PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma.

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier-penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.

Repurposing Azacitidine and Carboplatin to Prime Immune Checkpoint Blockade-resistant Melanoma for Anti-PD-L1 Rechallenge.

Purpose: Drug repurposing offers the opportunity for chemotherapy to be used to reestablish sensitivity to immune checkpoint blockade (ICB) therapy. Here we investigated the clinical and translational aspects of an early phase II study of azacitidine and carboplatin priming for anti-PDL1 immunotherapy (avelumab) in patients with advanced ICB-resistant melanoma. Experimental Design: A total of 20 participants with ICB-resistant metastatic melanoma received 2 × 4-week cycles of azacitidine and carboplatin followed by ICB rechallenge with anti-PD-L1 avelumab. The primary objective was overall response rate after priming and ICB rechallenge. Secondary objectives were clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS). Translational correlation analysis of HLA-A and PD-L1 expression, RNA sequencing, and reduced representation bisulfite sequencing of biopsies at baseline, after priming and after six cycles of avelmuab was performed. Results: The overall response rate (ORR) determined after azacitidine and carboplatin priming was 10% (2/20) with two partial responses (PR). The ORR determined after priming followed by six cycles of avelumab (week 22) was 10%, with 2 of 20 participants achieving immune partial response (iPR). The CBR for azacitidine and carboplatin priming was 65% (13/20) and after priming followed by six cycles of avelumab CBR was 35% (n = 7/20). The median PFS was 18.0 weeks [95% confidence interval (CI): 14.87-21.13 weeks] and the median OS was 47.86 weeks (95% CI: 9.67-86.06 weeks). Translational correlation analysis confirmed HLA-A generally increased after priming with azacitidine and carboplatin, particularly if it was absent at the start of treatment. Average methylation of CpGs across the HLA-A locus was decreased after priming and T cells, in particular CD8+, showed the greatest increase in infiltration. Conclusions: Priming with azacitidine and carboplatin can induce disease stabilization and resensitization to ICB for metastatic melanoma. Significance: There are limited treatments for melanoma once resistance to ICB occurs. Chemotherapy induces immune-related responses and may be repurposed to reinstate the response to ICB. This study provides the first evidence that chemotherapy can provide clinical benefit and increase OS for ICB-resistant melanoma.

Confocal microscopy, dermoscopy, and histopathology features of atypical intraepidermal melanocytic proliferations associated with evolution to melanoma in situ.

BACKGROUND: Atypical intraepidermal melanocytic proliferations (AIMP) is a descriptive term sometimes applied to biopsies that do not fulfill diagnostic criteria of melanoma. They are common on sun-damaged skin, but their definition and management are controversial. OBJECTIVE: To describe dermoscopic (DS), reflectance confocal microscopic (RCM) and histopathological features of AIMP and identify features associated with subsequent melanoma in situ (MIS). METHODS: A retrospective analysis of AIMP lesions correlated with patient outcome at two melanoma tertiary centers between 2005 and 2015. RESULTS: Thirty-four patients were included. Nine (26%) patients had MIS in subsequent biopsies. Predictors of later MIS were target-like pattern (OR:12.0 [CI: 1.23, 117.41]; P = 0.032) and high-density vascular network (OR:12 [CI: 1.23-117.41], P: 0.032) on DS, and presence of dendritic cells touching each other (OR:9.1 [CI: 1.54, 54.59], P = 0.014) on RCM. Clinical predictors of worse outcome included a previous history of MIS at the same site. Radiotherapy for AIMP had a high failure rate (all patients presented with recurrent disease, three as AIMP and two as MIS). CONCLUSIONS: Considering that most cases in this series received non-surgical treatment at baseline, we recommend close monitoring for lesions with target-like pattern and density vascular network on DS and treatment for lesions with progression of atypia and/or with "confluent" dendritic cells on RCM. Although the number of patients in this series is very low, early surgery is recommended for MIS cases that recur as AIMP.

BRAF mutation testing for patients diagnosed with stage III or stage IV melanoma: practical guidance for the Australian setting.

