primary orbital lesion. Local recurrence is frequent after primary surgical excision and the role of adjuvant radiotherapy is ill-defined. We describe a case of orbital MAC treated successfully with radiotherapy after incomplete margin clearance post exenteration surgery and review the associated literature.

Melanoma research, Jan 12, 2018

The aims of this study were to investigate, in cutaneous malignant melanoma (MM), the integrity o... more The aims of this study were to investigate, in cutaneous malignant melanoma (MM), the integrity of nuclear vitamin D receptor (VDR) signalling, as implied by VDR subcellular location; to investigate the relationship between VDR and tumour progression and the inhibitory effect on VDR by mitogen-activated protein kinase (MAPK) overactivity. Archived tissue from 34 benign melanocytic naevi, 149 MMs and 44 matched metastases were stained by immunohistochemistry for VDR and a subset of primary MMs were stained for phosphorylated-extracellular signal-regulated kinase as a marker of MAPK activity. MM cell lines were investigated to show the subcellular location of VDR and cell viability in response to ligand±MAPK inhibitor. Benign melanocytic naevi showed mainly a strong nuclear VDR staining in contrast to MM where decreased nuclear and emergent cytoplasmic VDRs were associated with malignant progression in terms of dermal invasion and metastasis. MMs that retained exclusive nuclear VDR at...

The American journal of surgical pathology, Jan 16, 2018

Histomorphologic prognostic biomarkers that can be measured using only an hematoxylin and eosin s... more Histomorphologic prognostic biomarkers that can be measured using only an hematoxylin and eosin stain are very attractive because they are simple and cheap. We conceived an entirely novel biomarker of this type, the Breslow density (BD), which measures invasive melanoma cell density at the site where Breslow thickness (BT) is measured. This study assessed BD's prognostic value. In this study, BD was measured in 1329 melanoma patients. Measurement accuracy and precision was assessed using intraclass correlation coefficient (ICC). Survival was assessed with a primary end-point of melanoma-specific survival (MSS) and also overall survival and metastasis-free survival. We found that BD measurement was accurate compared with gold standard image analysis (ICC, 0.84). Precision was excellent for 3 observers with different experience (ICC, 0.93) and for an observer using only written instructions (ICC, 0.93). BD was a highly significant predictor in multivariable analysis for overall su...

Orbit, 2017

A 49-year-old male presented with a 4 month history of dysaesthesia in the left periorbital regio... more A 49-year-old male presented with a 4 month history of dysaesthesia in the left periorbital region. A CT scan showed a lacrimal gland mass with areas of dense calcification. Biopsies of the left lacrimal gland revealed a silver material with associated chronic granulomatous inflammation and secondary calcification. The histological specimen was sent for X-ray microanalysis. This confirmed the silver material to be aluminium. The metal fragments were subsequently discovered to be from a car aerial, which caused an orbital impaling injury 20 years prior to presentation. This is the first reported case of lacrimal gland calcification secondary to chronic metal foreign body exposure. The case is presented alongside literature reviews of lacrimal gland calcification and associated radiological findings.

American Journal of Surgical Pathology, 2017

The purpose of this study was to evaluate the prognostic value of tumor-infiltrating lymphocytes ... more The purpose of this study was to evaluate the prognostic value of tumor-infiltrating lymphocytes (TILs) in melanoma and to determine whether a simpler numerical scoring system would be more effective. In total, 655 patients presenting to a UK teaching hospital with primary invasive melanoma were analyzed. TILs were rescored using the standard Clark’s method and univariable and multivariable analyses of the effect of TILs on overall survival (OS), disease-specific survival (DSS), and metastasis-free survival (MFS) was assessed using Cox regression. In total, 30 (5%) melanomas showed absent, 464 (71%) nonbrisk, and 161 (24%) brisk TILs. There was a statistically significant relationship between TILs and Breslow thickness, age, melanoma type, mitotic rate, and histologic regression. TIL grade was a significant predictor of MFS in multivariable analysis (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.25-0.77) but was not significant for OS or DSS. By contrast, when a simple nu...

