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Purpose: This study compares immunohistochemical (IHC) versus molecular subtyping (BluePrint and MammaPrint) in the population of patients enrolled in MINDACT and outcome based on molecular subtyping (MS) versus surrogate pathological subtyping (PS) as defined by the 2013 St. Gallen guidelines., Methods: MS classified patients in the following subtypes: Luminal A, Luminal B, HER-2-, and Basal-type. IHC/FISH for pathological subtyping (ER, PgR, HER-2, and Ki67) was centrally assessed in the European Institute of Oncology (n = 5806). Hazard ratios for distant-metastasis-free survival (DMFS) by subtype were adjusted for chemotherapy and endocrine therapy administration and thus independent of adjuvant treatment allocation., Results: PS Luminal cancers classified as HER-2+ or Basal-type by MS did not have a significantly lower DMFS than the Luminal-type cancers by MS (95.9%): HR = 1.40, 95% CI 0.75-2.60 (p = 0.294). More patients were identified with Luminal A disease by MS (63%) as compared with PS (47%) with comparable 5-year DMFS (≥96.0%). Among the 500 patients with PS TN cancers, MS identified 24 (5%) patients as Luminal-type with 5-year DMFS estimated at 100% versus 71.4% for MS HER-2+ or 90.1% for MS Basal-type., Conclusions: MS was able to re-stratify 54% of patients with a Luminal-B PS subtype to a low-risk Luminal A-type group with comparable outcome. Among TN EBC, 5% were classified as Luminal by MS with Luminal-like outcome. Molecular classification can help to identify a larger group of patients with low risk of recurrence compared with the more contemporarily used classification methodology including high-quality assessed Ki67.

Purpose: Hormone receptor-positive (HR+) tumors have heterogeneous biology and present a challenge for determining optimal treatment. In the Neoadjuvant Breast Registry Symphony Trial (NBRST) patients were classified according to MammaPrint/BluePrint subtyping to provide insight into the response to neoadjuvant endocrine therapy (NET) or neoadjuvant chemotherapy (NCT)., Objective: The purpose of this predefined substudy was to compare MammaPrint/BluePrint with conventional 'clinical' immunohistochemistry/fluorescence in situ hybridization (IHC/FISH) subtyping in 'clinical luminal' [HR+/human epidermal growth factor receptor 2-negative (HER2-)] breast cancer patients to predict treatment sensitivity., Methods: NBRST IHC/FISH HR+/HER2- breast cancer patients (n = 474) were classified into four molecular subgroups by MammaPrint/BluePrint subtyping: Luminal A, Luminal B, HER2, and Basal type. Pathological complete response (pCR) rates were compared with conventional IHC/FISH subtype., Results: The overall pCR rate for 'clinical luminal' patients to NCT was 11 %; however, 87 of these 474 patients were reclassified as Basal type by BluePrint, with a high pCR rate of 32 %. The MammaPrint index was highly associated with the likelihood of pCR (p < 0.001). Fifty-three patients with BluePrint Luminal tumors received NET with an aromatase inhibitor and 36 (68 %) had a clinical response., Conclusions: With BluePrint subtyping, 18 % of clinical 'luminal' patients are classified in a different subgroup, compared with conventional assessment, and these patients have a significantly higher response rate to NCT compared with BluePrint Luminal patients. MammaPrint/BluePrint subtyping can help allocate effective treatment to appropriate patients. In addition, accurate identification of subtype biology is important in the interpretation of neoadjuvant treatment response since lack of pCR in luminal patients does not portend the worse prognosis associated with residual disease in Basal and HER2 subtypes.