Targeted therapy (BRAF inhibitor plus MEK inhibitor) is now among the possible treatment options for patients with BRAF mutation-positive stage III or stage IV melanoma. This makes prompt BRAF mutation testing an important step in the management of patients diagnosed with stage III or IV melanoma; one that can help better ensure that the optimal choice of systemic treatment is initiated with minimal delay. This article offers guidance about when and how BRAF mutation testing should be conducted when patients are diagnosed with melanoma in Australia. Notably, it recommends that pathologists reflexively order BRAF mutation testing whenever a patient is found to have American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) stage III or IV melanoma (i.e., any metastatic spread beyond the primary tumour) and that patient's BRAF mutation status is hitherto unknown, even if BRAF mutation testing has not been specifically requested by the treating clinician (in Australia, Medicare-subsidised BRAFV600 mutation testing does not need to be requested by the treating clinician). When performed in centres with appropriate expertise and experience, immunohistochemistry (IHC) using the anti-BRAF V600E monoclonal antibody (VE1) can be a highly sensitive and specific means of detecting BRAFV600E mutations, and may be used as a rapid and relatively inexpensive initial screening test. However, VE1 immunostaining can be technically challenging and difficult to interpret, particularly in heavily pigmented tumours; melanomas with weak, moderate or focal BRAFV600E immunostaining should be regarded as equivocal. It must also be remembered that other activating BRAFV600 mutations (including BRAFV600K), which account for ∼10-20% of BRAFV600 mutations, are not detected with currently available IHC antibodies. For these reasons, if available and practicable, we recommend that DNA-based BRAF mutation testing always be performed, regardless of whether IHC-based testing is also conducted. Advice about tissue/specimen selection for BRAF mutation testing of patients diagnosed with stage III or IV melanoma is also offered in this article; and potential pitfalls when interpreting BRAF mutation tests are highlighted.

Low tumour-infiltrating lymphocyte density in primary and recurrent glioblastoma.

Immunotherapies targeting tumour-infiltrating lymphocytes (TILs) that express the immune checkpoint molecule programmed cell death-1 (PD-1) have shown promise in preclinical glioblastoma models but have had limited success in clinical trials. To assess when glioblastoma is most likely to benefit from immune checkpoint inhibitors we determined the density of TILs in primary and recurrent glioblastoma. Thirteen cases of matched primary and recurrent glioblastoma tissue were immunohistochemically labelled for CD3, CD8, CD4 and PD-1, and TIL density assessed. CD3+ TILs were observed in all cases, with the majority of both primary (69.2%) and recurrent (61.5%) tumours having low density of TILs present. CD8+ TILs were observed at higher densities than CD4+ TILs in both tumour groups. PD-1+ TILs were sparse and present in only 25% of primary and 50% of recurrent tumours. Quantitative analysis of TILs demonstrated significantly higher CD8+ TIL density at recurrence (p = 0.040). No difference was observed in CD3+ (p = 0.191), CD4+ (p = 0.607) and PD-1+ (p = 0.070) TIL density between primary and recurrent groups. This study shows that TILs are present at low densities in both primary and recurrent glioblastoma. Furthermore, PD-1+ TILs were frequently absent, which may provide evidence as to why anti-PD-1 immunotherapy trials have been largely unsuccessful in glioblastoma.

Pilot early phase II study of decitabine and carboplatin in patients with advanced melanoma.