Modern Pathology, 2016

Outcomes for melanoma patients vary within cancer stage. Prognostic biomarkers are potential adju... more Outcomes for melanoma patients vary within cancer stage. Prognostic biomarkers are potential adjuncts to provide more precise prognostic information. Simple, low-cost biomarker assays, such as those based on immunohistochemistry, have strong translational potential. 5-hydroxymethylcytosine (5 hmC) shows prognostic potential in melanoma but prior studies were small. We, therefore, analysed 5 hmC in a retrospective cohort to provide external validation of its prognostic value. Two hundred primary melanomas were evaluated for 5 hmC expression using immunohistochemistry. The primary objective was to assess the effect on overall survival while controlling for important confounders. Univariable and multivariable analyses were performed. REMARK guidelines were followed. The 5 hmC immunohistochemistry scoring showed very strong inter-observer agreement (ICC 0.88) and expression was significantly related to age, site, Breslow thickness, ulceration, mitotic rate, and stage. Kaplan-Meier analysis showed 5 hmC was associated with metastasis-free, melanomaspecific, and overall survival, Po0.0001 for each. In univariable Cox proportional hazards models, 5 hmC hazard ratios were significant and remained so in a multivariable model. A two-step cox model was created using stage and 5 hmC, as stage is the gold standard for clinical practice. The addition of 5 hmC produced significant improvement in the model and 5 hmC and stage were independent significant predictors. This is the largest study of the prognostic value of 5 hmC immunohistochemistry in melanoma. The 5 hmC scoring was easily and reproducibly performed and it was an independent predictor of metastasis-free survival, melanoma-specific survival, and overall survival. This work supports further development of 5 hmC as a prognostic biomarker and suggests that it could add more precision to American Joint Committee on Cancer staging.

Histopathology, 2016

Aims-In 1970, Breslow described his eponymously named thickness measurement. No-one has sought to... more Aims-In 1970, Breslow described his eponymously named thickness measurement. No-one has sought to enhance Breslow thickness (BT). This purpose of this study was to demonstrate proof of concept that the density of melanoma cells at the position where Breslow thickness is measured is a morphological prognostic biomarker, which we name Breslow density (BD). The hypothesis was that BD has prognostic value for overall survival (OS) and is independent of BT. Methods and results-We analysed 100 cutaneous melanomas and followed REMARK guidelines. BD was the estimated percentage dermal area occupied by melanoma cells in a specified location. BT and BD had a strong correlation (p = 2.1 x 10-11) but despite this they were independent prognostic factors for OS in Cox regression (BD: HR 1.03, p=0.001849 and BT: HR 1.09, p=0.000146). This was corroborated by an independent effect on melanoma specific survival. We assessed whether BT and BD could be combined into a Breslow score. A prognostic index based on Cox regression coefficients was used and this showed a marginal improvement in predicted 5-year survival compared to BT alone (are under curve 94.8% v 96.7%). Conclusions-We show proof of concept that BD represents a novel morphological prognostic biomarker that is independent of BT and that there is potential to combine these into a Breslow score. Larger studies are needed to validate BD, but the simplicity of this biomarker makes it a strong candidate for translation to clinical practice.

Modern Pathology, 2016

Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disea... more Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disease has been revolutionized by new agents. More therapeutic options, coupled with the desire to extend treatment to the adjuvant setting mean that prognostic biomarkers that can be assayed from formalin-fixed paraffin-embedded clinical would be valuable. microRNAs have potential to fill this need. We analyzed 377 microRNAs in 79 primary melanomas and 32 metastases using a split sample discovery strategy. From a discovery analysis using 40 thick primary melanomas (20 cases with metastasis and 20 controls without metastasis at 5 years), microRNA expression was measured by quantitative RT-PCR (QRT-PCR). MiR-10b emerged as a candidate prognostic microRNA. This was confirmed in an independent validation set of thick primary melanomas (20 cases with metastasis and 19 controls without metastasis at 5 years). In the combined discovery and validation cohorts (n = 79), miR-10b expression showed a 3.7-fold increase in expression between cases and controls (P = 0.005) and showed a trend of increasing expression between primary melanomas and their matched metastases (Po 0.001). In situ hybridization showed expression was in melanoma cells and correlated with expression measured by QRT-PCR (P = 0.0005). We used the combined discovery and validation samples to verify the prognostic value of additional candidate microRNAs identified from other studies, and proceeded to analyze miR-200b. We demonstrated that miR-10b and miR-200b showed independent prognostic value (P = 0.002 and 0.047, respectively) in multivariable analysis alongside known clinico-pathological prognostic features (eg, Breslow thickness) using a Cox proportional hazards regression model. Furthermore, the addition of these microRNAs to the clinico-pathological features led to an improved regression model with better identification of aggressive thick melanomas. Taken together, these data suggest that miR-10b is a new prognostic microRNA for melanoma and that there could be a place for microRNA analysis in stratifying melanoma for therapy.