Background: The prospective Neoadjuvant Breast Symphony Trial (NBRST) study found that MammaPrint/BluePrint functional molecular subtype is superior to conventional immunohistochemistry/fluorescence in situ hybridization subtyping for predicting pathologic complete response (pCR) to neoadjuvant chemotherapy. The purpose of this substudy was to determine if the rate of pCR is affected by tumor size., Methods: The NBRST study includes breast cancer patients who received neoadjuvant chemotherapy. MammaPrint/BluePrint subtyping classified patients into four molecular subgroups: Luminal A, Luminal B, HER2 (human epidermal growth factor receptor 2), and Basal type. Probability of pCR (ypT0/isN0) as a function of tumor size and molecular subgroup was evaluated., Results: A total of 608 patients were evaluable with overall pCR rates of 28.5 %. Luminal A and B patients had significantly lower rates of pCR (6.1 and 8.7 %, respectively) than either basal (37.1 %) or HER2 (55.0 %) patients (p < 0.001). The probability of pCR significantly decreased with tumor size >5 cm [p = 0.022, odds ratio (OR) 0.58, 95 % confidence interval (CI) 0.36, 0.93]. This relationship was statistically significant in the Basal (p = 0.026, OR 0.46, 95 % CI 0.23, 0.91) and HER2 (p = 0.039, OR 0.36, 95 % CI 0.14, 0.95) subgroups. In multivariate logistic regression analyses, the dichotomized tumor size variable was not significant in any of the molecular subgroups., Discussion: Even though tumor size would intuitively be a clinical determinant of pCR, the current analysis showed that the adjusted OR for tumor size was not statistically significant in any of the molecular subgroups. Factors significantly associated with pCR were PR status, grade, lymph node status, and BluePrint molecular subtyping, which had the strongest correlation.

Accurate identification of breast cancer patients most likely to benefit from adjuvant systemic therapies is crucial. Better understanding of differences between methods can lead to an improved ER, PgR, and HER-2 assessment. The purpose of this preplanned translational research is to investigate the correlation of central IHC/FISH assessments with microarray mRNA readouts of ER, PgR, and HER-2 status in the MINDACT trial and to determine if any discordance could be attributed to intratumoral heterogeneity or the DCIS and normal tissue components in the specimens. MINDACT is an international, prospective, randomized, phase III trial investigating the clinical utility of MammaPrint in selecting patients with early breast cancer for adjuvant chemotherapy (n = 6694 patients). Gene-expression data were obtained by TargetPrint; IHC and/or FISH were assessed centrally (n = 5788; 86 %). Macroscopic and microscopic evaluation of centrally submitted FFPE blocks identified 1427 cases for which the very same sample was submitted for gene-expression analysis. TargetPrint ER had a positive agreement of 98 %, and a negative agreement of 95 % with central pathology. Corresponding figures for PgR were 85 and 94 % and for HER-2 72 and 99 %. Agreement of mRNA versus central protein was not different when the same or a different portion of the tumor tissue was analyzed or when DCIS and/or normal tissue was included in the sample subjected to mRNA assays. This is the first large analysis to assess the discordance rate between protein and mRNA analysis of breast cancer markers, and to look into intratumoral heterogeneity, DCIS, or normal tissue components as a potential cause of discordance. The observed difference between mRNA and protein assessment for PgR and HER-2 needs further research; the present analysis does not support intratumoral heterogeneity or the DCIS and normal tissue components being likely causes of the discordance.

The 70-gene signature (MammaPrint) has been developed to predict the risk of distant metastases in breast cancer and select those patients who may benefit from adjuvant treatment. Given the strong association between locoregional and distant recurrence, we hypothesize that the 70-gene signature will also be able to predict the risk of locoregional recurrence (LRR). 1,053 breast cancer patients primarily treated with breast-conserving treatment or mastectomy at the Netherlands Cancer Institute between 1984 and 2006 were included. Adjuvant treatment consisted of radiotherapy, chemotherapy, and/or endocrine therapy as indicated by guidelines used at the time. All patients were included in various 70-gene signature validation studies. After a median follow-up of 8.96 years with 87 LRRs, patients with a high-risk 70-gene signature (n = 492) had an LRR risk of 12.6% (95% CI 9.7-15.8) at 10 years, compared to 6.1% (95% CI 4.1-8.5) for low-risk patients (n = 561; P < 0.001). Adjusting the 70-gene signature in a competing risk model for the clinicopathological factors such as age, tumour size, grade, hormone receptor status, LVI, axillary lymph node involvement, surgical treatment, endocrine treatment, and chemotherapy resulted in a multivariable HR of 1.73 (95% CI 1.02-2.93; P = 0.042). Adding the signature to the model based on clinicopathological factors improved the discrimination, albeit non-significantly [C-index through 10 years changed from 0.731 (95% CI 0.682-0.782) to 0.741 (95% CI 0.693-0.790)]. Calibration of the prognostic models was excellent. The 70-gene signature is an independent prognostic factor for LRR. A significantly lower local recurrence risk was seen in patients with a low-risk 70-gene signature compared to those with high-risk 70-gene signature.