INTRODUCTION: Resistance to targeted and immune checkpoint blockade treatment remains a major problem in patients with advanced metastatic melanoma. To overcome this problem, there needs to be a decrease in burden of disease as well as re-establishing of immune sensitivity. The aim of this early phase 2 clinical trial is to investigate a novel way of sequencing and combining decitabine and carboplatin to decrease methylation and increase DNA repair resulting in a decrease in the disease burden and to re-establish sensitivity to the immune response. METHODS AND ANALYSIS: This single-site early phase 2 clinical trial will be conducted in 30 patients with metastatic melanoma that are resistant to all approved therapies. Patients will receive 2 × 4-week cycles of decitabine 7 mg/m IVI/day for 5 days (D1-D5) followed by Carboplatin AUC 5 IVI on D8; Week 3 and Week 4 no treatment. The primary objective is to determine DNA methylation and DNA repair levels before, and immediately after treatment; quantify immune-response markers (PDL-1, PD-1, CD4/CD8, and CD68) in blood, tumor and microenvironment before treatment and after 2 cycles. The secondary outcome objective is to quantify response rate (RR) to administration of 2 cycles of decitabine and carboplatin cycle using response evaluation criteria in solid tumors (RECIST 1.1) criteria. This data will be used to calculate sample size and determine statistical analysis plan for larger Phase 2 study. ETHICS AND DISSEMINATION: Protocol version 1.1 was reviewed and approved by the Hunter New England Health Human Research Ethics Committee (Reference No: 15/12/16/3.08, NSW HREC Reference No: HREC/15/HNE/505) and site-specific approval from the Calvary Mater Hospital Newcastle, NSW, Australia (NSW SSA Reference No: SSA/16/HNE/224). Primary and secondary outcomes and safety data will be disseminated through publications. TRIAL REGISTRATION DETAILS: Australian New Zealand Clinical Trial Registry ACTRN12616000440426.

The prognostic value of tumor mitotic rate in children and adolescents with cutaneous melanoma: A retrospective cohort study.

BACKGROUND: Mitotic rate is a strong predictor of outcome in adult patients with primary cutaneous melanoma, but for children and adolescent patients this is unknown. OBJECTIVE: We sought to assess the prognostic value of primary tumor mitotic rate in children and adolescents with primary melanoma. METHODS: This was a cohort study of 156 patients who were <20 years of age and who had clinically localized cutaneous melanoma. Patients <12 years of age were classified as children and those 12 to 19 years of age as adolescents. Clinicopathologic and outcome data were collected. Recurrence-free and melanoma-specific survival were calculated. Univariable and multivariable analyses were performed using Cox proportional hazard models. RESULTS: Thirteen of 156 patients (8%) were children. The mitotic rate was ≥1/mm2 in 104 patients (67%) and correlated with increasing Breslow thickness. A positive sentinel node was found in 23 of 61 patients (38%) in whom a sentinel lymph node biopsy specimen was obtained. The median follow-up was 61 months. Five-year melanoma-specific and recurrence-free survival rates were 91% and 84%, respectively. Mitotic rate was a stronger predictor of outcome than tumor thickness and was the only factor independently associated with recurrence-free survival. LIMITATIONS: This research was conducted at a single institution and the sample size was small. CONCLUSION: Mitotic rate is an independent predictor of recurrence-free survival in children and adolescents with clinically localized melanoma.

Monitoring Patient Response to Pembrolizumab With Peripheral Blood Exhaustion Marker Profiles.

Exhausted T cells are effector T cells that are silenced due to continuous T cell receptor (TCR) stimulation from persistent antigens. Characteristics of exhaustion include the increased expression of multiple inhibitory receptors such as programme death-1[PD-1], lymphocyte activation gene 3 [LAG-3], T cell Ig and mucin domain [TIM-3], the loss of effector cytokine secretion and altered transcriptional profile. The PD-1/PD-L1 interaction induces functional exhaustion of tumor-reactive cytotoxic T cells and interferes with anti-tumor T cell immunity. T cell exhaustion has been observed in metastatic melanoma patients where the exhaustion of tumor specific T cells suggests that tumor clearance has been impeded and contributed to tumor immune escape. Checkpoint immunotherapies are antibodies designed to block the interaction between the inhibitory receptors expressed on T cells and their respective ligands. Therapies such as anti-PD-1 (Pembrolizumab and Nivolumab) block these inhibitory receptors and are associated with a significant improvement in overall survival and progression free survival. However, only 20-40% of metastatic melanoma patients experience long-term benefit. In a cohort of 16 metastatic melanoma patients receiving pembrolizumab, blood was serially collected before each infusion (mean 8.3; range 1-12 cycles). The presence of inhibitory markers LAG-3, TIM-3, and PD-1 on the surface of T cells was examined and assessed in relation to patient response to identify if inhibitory markers can be used to differentiate responders from non-responders for Pembrolizumab. We confirmed that across a range of cycles (range 1-26) of pembrolizumab, PD-1 expression was significantly higher on CD4+ T cells from non-responders compared to responders and TIM-3 expressed on the surface of CD8+ T cells was significantly higher in non-responders compared to responders. This longitudinal data confirms previous studies that assessed single timepoints. This study provides preliminary evidence that PD-1 and TIM-3 may be predictive of non-responders when assessed over multiple treatment cycles.