Melanoma research, 2016

Identification of prognostic biomarkers is timely for melanoma as clinicians seek ways to stratif... more Identification of prognostic biomarkers is timely for melanoma as clinicians seek ways to stratify patients for molecular therapy. MicroRNAs are promising as tissue biomarkers because they can be assayed directly from formalin-fixed paraffin-embedded clinical samples. We previously reported that microRNA-21 (miR-21) was strongly expressed in melanoma relative to naevi and now sought to further assess the significance of this by assessing its relationship with its putative target, PTEN. Clinical melanoma samples were analysed by immunohistochemical analysis for PTEN, stem-loop qRT-PCR for miR-21 and PCR for BRAF/NRAS mutation status. Cell lines were investigated for the effect of anti-miR-21 on PTEN. A total of 81 clinical melanocytic tumour samples were investigated, with uniformly high PTEN expression in the nucleus and cytoplasm of naevi and with preferential loss of PTEN expression in the nucleus of melanoma cells. miR-21 expression was inversely associated with nuclear PTEN expr...

The Journal of molecular diagnostics : JMD, Jan 29, 2015

Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical p... more Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCR-based assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. The reproducibility and accuracy of the DRTs were demonstrated and appropriate correction factors for DNA quality calculated for each assay, based on the results of 108 diploid samples. As a panel, seven DRTs were able to differentiate unambiguous cases of melanoma and nevi with a sensitivity of 87% (95% CI, 83%-91%) and a specificity of 88% (95% CI, 84%-92%) in a series of 145 melanomas and 123 nevi. The DRT scores for 20 nonmetastasizing primary melanomas a...

Cancer Research, 2013

Embryonic transcription factors (ETFs) of the Snail, Zeb and Twist families are essential mediato... more Embryonic transcription factors (ETFs) of the Snail, Zeb and Twist families are essential mediators of EMT programs (Epithelial to Mesenchymal Transition) and play central roles in cell determination and cell plasticity. The aberrant expression of ETFs is frequently observed in various cancer types, particularly in carcinomas, and is associated with poor prognosis and high risk of metastasis. We and others have demonstrated that their oncogenic potential relies on their ability to enable escape from oncogene-induced failsafe programs, senescence and apoptosis. Therefore, EMT-resembling processes initiated by ETFs contribute to neoplastic transformation in vitro and accelerate tumor progression in vivo in an epithelial context. We now unveil a completely different scenario in melanoma, a highly metastatic neural crest-derived cancer. Consistent with their role in melanoblasts determination, SNAIL2 and ZEB2 are already expressed in melanocytes and naevi, but their expression is down-r...

Clinics in laboratory medicine, 2011

Melanoma is the most serious type of skin cancer. Unfortunately, treatment has progressed little ... more Melanoma is the most serious type of skin cancer. Unfortunately, treatment has progressed little and advanced melanoma has appalling survival rates. A goal of molecular analysis is to fully describe the alterations that underpin melanoma's clinical phenotype so that diagnosis can be more accurate, outcome can be predicted with greater confidence, and treatment that is tailored to the patient can be given. This article describes the handful of "signature" changes that are known to occur, describes how some recent studies have shed light on changes beyond this signature, and finally discusses the impact of molecular pathology for practicing histopathologists.