Purpose: The purpose of the NBRST study is to compare a multigene classifier to conventional immunohistochemistry (IHC)/fluorescence in situ hybridization (FISH) subtyping to predict chemosensitivity as defined by pathological complete response (pCR) or endocrine sensitivity as defined by partial response., Methods: The study includes women with histologically proven breast cancer, who will receive neoadjuvant chemotherapy (NCT) or neoadjuvant endocrine therapy. BluePrint in combination with MammaPrint classifies patients into four molecular subgroups: Luminal A, Luminal B, HER2, and Basal., Results: A total of 426 patients had definitive surgery. Thirty-seven of 211 (18 %) IHC/FISH hormone receptor (HR)+/HER2- patients were reclassified by Blueprint as Basal (n = 35) or HER2 (n = 2). Fifty-three of 123 (43 %) IHC/FISH HER2+ patients were reclassified as Luminal (n = 36) or Basal (n = 17). Four of 92 (4 %) IHC/FISH triple-negative (TN) patients were reclassified as Luminal (n = 2) or HER2 (n = 2). NCT pCR rates were 2 % in Luminal A and 7 % Luminal B patients versus 10 % pCR in IHC/FISH HR+/HER2- patients. The NCT pCR rate was 53 % in BluePrint HER2 patients. This is significantly superior (p = 0.047) to the pCR rate in IHC/FISH HER2+ patients (38 %). The pCR rate of 36 of 75 IHC/FISH HER2+/HR+ patients reclassified as BPLuminal is 3 %. NCT pCR for BluePrint Basal patients was 49 of 140 (35 %), comparable to the 34 of 92 pCR rate (37 %) in IHC/FISH TN patients., Conclusions: BluePrint molecular subtyping reclassifies 22 % (94/426) of tumors, reassigning more responsive patients to the HER2 and Basal categories while reassigning less responsive patients to the Luminal category. These findings suggest that compared with IHC/FISH, BluePrint more accurately identifies patients likely to respond (or not respond) to NCT.

Background: To investigate the correlation of TargetPrint with local and central immunohistochemistry/fluorescence in situ hybridization assessment of estrogen (ER), progesterone (PgR), and human epidermal growth factor receptor 2 (HER2) in the first 800 patients enrolled in the MINDACT trial., Patients and Methods: Data from local (N = 800) and central (N = 626) assessments of receptor status were collected and compared with TargetPrint results., Results: For ER, the positive agreement (the percentage of central pathology positive assessments that were also TargetPrint/local laboratory positive) for TargetPrint in comparison to centralized assessment was 98% with a negative agreement (the percentage of central pathology negative assessments that were also TargetPrint/local laboratory negative) of 96%. For PgR, the positive agreement was 83% with a negative agreement of 92%. For HER2, the positive agreement was 75% with a negative agreement of 99%. Even though the local assessment showed higher positive agreement for PgR (89%) and higher positive agreement for HER2 (85%), the range of discordant local versus central assessments were as high as 6.7% for ER, 12.9% for PgR, and 4.3% for HER2., Conclusion: TargetPrint and local assessment of ER, PgR, and HER2 show high concordance with central assessment in the first 800 MINDACT patients. However, there are concerns about the higher discordance rates for some local sites. TargetPrint can improve the reliability of hormone receptor and HER2 testing for those centers with a lower rate of concordance with the reference laboratory, with the limitation of a positive agreement of 75% for HER2. TargetPrint consequently has important implications for treatment decisions in clinical practice and is a reliable alternative to local assessment for ER., Clinical Trials Number: NCT00433589.

MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, has been available for fresh tissue. Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Here, we describe method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrint-fresh, was performed on an independent series of matched tissue from five hospitals (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, interlaboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue., (Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Lifetime melanoma risk of mutation carriers from families with a germline mutation in the CDKN2A gene is estimated to be 67%. The necessity to include family members in a melanoma surveillance program is widely endorsed, but there is no consensus on which family members should be invited., Methods: In a retrospective follow-up study, we investigated the yield of surveillance of first- and second-degree relatives of melanoma and pancreatic cancer patients from 21 families with the "p16-Leiden" CDKN2A mutation. Melanoma incidence rates were compared with the general population., Results: Three-hundred and fifty-four first-degree relatives and 391 second-degree relatives were included. Forty-five first-degree relatives and 11 second-degree relatives were diagnosed with melanoma. Most (72%) of second-degree relatives diagnosed with melanoma had become a first-degree relative before diagnosis, due to the occurrence of a melanoma in a parent or sibling. Overall, melanoma incidence rate was 2.1 per 1,000 person years [95% confidence interval (CI), 1.2-3.8] in family members still being second-degree relatives at diagnosis, compared with 9.9 per 1,000 person years (95% CI, 7.4-13.3) in first-degree relatives. The standardized morbidity ratio for melanoma of second-degree relatives compared with the general population was 12.9 (95% CI, 7.2-23.4)., Conclusion: Second-degree relatives from families with the p16-Leiden mutation in CDKN2A have a considerably increased melanoma risk compared with the general population., Impact: This study provides justification for the surveillance of second-degree relatives from families with a CDKN2A germline mutation.

Purpose: To compare breast cancer subtyping with the three centrally assessed microarray-based assays BluePrint, MammaPrint, and TargetPrint with locally assessed clinical subtyping using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)., Methods: BluePrint, MammaPrint, and TargetPrint were all performed on fresh tumor samples. Microarray analysis was performed at Agendia Laboratories, blinded for clinical and pathological data. IHC/FISH assessments were performed according to local practice at each institution; estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) assessments were performed on 132 samples, and Ki-67 on 79 samples., Results: The concordance between BluePrint and IHC/FISH subtyping was 94 % for the Luminal-type, 95 % for the HER2-type, and 94 % for the Basal-type subgroups. The concordance of BluePrint with subtyping using mRNA single gene readout (TargetPrint) was 96 % for the Luminal-type, 97 % for the HER2-type, and 98 % for the Basal-type subgroups. The concordance for substratification into Luminal A and B using MammaPrint and Ki-67 was 68 %. The concordance between TargetPrint and IHC/FISH was 97 % for ER, 80 % for PR, and 95 % for HER2., Conclusions: The implementation of multigene assays such as TargetPrint, BluePrint, and MammaPrint may improve the clinical management of breast cancer patients. High discordance between Luminal A and B substratification based on MammaPrint versus locally assessed Ki-67 or grade indicates that chemotherapy decisions should not be based on the basis of Ki-67 readout or tumor grade alone. TargetPrint serves as a second opinion for those local pathology settings where high-quality standardization is harder to maintain.

Classification of breast cancer into molecular subtypes maybe important for the proper selection of therapy, as tumors with seemingly similar histopathological features can have strikingly different clinical outcomes. Herein, we report the development of a molecular subtyping profile (BluePrint), that enables rationalization in patient selection for either chemotherapy or endocrine therapy prescription. An 80-Gene Molecular Subtyping Profile (BluePrint) was developed using 200 breast cancer patient specimens and confirmed on four independent validation cohorts (n = 784). Additionally, the profile was tested as a predictor of chemotherapy response in 133 breast cancer patients, treated with T/FAC neoadjuvant chemotherapy. BluePrint classification of a patient cohort that was treated with neoadjuvant chemotherapy (n = 133) shows improved distribution of pathological Complete Response (pCR), among molecular subgroups compared with local pathology: 56% of the patients had a pCR in the Basal-type subgroup, 3% in the MammaPrint Low-risk, Luminal-type subgroup, 11% in the MammaPrint High-risk, Luminal-type subgroup, and 50% in the HER2-type subgroup. The group of genes identifying Luminal-type breast cancer is highly enriched for genes having an Estrogen Receptor binding site proximal to the promoter-region, suggesting that these genes are direct targets of the Estrogen Receptor. Implementation of this profile may improve the clinical management of breast cancer patients, by enabling the selection of patients who are most likely to benefit from either chemotherapy or from endocrine therapy.