Nucleotide excision repair protein ERCC1 and tumour-infiltrating lymphocytes are potential biomarkers of neoadjuvant platinum resistance in high grade serous ovarian cancer.

OBJECTIVE: ERCC1 is a nucleotide excision repair protein that may have a role in drug resistance in high grade serous ovarian cancer (HGSOC). We hypothesized that ERCC1 expression and tumour infiltrating lymphocytes (TILS) are induced by chemotherapy in HGSOC, which may be prognostically useful. METHODS: 115 HGSOC patients were used for this study. 92 (80%) of the tissue analysed had not been exposed to platinum chemotherapy. The remaining 20% (n = 23) of cases received combination or monotherapy with carboplatin before tissue was collected. Immunohistochemistry was used to score for ERCC1 expression and morphology to score for TILs. Correlation analysis of all clinical parameters, TILs and ERCC1 and Kaplan-Meier survival analysis was performed using the ERCC1 and TILs scoring parameters (0, 1, 2 or 3). RESULTS: ERCC1 expression was 2-fold higher in the neoadjuvant chemotherapy group compared to the primary cytoreductive surgery group (p < 0.0001). The mean overall survival for the neoadjuvant group with high ERCC1 was 141.6 ±â€¯20.2 months which was significantly longer than absent ERCC1 survival of 61 + 22.6 months (p = 0.028). ERCC1 score strongly correlated with TILs score across the whole cohort (0.349, p = 1.3 × 10-4) suggesting there is a relationship between ERCC1 expression and TILs, but this requires further investigation. CONCLUSION: In conclusion, ERCC1 was identified as a potential biomarker of platinum response overall survival in HGSOC undergoing neoadjuvant HGSOC treatment.

Whole-genome landscapes of major melanoma subtypes.

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.

Dynamic Changes in PD-L1 Expression and Immune Infiltrates Early During Treatment Predict Response to PD-1 Blockade in Melanoma.

Purpose: Disruption of PD-L1/cytotoxic T-cell PD-1 signaling by immune checkpoint inhibitors improves survival in cancer patients. This study sought to identify changes in tumoral PD-L1 expression and tumor-associated immune cell flux with anti-PD-1 therapies in patients with melanoma, particularly early during treatment, and correlate them with treatment response.Experimental Design: Forty-six tumor biopsies from 23 patients with unresectable AJCC stage III/IV melanoma receiving pembrolizumab/nivolumab were analyzed. Biopsies were collected prior to (PRE, n = 21), within 2 months of commencing treatment (EDT, n = 20) and on disease progression after previous response (PROG, n = 5). Thirteen patients responded (defined as CR, PR, or durable SD by RECIST/irRC criteria), and 10 did not respond.Results: PRE intratumoral and peritumoral PD-1+ T-cell densities were sevenfold (P = 0.006) and fivefold higher (P = 0.011), respectively, in responders compared with nonresponders and correlated with degree of radiologic tumor response (r = -0.729, P = 0.001 and r = -0.725, P = 0.001, respectively). PRE PD-L1 expression on tumor and macrophages was not significantly different between the patient groups, but tumoral PD-L1 and macrophage PD-L1 expression was higher in the EDT of responders versus nonresponders (P = 0.025 and P = 0.033). Responder EDT biopsies (compared with PRE) also showed significant increases in intratumoral CD8+ lymphocytes (P = 0.046) and intratumoral CD68+ macrophages (P = 0.046).Conclusions: Higher PRE PD-1+ T cells in responders suggest active suppression of an engaged immune system that is disinhibited by anti-PD-1 therapies. Furthermore, immunoprofiling of EDT biopsies for increased PD-L1 expression and immune cell infiltration showed greater predictive utility than PRE biopsies and may allow better selection of patients most likely to benefit from anti-PD-1 therapies and warrants further evaluation. Clin Cancer Res; 23(17); 5024-33. ©2017 AACR.