The Journal of Molecular Diagnostics, 2013

A minority of melanocytic lesions cannot confidently be classified as benign or malignant on hist... more A minority of melanocytic lesions cannot confidently be classified as benign or malignant on histopathological examination, causing diagnostic uncertainty. DNA copy number changes can be used to distinguish nevi from melanoma, although the use of FFPE tissue can pose technical challenges. DNA copy number assays, called duplex ratio tests, have been developed with duplex real-time PCR, using a simple method with potential for high throughput. Five duplex ratio test assays targeting loci with common DNA copy number changes in melanoma were designed and tested using DNA extracted from FFPE samples microdissected from melanoma, common nevi, benign tonsil (10 each), and two melanoma cell lines. The assays proved accurate when DNA extracted from fresh and FFPE melanoma cell lines were compared (intraclass correlation coefficient, 0.99) and gave precise results when repeated on DNA from FFPE tissue (intraclass correlation coefficient range, 0.90 to 0.96). In combination, duplex ratio test values from three of the assays distinguished between the nevi and melanomas with 100% sensitivity (95% CI, 69.1% to 100%) and 100% specificity (95% CI, 69.1% to 100%). Duplex ratio test assays have been shown to be accurate and precise and can distinguish melanomas from common nevi using DNA from FFPE tissue. Appropriately designed assays could have value in assessment of other cancers.

Journal of Investigative Dermatology, 2013

International Journal of Cancer, 2004

Cutaneous melanoma (CM) is the most lethal form of skin cancer. Along with some benign melanocyti... more Cutaneous melanoma (CM) is the most lethal form of skin cancer. Along with some benign melanocytic tumors, the majority shows BRAF or NRAS mutation, but it is not known whether these are essential to all forms of melanocytic neoplasia. We screened 79 melanocytic tumors of different types for BRAF and NRAS mutations and looked at MAPK pathway activity using immunohistochemistry in a subset. Significant differences in BRAF exon 15 mutation frequency were found: 14/16 (87.5%) in common acquired naevi (CANs), 9/12 (75%) in CMs, 0/26 in Spitz naevi and 3/25 (12%) in blue naevi (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01). We looked at whether Spitz and blue naevi showed a compensatory increase in BRAF exon 11 and/or NRAS exons 1 and 2 mutations to account for the low BRAF exon 15 mutation frequency. NRAS mutations were found in only 1/16 (6.3%) Spitz naevi and 0/15 blue naevi. In addition, NRAS mutations were found in 2/11 (18.2%) CANs and 3/12 (25%) CMs. None of the tumors showed BRAF exon 11 mutations. Despite their low combined BRAF and NRAS mutation frequency, Spitz naevi showed strong MAPK pathway activation as measured by cytoplasmic expression of dually phosphorylated ERK1/2, while blue naevi had weak pathway activation. We conclude that BRAF and NRAS mutations are not necessary for melanocytic tumor development and that some types of tumor must arise by alternative mechanisms.

Journal of Cutaneous Pathology, 2022

BackgroundInvasive width, the distance between the most peripheral invasive melanoma cells on the... more BackgroundInvasive width, the distance between the most peripheral invasive melanoma cells on the section where Breslow thickness (BT) was measured, was recently identified as a prognostic feature. It is unclear whether a routine measurement is justified, given that macroscopic width is already included in many melanoma histopathology reports and may itself be a prognostic feature. This study sought to investigate this.MethodsA retrospective cohort of 718 melanoma patients in which macroscopic width had been stated in the original histopathology report was used. Survival analysis was performed.ResultsMacroscopic and invasive widths were positively correlated (p < 0.001). Invasive width was typically smaller than the paired macroscopic width (median difference 3.7 mm, p < 0.001), a difference seen across all T groups. Both macroscopic and invasive widths were significantly associated with melanoma survival in Kaplan–Meier analysis, including overall survival, but invasive width...