Cutaneous melanoma, a type of skin tumor originating from melanocytes, often develops from premalignant naevoid lesions via a gradual transformation process driven by an accumulation of (epi)genetic lesions. These dysplastic naevi display altered morphology and often proliferation of melanocytes. Additionally, melanocytes in dysplastic naevi show structural mitochondrial and melanosomal alterations and have elevated reactive oxygen species (ROS) levels. For this study we performed genome-wide expression and proteomic analysis of melanocytes from dysplastic naevus (DNMC) and adjacent normal skin (MC) from 18 patients. Whole genome expression profiles of the DNMC and MC of each individual patient subjected to GO-based comparative statistical analysis yielded significantly differentially expressed GO classes including "organellar ribosome," "mitochondrial ribosome," "hydrogen ion transporter activity," and "prefoldin complex." Validation of 5 genes from these top GO classes revealed a heterogeneous differential expression pattern. Proteomic analysis demonstrated differentially expressed proteins in DNMC that are involved in cellular metabolism, detoxification, and cytoskeletal organization processes, such as GTP-binding Rho-like protein CDC42, glutathione-S-transferase omega-1 and prolyl 4-hydroxylase. Collectively these results point to deregulation of cellular processes, such as metabolism and protein synthesis, consistent with the observed elevated oxidative stress levels in DNMC potentially resulting in oxidative DNA damage in these cells.

Background: For more than 25 years families with an increased susceptibility to melanoma have been under surveillance at our institution., Objective: We sought to investigate the effectiveness of surveillance for CDKN2A-mutated families and causes for failure of the program in patients with more advanced tumors., Methods: In a retrospective case-control study, Breslow thickness of melanomas diagnosed in relatives enrolled in the surveillance program were compared with melanomas of unscreened index patients. We investigated the influence of mode of detection and length of surveillance interval on outcome., Results: Surveillance melanomas (n = 226, median thickness: 0.50 mm) had a significantly lower Breslow thickness (multiplication factor: 0.61 [95% confidence interval 0.47-0.80], P < .001) than index melanomas (n = 40, median thickness: 0.98 mm). Index melanomas were more likely diagnosed with a Breslow thickness greater than 1.0 mm (odds ratio: 3.1 [95% confidence interval 1.2-8.1], P = .022). In all, 53% of surveillance melanomas were diagnosed during regular screens, 7% during patients' first screen, 20% between regular screens, and 20% in patients who were noncompliant with the surveillance schedule. The majority of surveillance melanomas (58%) were detected within 6 months after the last screen. There was no correlation between tumor thickness and the length of the screening interval for tumors diagnosed within 24 months since the last screen., Limitations: The study is retrospective., Conclusions: Surveillance was associated with earlier detection of melanomas. Noncompliance was an important cause for failing surveillance. Shortening surveillance intervals may advance detection of tumors, but may paradoxically have little impact on prognosis., (Copyright © 2010 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

Background: About 10% of cutaneous malignant melanomas (CMM) occur in individuals with a family history of melanoma. In 20% to 40% of melanoma families germline mutations in CDKN2A are detected. Knowledge of the clinicohistologic characteristics of melanomas and patients from these families is important for optimization of management strategies, and may shed more light on the complex interplay of genetic and environmental factors in the pathogenesis of melanoma., Objective: We sought to investigate the clinical and histologic characteristics of CMM in CDKN2A-mutated families., Methods: Clinical and histologic characteristics of 182 patients with 429 CMM from families with a founder mutation in CDKN2A (p16-Leiden mutation) were compared with 7512 patients with 7842 CMM from a population-based cancer registry., Results: Patients with p16-Leiden had their first melanoma 15.3 years younger than control patients. The 5-year cumulative incidence of second primary CMM was 23.4% for patients with p16-Leiden compared with 2.3% for control patients. The risk of a second melanoma was twice as high for patients with p16-Leiden who had their first melanoma before age 40 years, compared with older patients with p16-Leiden. Unlike control patients, there was no body site concordance of the first and second melanoma in patients with p16-Leiden and multiple primary melanomas. Patients with p16-Leiden had significantly more superficial spreading, and less nodular and lentiginous melanomas., Limitations: Ascertainment of patients with p16-Leiden was family based. The study was performed in families with a founder mutation, the p16-Leiden mutation., Conclusion: Our findings are consistent with a pathogenic pathway of melanoma development from nevi, starting early and ongoing throughout life, and not related to chronic sun exposure., (Copyright © 2010 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