Programmed Death-Ligand 1 Expression in a Large Cohort of Pediatric Patients With Solid Tumor and Association With Clinicopathologic Features in Neuroblastoma.

PURPOSE: Programmed death-ligand 1 (PD-L1) expression represents a potential predictive biomarker of immune checkpoint blockade response. However, literature about the prevalence of PD-L1 expression in the pediatric cancer setting is discordant. METHODS: PD-L1 expression was analyzed using immunohistochemistry in 500 pediatric tumors (including neuroblastoma, sarcomas, and brain cancers). Tumors with ≥ 1% cells showing PD-L1 membrane staining of any intensity were scored as positive. Positive cases were further characterized, with cases with weak intensity PD-L1 staining reported as having low PD-L1 expression and cases with a moderate or strong intensity of staining considered to have high PD-L1 expression. RESULTS: PD-L1-positive staining was identified in 13% of cases, whereas high PD-L1 expression was found in 3% of cases. Neuroblastoma (n = 254) showed PD-L1 expression of any intensity in 18.9% of cases and was associated with longer overall survival (P = .045). However, high PD-L1 expression in neuroblastoma (3.1%) was significantly associated with an increased risk of relapse (P = .002). Positive PD-L1 staining was observed more frequently in low- and intermediate-risk patients (P = .037) and in cases lacking MYCN amplification (P = .002). CONCLUSION: In summary, high PD-L1 expression in patients with neuroblastoma may represent an unfavorable prognostic factor associated with a higher risk of cancer relapse. This work proposes PD-L1 immunohistochemical assessment as a novel parameter for identifying patients with an increased likelihood of cancer recurrence.

5-Hydroxymethylcytosine is a nuclear biomarker to assess biological potential in histologically ambiguous heavily pigmented melanocytic neoplasms.

BACKGROUND: 5-Hydroxymethylcytosine (5-hmC) is an epigenetic marker detectable through immunohistochemistry (IHC) that has been shown to distinguish benign nevi from melanoma with high sensitivity and specificity. The purpose of the study was to explore its diagnostic utility in a subset of histologically challenging, heavily pigmented cutaneous melanocytic neoplasms. METHODS: 5-hmC IHC was performed on 54 heavily pigmented melanocytic tumors. Semi-quantitative analysis of immunoreactivity was correlated with clinical, pathologic and follow-up data. RESULTS: Benign melanocytic neoplasms (4 of 4 blue nevi with epithelioid change; 12 of 12 combined nevi; 5 of 5 deep penetrating nevi, DPN) exhibited strong 5-hmC nuclear reactivity. Eight heavily pigmented blue nevus-like melanomas and 7 of 8 pigmented epithelioid melanocytomas (PEM) showed significant 5-hmC loss. Five of 7 atypical DPN cases and 8 of 10 melanocytic tumors of uncertain malignant potential (MELTUMP) showed low to intermediate 5-hmC immunoreactivity. These differences were statistically significant (P-value <.0001). CONCLUSIONS: Loss of 5-hmC may be helpful in differentiating benign, diagnostically challenging, heavily pigmented melanocytic tumors from those with malignant potential. The intermediate to low 5-hmC immunoreactivity in atypical DPNs, PEMs and so-called MELTUMP categories further underscores the need to consider these neoplasms as having some potential for lethal biological behavior.

PD-L1 expression in tonsillar cancer is associated with human papillomavirus positivity and improved survival: implications for anti-PD1 clinical trials.

In this study, we examined PD-L1 expression by immunohistochemistry in 99 patients with tonsillar cancer and known human papillomavirus (HPV) status to assess its clinical significance. We showed that the pattern of PD-L1 expression is strongly related to HPV status. The PD-L1 positivity rate was 83.3% in HPV-positive cases and 56.9% in HPV-negative cases (p < 0.05). Patients with HPV-positive/PD-L1-positive cancer had significantly better event free survival and overall survival compared with patients with HPV-negative/PD-L1-negative cancer. Relative to those patients with HPV-negative/PD-L1-negative disease who had the highest risk of death, patients with HPV-positive/PD-L1-positive cancers had a 2.85 fold lower risk of developing an event (HR 0.35, 95% CI: 0.16-0.79) and a 4.5 fold lower risk of death (HR =0.22, 95% CI: 0.09-0.53). Our findings will help to guide future clinical trial design in immunotherapy based on PD-L1 expression in tonsillar cancer.