Social Science Research Network, 2019

The Hedgehog signalling pathway and its role in cancer Gerald Saldanha Mutations in Hedgehog (Hh)... more The Hedgehog signalling pathway and its role in cancer Gerald Saldanha Mutations in Hedgehog (Hh) pathway genes such as Patched1 are of established importance in Basal cell carcinoma (BCC). However, BCC is unusual because it has indolent behaviour and rarely metastasises. Hh pathway gene mutations are rare in cancers with more typical malignant behaviour, leading to the hypothesis that the pathway plays an important role in BCC but has limited importance in cancers with typical malignant behaviour. This has been addressed in three ways. First, evidence that superficial BCC is a clonal proliferation driven by alterations at the Patched1 locus was sought. Second, evidence of Wnt pathway activation in BCC was sought by looking for nuclear accumulation of p-catenin, because Wnt is a putative Hedgehog pathway target gene. Third, to determine whether the Hh pathway was important in a cancer with typical malignant behaviour, breast cancer was studied. In superficial BCC, microdissection of...

JCO Precision Oncology, 2019

Successful treatment of primary cutaneous diffuse large B cell lymphoma leg type with single agen... more Successful treatment of primary cutaneous diffuse large B cell lymphoma leg type with single agent venetoclax

Orbit (Amsterdam, Netherlands), Jan 20, 2018

Microcystic adnexal carcinoma (MAC) is a very rare and locally aggressive sweat gland tumour. MAC... more Microcystic adnexal carcinoma (MAC) is a very rare and locally aggressive sweat gland tumour. MAC has been well reported presenting as a periocular cutaneous lesion, rarely with subsequent direct orbital invasion and only once as a primary orbital lesion. Local recurrence is frequent after primary surgical excision and the role of adjuvant radiotherapy is ill-defined. We describe a case of orbital MAC treated successfully with radiotherapy after incomplete margin clearance post exenteration surgery and review the associated literature.

Melanoma research, Jan 12, 2018

The aims of this study were to investigate, in cutaneous malignant melanoma (MM), the integrity o... more The aims of this study were to investigate, in cutaneous malignant melanoma (MM), the integrity of nuclear vitamin D receptor (VDR) signalling, as implied by VDR subcellular location; to investigate the relationship between VDR and tumour progression and the inhibitory effect on VDR by mitogen-activated protein kinase (MAPK) overactivity. Archived tissue from 34 benign melanocytic naevi, 149 MMs and 44 matched metastases were stained by immunohistochemistry for VDR and a subset of primary MMs were stained for phosphorylated-extracellular signal-regulated kinase as a marker of MAPK activity. MM cell lines were investigated to show the subcellular location of VDR and cell viability in response to ligand±MAPK inhibitor. Benign melanocytic naevi showed mainly a strong nuclear VDR staining in contrast to MM where decreased nuclear and emergent cytoplasmic VDRs were associated with malignant progression in terms of dermal invasion and metastasis. MMs that retained exclusive nuclear VDR at...

The American journal of surgical pathology, Jan 16, 2018

Histomorphologic prognostic biomarkers that can be measured using only an hematoxylin and eosin s... more Histomorphologic prognostic biomarkers that can be measured using only an hematoxylin and eosin stain are very attractive because they are simple and cheap. We conceived an entirely novel biomarker of this type, the Breslow density (BD), which measures invasive melanoma cell density at the site where Breslow thickness (BT) is measured. This study assessed BD's prognostic value. In this study, BD was measured in 1329 melanoma patients. Measurement accuracy and precision was assessed using intraclass correlation coefficient (ICC). Survival was assessed with a primary end-point of melanoma-specific survival (MSS) and also overall survival and metastasis-free survival. We found that BD measurement was accurate compared with gold standard image analysis (ICC, 0.84). Precision was excellent for 3 observers with different experience (ICC, 0.93) and for an observer using only written instructions (ICC, 0.93). BD was a highly significant predictor in multivariable analysis for overall su...

Orbit, 2017

A 49-year-old male presented with a 4 month history of dysaesthesia in the left periorbital regio... more A 49-year-old male presented with a 4 month history of dysaesthesia in the left periorbital region. A CT scan showed a lacrimal gland mass with areas of dense calcification. Biopsies of the left lacrimal gland revealed a silver material with associated chronic granulomatous inflammation and secondary calcification. The histological specimen was sent for X-ray microanalysis. This confirmed the silver material to be aluminium. The metal fragments were subsequently discovered to be from a car aerial, which caused an orbital impaling injury 20 years prior to presentation. This is the first reported case of lacrimal gland calcification secondary to chronic metal foreign body exposure. The case is presented alongside literature reviews of lacrimal gland calcification and associated radiological findings.