Approximately 5% to 10% of melanoma may be hereditary in nature, and about 2% of melanoma can be specifically attributed to pathogenic germline mutations in cyclin-dependent kinase inhibitor 2A (CDKN2A). To appropriately identify the small proportion of patients who benefit most from referral to a genetics specialist for consideration of genetic testing for CDKN2A, we have reviewed available published studies of CDKN2A mutation analysis in cohorts with invasive, cutaneous melanoma and found variability in the rate of CDKN2A mutations based on geography, ethnicity, and the type of study and eligibility criteria used. Except in regions of high melanoma incidence, such as Australia, we found higher rates of CDKN2A positivity in individuals with 3 or more primary invasive melanomas and/or families with at least one invasive melanoma and two or more other diagnoses of invasive melanoma and/or pancreatic cancer among first- or second-degree relatives on the same side of the family. The work summarized in this review should help identify individuals who are appropriate candidates for referral for genetic consultation and possible testing.

Purpose: We report the largest study to date analyzing the risk of cancers other than melanoma in melanoma families positive for the same CDKN2A mutation., Experimental Design: We studied family members of 22 families positive for the p16-Leiden founder mutation who had attended a surveillance clinic or were their close relatives. Within this cohort, observed and expected rates of cancer were computed by mutation status consisting of 221 (proven plus obligate) carriers, 639 (proven plus obligate) noncarriers, and 668 first-degree relatives whose carrier risk was estimated from the relationship to known carriers and the age and melanoma status of that person and their relatives., Results: Our analysis shows a relative risk (RR) of cancer other than melanoma and nonmelanoma skin cancer of 4.4 [95% confidence interval (95% CI), 3.3-5.6], predominantly attributable to the increased risk for pancreatic cancer (RR, 46.6; 95% CI, 24.7-76.4), but also for other cancers. We provide substantial proof for pancreatic cancer being a key component of the p16-Leiden phenotype. Inclusion of this cancer in a penetrance analysis leads to an estimated RR of pancreatic cancer for mutation carriers of 47.8 (95% CI, 28.4-74.7)., Conclusions: This study shows clear evidence of increased risk of cancers other than melanoma in CDKN2A families carrying the p16-Leiden mutation. Further research is necessary to determine if similar risks apply to families with CDKN2A mutations other than p16-Leiden.

In most Dutch melanoma families, a founder deletion in the melanoma susceptibility gene CDKN2A (which encodes p16) is present. This founder deletion (p16-Leiden) accounts for a significant proportion of the increased melanoma risk. However, it does not account for the Atypical Nevus (AN) phenotype that segregates in both p16-Leiden carriers and non-carriers. The AN-affected p16-Leiden family members are therefore a unique valuable resource for unraveling the genetic etiology of the AN phenotype, which is considered both a risk factor and a precursor lesion for melanoma. In this study, we performed a genome-wide scan for linkage in four p16-Leiden melanoma pedigrees, classifying family members with five or more AN as affected. The strongest evidence for an atypical nevus susceptibility gene was mapped to chromosome band 7q21.3 (two-point LOD score=2.751), a region containing candidate gene CDK6.

Objective: We report on the uptake and psychological impact of p16-Leiden genetic testing to contribute to a greater understanding of counseling melanoma families., Methods: Within a defined research setting, genetic counseling and testing were offered to members of p16-Leiden-positive melanoma pedigrees, at risk of carrying a gene defect associated with an increased risk of melanoma and pancreatic cancer., Results: One hundred and eighty-four individuals sought counseling, of which 141 (77%) opted for genetic testing. Uptake of genetic counseling and testing, and psychological motivation was evaluated in 94 (57%) individuals. Higher pre-test risk of carrying the mutation and older age proved significantly predictive for counseling uptake. Age was predictive for test acceptance, whereas fearful test expectancies predicted test decline. Counselees had lower distress levels than those reported in other oncogenetic testing settings., Conclusion: We are the first to report on genetic testing for familial melanoma. Following the first counseling session, we found a relatively high uptake rate for p16-Leiden testing and no clinically worrisome levels of distress.

Background: Atypical nevi (AN), present in either a familial or a sporadic setting, are strong indicators of increased melanoma risk., Objective: To estimate the extent of this risk and the extent of reclassification of sporadic to familial cases during follow-up., Methods: We studied 167 sporadic patients with AN (>or=5). At the end of follow-up we updated the family history regarding melanoma and performed germline mutation analysis of the known melanoma susceptibility genes., Results: We found a relative risk for melanoma of 46.1 (95% confidence interval 21.0-87.5). Six of 167 patients were carriers of a CDKN2A mutation. At the end of follow-up, 10 of 136 patients with sporadic AN reported being a member of a melanoma family., Limitations: This study was conducted in an area with a founder mutation in many of its melanoma families; therefore the results may not be applicable to other populations., Conclusion: We report a high relative risk of 46.1 of melanoma development in patients with sporadic AN. A significant proportion of this Dutch cohort reported additional cases in their families over time.

GenoMEL, comprising major familial melanoma research groups from North America, Europe, Asia, and Australia has created the largest familial melanoma sample yet available to characterize mutations in the high-risk melanoma susceptibility genes CDKN2A/alternate reading frames (ARF), which encodes p16 and p14ARF, and CDK4 and to evaluate their relationship with pancreatic cancer (PC), neural system tumors (NST), and uveal melanoma (UM). This study included 466 families (2,137 patients) with at least three melanoma patients from 17 GenoMEL centers. Overall, 41% (n = 190) of families had mutations; most involved p16 (n = 178). Mutations in CDK4 (n = 5) and ARF (n = 7) occurred at similar frequencies (2-3%). There were striking differences in mutations across geographic locales. The proportion of families with the most frequent founder mutation(s) of each locale differed significantly across the seven regions (P = 0.0009). Single founder CDKN2A mutations were predominant in Sweden (p.R112&#95;L113insR, 92% of family's mutations) and the Netherlands (c.225&#95;243del19, 90% of family's mutations). France, Spain, and Italy had the same most frequent mutation (p.G101W). Similarly, Australia and United Kingdom had the same most common mutations (p.M53I, c.IVS2-105A>G, p.R24P, and p.L32P). As reported previously, there was a strong association between PC and CDKN2A mutations (P < 0.0001). This relationship differed by mutation. In contrast, there was little evidence for an association between CDKN2A mutations and NST (P = 0.52) or UM (P = 0.25). There was a marginally significant association between NST and ARF (P = 0.05). However, this particular evaluation had low power and requires confirmation. This GenoMEL study provides the most extensive characterization of mutations in high-risk melanoma susceptibility genes in families with three or more melanoma patients yet available.

This review aims to provide an up-to-date view on our understanding of the molecular genetics of melanoma development. It gives an overview of genes (and loci) currently known to be substantially involved in melanoma predisposition and progression. Broadly, the review falls into 3 sections: genes/loci involved in melanoma susceptibility through germline mutation, tumor suppressor genes somatically mutated or deleted in melanoma, and oncogenes mutated somatically in melanoma. The main cellular pathways in which these genes are involved are summarized and discussed. From this it is evident that abberations of cell cycle regulation, DNA repair and receptor-mediated signal transduction are important for melanocytic neoplasia.