American Journal of Surgical Pathology, 2017

The purpose of this study was to evaluate the prognostic value of tumor-infiltrating lymphocytes ... more The purpose of this study was to evaluate the prognostic value of tumor-infiltrating lymphocytes (TILs) in melanoma and to determine whether a simpler numerical scoring system would be more effective. In total, 655 patients presenting to a UK teaching hospital with primary invasive melanoma were analyzed. TILs were rescored using the standard Clark’s method and univariable and multivariable analyses of the effect of TILs on overall survival (OS), disease-specific survival (DSS), and metastasis-free survival (MFS) was assessed using Cox regression. In total, 30 (5%) melanomas showed absent, 464 (71%) nonbrisk, and 161 (24%) brisk TILs. There was a statistically significant relationship between TILs and Breslow thickness, age, melanoma type, mitotic rate, and histologic regression. TIL grade was a significant predictor of MFS in multivariable analysis (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.25-0.77) but was not significant for OS or DSS. By contrast, when a simple nu...

Modern Pathology, 2016

Outcomes for melanoma patients vary within cancer stage. Prognostic biomarkers are potential adju... more Outcomes for melanoma patients vary within cancer stage. Prognostic biomarkers are potential adjuncts to provide more precise prognostic information. Simple, low-cost biomarker assays, such as those based on immunohistochemistry, have strong translational potential. 5-hydroxymethylcytosine (5 hmC) shows prognostic potential in melanoma but prior studies were small. We, therefore, analysed 5 hmC in a retrospective cohort to provide external validation of its prognostic value. Two hundred primary melanomas were evaluated for 5 hmC expression using immunohistochemistry. The primary objective was to assess the effect on overall survival while controlling for important confounders. Univariable and multivariable analyses were performed. REMARK guidelines were followed. The 5 hmC immunohistochemistry scoring showed very strong inter-observer agreement (ICC 0.88) and expression was significantly related to age, site, Breslow thickness, ulceration, mitotic rate, and stage. Kaplan-Meier analysis showed 5 hmC was associated with metastasis-free, melanomaspecific, and overall survival, Po0.0001 for each. In univariable Cox proportional hazards models, 5 hmC hazard ratios were significant and remained so in a multivariable model. A two-step cox model was created using stage and 5 hmC, as stage is the gold standard for clinical practice. The addition of 5 hmC produced significant improvement in the model and 5 hmC and stage were independent significant predictors. This is the largest study of the prognostic value of 5 hmC immunohistochemistry in melanoma. The 5 hmC scoring was easily and reproducibly performed and it was an independent predictor of metastasis-free survival, melanoma-specific survival, and overall survival. This work supports further development of 5 hmC as a prognostic biomarker and suggests that it could add more precision to American Joint Committee on Cancer staging.

Histopathology, 2016

Aims-In 1970, Breslow described his eponymously named thickness measurement. No-one has sought to... more Aims-In 1970, Breslow described his eponymously named thickness measurement. No-one has sought to enhance Breslow thickness (BT). This purpose of this study was to demonstrate proof of concept that the density of melanoma cells at the position where Breslow thickness is measured is a morphological prognostic biomarker, which we name Breslow density (BD). The hypothesis was that BD has prognostic value for overall survival (OS) and is independent of BT. Methods and results-We analysed 100 cutaneous melanomas and followed REMARK guidelines. BD was the estimated percentage dermal area occupied by melanoma cells in a specified location. BT and BD had a strong correlation (p = 2.1 x 10-11) but despite this they were independent prognostic factors for OS in Cox regression (BD: HR 1.03, p=0.001849 and BT: HR 1.09, p=0.000146). This was corroborated by an independent effect on melanoma specific survival. We assessed whether BT and BD could be combined into a Breslow score. A prognostic index based on Cox regression coefficients was used and this showed a marginal improvement in predicted 5-year survival compared to BT alone (are under curve 94.8% v 96.7%). Conclusions-We show proof of concept that BD represents a novel morphological prognostic biomarker that is independent of BT and that there is potential to combine these into a Breslow score. Larger studies are needed to validate BD, but the simplicity of this biomarker makes it a strong candidate for translation to clinical practice.