Background: Since p16-Leiden presymptomatic testing for hereditary melanoma has become available in the Netherlands, the benefits and risks of offering such testing are evaluated. The current paper investigated why the non-participants were reluctant to participate in genetic testing., Methods: Sixty six eligible individuals, who were knowledgeable about the test but had not participated in genetic testing by January 2003, completed a self-report questionnaire assessing motivation, anxiety, family dynamics, risk knowledge and causal attributions., Results: Non-participants reported anxiety levels below clinical significance. A principal components analysis on reasons for non-participation distinguished two underlying motives: emotional and rational motivation. Rational motivation for non-participation was associated with more accurate risk knowledge, the inclination to preselect mutation carriers within the family and lower scores on anxiety. Emotional motivation for non-participation was associated with disease misperceptions, hesitation to communicate unfavourable test results within the family and higher scores on anxiety., Conclusion: Rational and emotional motivation for non-participation in the genetic test for hereditary melanoma was found. Emotionally motivated individuals may be reluctant to disseminate genetic risk information. Rationally motivated individuals were better informed than emotionally motivated individuals. It is suggested that a leaflet is added to the invitation letter to enhance informed decision-making about genetic testing.

Germline mutations of CDKN2A that affect the p16INK4a transcript have been identified in numerous melanoma pedigrees worldwide. In the UK, over 50% of pedigrees with three or more cases of melanoma have been found to carry mutations of CDKN2A. Mutations that affect p14ARF exon 1beta exclusively are very rare. This has led to the suggestion that it is p16INK4a and not p14ARF that plays the critical role in melanoma predisposition. We report the identification of a cluster of five different germline mutations at the p14ARF exon 1beta splice donor site in melanoma pedigrees. All the five splice site variants showed evidence of being causal mutations. Three of the variants were demonstrated to result in aberrant splicing of the p14ARF mRNA, confirming their role in melanoma predisposition. No other point mutations were identified in the coding region of p14ARF. The p14ARF transcript of CDKN2A is clearly important in disease predisposition in a subset of melanoma pedigrees. Curiously, the only mutations so far reported to affect p14ARF exon 1beta exclusively have been knockout mutations. Further investigation into the spectrum of mutations observed in this gene may help clarify the exact role of p14ARF in melanoma predisposition.

Purpose: The purpose is to document the clinical, pathological, and genetic features of pancreatic carcinoma (PC) in carriers of a specific p16-Leiden mutation (a 19-bp deletion in exon 2 of the CDKN2A gene)., Experimental Design: Clinical data and paraffin embedded tissue were obtained from 12 patients of p16-Leiden-positive families with PC. Because of the known 19-bp germ-line deletion, we could specifically analyze the genotype of the wild-type allele for loss of heterozygosity. K-ras codon 12 mutations were determined and immunohistochemical testing for p16, Tp53, Smad4, and cyclooxygenase 2 was performed., Results: The average age of subjects that developed PC (8 males) was 58 years (range, 43-74 years). Histology was considered as conventional ductal adenocarcinoma in 11 of 12 and neuroendocrine carcinoma (1 of 12). The carcinomas were located in the head (10 of 12), corpus (1 of 12), and tail (1 of 12) of the pancreas. The specific p16-Leiden mutation was confirmed in the tissue of all subjects. Loss of heterozygosity of the wild-type allele was present in 2 of 7 tumors analyzed. Immunostaining for p16 was negative in 10 of 10. Tp53 mutations were detected in 5 of 12. Smad4 was negative in 5 of 12 and cyclooxygenase 2 was overexpressed in 11 of 12. K-ras codon 12 mutations were present in 9 of 10 and in three precursor lesions even before abrogation of p16 protein expression was seen (one of three)., Conclusions: The p16-Leiden deletion was associated with progression toward conventional ductal adenocarcinomas in all cases but one. Our observations might support the feasibility of early diagnosis of PC in p16-Leiden mutation carriers and might also indicate that chemoprevention needs consideration.

The initial enthusiasm generated by the discovery of the first susceptibility gene found for melanoma has slightly dampened over recent years. For the majority of melanoma families the underlying gene defect is still not known, so the search for other melanoma genes is continuing. Also, the increased risk of melanoma does not seem to be restricted to mutation carriers, but is present even in non-mutation carriers in melanoma families. The underlying defect of familial melanoma is less straightforward than previously thought; both environmental and hereditary risk modifiers intermingle in a perplexing way. This makes familial melanoma a complex disorder which deserves the close attention of both clinicians and researchers, especially as the opinion on gene testing in familial melanoma has not yet achieved consensus. On the one hand, there is a rising demand from families for genetic testing; on the other hand, there is the clinicians' concern about the value of such testing.