Modern Pathology, 2016

Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disea... more Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disease has been revolutionized by new agents. More therapeutic options, coupled with the desire to extend treatment to the adjuvant setting mean that prognostic biomarkers that can be assayed from formalin-fixed paraffin-embedded clinical would be valuable. microRNAs have potential to fill this need. We analyzed 377 microRNAs in 79 primary melanomas and 32 metastases using a split sample discovery strategy. From a discovery analysis using 40 thick primary melanomas (20 cases with metastasis and 20 controls without metastasis at 5 years), microRNA expression was measured by quantitative RT-PCR (QRT-PCR). MiR-10b emerged as a candidate prognostic microRNA. This was confirmed in an independent validation set of thick primary melanomas (20 cases with metastasis and 19 controls without metastasis at 5 years). In the combined discovery and validation cohorts (n = 79), miR-10b expression showed a 3.7-fold increase in expression between cases and controls (P = 0.005) and showed a trend of increasing expression between primary melanomas and their matched metastases (Po 0.001). In situ hybridization showed expression was in melanoma cells and correlated with expression measured by QRT-PCR (P = 0.0005). We used the combined discovery and validation samples to verify the prognostic value of additional candidate microRNAs identified from other studies, and proceeded to analyze miR-200b. We demonstrated that miR-10b and miR-200b showed independent prognostic value (P = 0.002 and 0.047, respectively) in multivariable analysis alongside known clinico-pathological prognostic features (eg, Breslow thickness) using a Cox proportional hazards regression model. Furthermore, the addition of these microRNAs to the clinico-pathological features led to an improved regression model with better identification of aggressive thick melanomas. Taken together, these data suggest that miR-10b is a new prognostic microRNA for melanoma and that there could be a place for microRNA analysis in stratifying melanoma for therapy.

Melanoma research, 2016

Identification of prognostic biomarkers is timely for melanoma as clinicians seek ways to stratif... more Identification of prognostic biomarkers is timely for melanoma as clinicians seek ways to stratify patients for molecular therapy. MicroRNAs are promising as tissue biomarkers because they can be assayed directly from formalin-fixed paraffin-embedded clinical samples. We previously reported that microRNA-21 (miR-21) was strongly expressed in melanoma relative to naevi and now sought to further assess the significance of this by assessing its relationship with its putative target, PTEN. Clinical melanoma samples were analysed by immunohistochemical analysis for PTEN, stem-loop qRT-PCR for miR-21 and PCR for BRAF/NRAS mutation status. Cell lines were investigated for the effect of anti-miR-21 on PTEN. A total of 81 clinical melanocytic tumour samples were investigated, with uniformly high PTEN expression in the nucleus and cytoplasm of naevi and with preferential loss of PTEN expression in the nucleus of melanoma cells. miR-21 expression was inversely associated with nuclear PTEN expr...

The Journal of molecular diagnostics : JMD, Jan 29, 2015

Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical p... more Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCR-based assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. The reproducibility and accuracy of the DRTs were demonstrated and appropriate correction factors for DNA quality calculated for each assay, based on the results of 108 diploid samples. As a panel, seven DRTs were able to differentiate unambiguous cases of melanoma and nevi with a sensitivity of 87% (95% CI, 83%-91%) and a specificity of 88% (95% CI, 84%-92%) in a series of 145 melanomas and 123 nevi. The DRT scores for 20 nonmetastasizing primary melanomas a...

Cancer Research, 2013

Embryonic transcription factors (ETFs) of the Snail, Zeb and Twist families are essential mediato... more Embryonic transcription factors (ETFs) of the Snail, Zeb and Twist families are essential mediators of EMT programs (Epithelial to Mesenchymal Transition) and play central roles in cell determination and cell plasticity. The aberrant expression of ETFs is frequently observed in various cancer types, particularly in carcinomas, and is associated with poor prognosis and high risk of metastasis. We and others have demonstrated that their oncogenic potential relies on their ability to enable escape from oncogene-induced failsafe programs, senescence and apoptosis. Therefore, EMT-resembling processes initiated by ETFs contribute to neoplastic transformation in vitro and accelerate tumor progression in vivo in an epithelial context. We now unveil a completely different scenario in melanoma, a highly metastatic neural crest-derived cancer. Consistent with their role in melanoblasts determination, SNAIL2 and ZEB2 are already expressed in melanocytes and naevi, but their expression is down-r...

Clinics in laboratory medicine, 2011

Melanoma is the most serious type of skin cancer. Unfortunately, treatment has progressed little ... more Melanoma is the most serious type of skin cancer. Unfortunately, treatment has progressed little and advanced melanoma has appalling survival rates. A goal of molecular analysis is to fully describe the alterations that underpin melanoma's clinical phenotype so that diagnosis can be more accurate, outcome can be predicted with greater confidence, and treatment that is tailored to the patient can be given. This article describes the handful of "signature" changes that are known to occur, describes how some recent studies have shed light on changes beyond this signature, and finally discusses the impact of molecular pathology for practicing histopathologists.

The Journal of Molecular Diagnostics, 2013

A minority of melanocytic lesions cannot confidently be classified as benign or malignant on hist... more A minority of melanocytic lesions cannot confidently be classified as benign or malignant on histopathological examination, causing diagnostic uncertainty. DNA copy number changes can be used to distinguish nevi from melanoma, although the use of FFPE tissue can pose technical challenges. DNA copy number assays, called duplex ratio tests, have been developed with duplex real-time PCR, using a simple method with potential for high throughput. Five duplex ratio test assays targeting loci with common DNA copy number changes in melanoma were designed and tested using DNA extracted from FFPE samples microdissected from melanoma, common nevi, benign tonsil (10 each), and two melanoma cell lines. The assays proved accurate when DNA extracted from fresh and FFPE melanoma cell lines were compared (intraclass correlation coefficient, 0.99) and gave precise results when repeated on DNA from FFPE tissue (intraclass correlation coefficient range, 0.90 to 0.96). In combination, duplex ratio test values from three of the assays distinguished between the nevi and melanomas with 100% sensitivity (95% CI, 69.1% to 100%) and 100% specificity (95% CI, 69.1% to 100%). Duplex ratio test assays have been shown to be accurate and precise and can distinguish melanomas from common nevi using DNA from FFPE tissue. Appropriately designed assays could have value in assessment of other cancers.

Journal of Investigative Dermatology, 2013

International Journal of Cancer, 2004

Cutaneous melanoma (CM) is the most lethal form of skin cancer. Along with some benign melanocyti... more Cutaneous melanoma (CM) is the most lethal form of skin cancer. Along with some benign melanocytic tumors, the majority shows BRAF or NRAS mutation, but it is not known whether these are essential to all forms of melanocytic neoplasia. We screened 79 melanocytic tumors of different types for BRAF and NRAS mutations and looked at MAPK pathway activity using immunohistochemistry in a subset. Significant differences in BRAF exon 15 mutation frequency were found: 14/16 (87.5%) in common acquired naevi (CANs), 9/12 (75%) in CMs, 0/26 in Spitz naevi and 3/25 (12%) in blue naevi (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01). We looked at whether Spitz and blue naevi showed a compensatory increase in BRAF exon 11 and/or NRAS exons 1 and 2 mutations to account for the low BRAF exon 15 mutation frequency. NRAS mutations were found in only 1/16 (6.3%) Spitz naevi and 0/15 blue naevi. In addition, NRAS mutations were found in 2/11 (18.2%) CANs and 3/12 (25%) CMs. None of the tumors showed BRAF exon 11 mutations. Despite their low combined BRAF and NRAS mutation frequency, Spitz naevi showed strong MAPK pathway activation as measured by cytoplasmic expression of dually phosphorylated ERK1/2, while blue naevi had weak pathway activation. We conclude that BRAF and NRAS mutations are not necessary for melanocytic tumor development and that some types of tumor must arise by alternative mechanisms.