Repair of UVB-induced DNA damage is reduced in melanoma due to low XPC and global genome repair.

UVB exposure leads to DNA damage, which when unrepaired induces C>T transitions. These mutations are found throughout the melanoma genome, particularly in non-transcribed regions. The global genome repair (GGR) branch of nucleotide excision repair (NER) is responsible for repairing UV-induced DNA damage across non-transcribed and silent regions of the genome. This study aimed to examine the relationship between UVB and GGR in melanoma. DNA repair capacity and relative expression of NER in melanocytes and melanoma cell lines before and after treatment with UVB was quantified. Transcript expression from 196 melanomas was compared to clinical parameters including solar elastosis and whole transcriptome data collected. Melanoma cell lines showed significantly reduced DNA repair when compared to melanocytes, most significantly in the S phase of the cell cycle. Expression of GGR components XPC, DDB1 and DDB2 was significantly lower in melanoma after UVB. In the melanoma tumours, XPC expression correlated with age of diagnosis and low XPC conferred significantly poorer survival. The same trend was seen in the TCGA melanoma dataset. Reduced GGR in melanoma may contribute to the UV mutation spectrum of the melanoma genome and adds further to the growing evidence of the link between UV, NER and melanoma.

Breslow Thickness Measurements of Melanomas Around American Joint Committee on Cancer Staging Cut-Off Points: Imprecision and Terminal Digit Bias Have Important Implications for Staging and Patient Management.

BACKGROUND: Breslow thickness is the most important prognostic factor in patients with clinically localized primary cutaneous melanomas, and its accuracy has important implications for staging and management. A review of the Melanoma Institute Australia database and population-based data for the state of New South Wales, Australia, found an unexpectedly large number of melanomas reported as being exactly 1.0 mm thick. We sought to determine possible causes for this biologically implausible finding. METHODS: The tumor thickness of 125 invasive cutaneous melanomas with a recorded Breslow thickness of 0.9-1.1 mm was remeasured and recorded by two pathologists. RESULTS: Concordance of measurements between the two pathologists was high (intraclass correlation coefficient 0.816, 95 % CI 0.733-0.873). The original measurements showed clustering at 0.9, 1.0, and 1.1 mm, whereas the review measurements did not. The original measurements staged 84 cases (72 %) as T1 and 33 (28 %) as T2, while the reviewed measurements staged 58 cases (50 %) as T1 and 59 (50 %) as T2 (p < 0.001). CONCLUSIONS: Our study demonstrated imprecision in Breslow thickness measurements and its significant impact on staging. Two potential sources of imprecision are failure to follow standardized thickness measurement guidelines and the phenomenon of terminal digit bias, not previously identified as a problem in this field. Educating pathologists about this phenomenon and the importance of utilizing ocular micrometers may improve the precision of melanoma thickness measurements around critical staging cut-off points. Clinicians must also be educated to appreciate that there is an inevitable margin of error with Breslow thickness measurements that should be considered when making management decisions.

The regenerating naevus.

The re-emergence of a melanocytic proliferation at the site of a previously excised pigmented lesion may not only cause great concern clinically but may also be amongst the most difficult of all melanocytic lesions for pathologists to assess. These lesions can adopt an appearance which may be impossible to confidently distinguish from a regressing or traumatised melanoma on histological grounds alone. For this reason, careful attention must be paid to the clinical context which has given rise to the lesion or a misdiagnosis may occur. In the absence of a corroborating history of prior surgery or trauma to the site, a diagnosis of a regenerating naevus may only be provisional. When considering a diagnosis of regenerating naevus, whenever possible, it is important to review and confirm the benign nature of the precursor lesion. Nevertheless, 50 years of research into this phenomenon has identified certain characteristic clinical features and histological patterns which provide clues both to clinicians and pathologists that will assist them to make the correct diagnosis and avoid over diagnosing as melanoma what is ultimately a benign process.

Primary and Metastatic Cutaneous Melanomas Express ALK Through Alternative Transcriptional Initiation.

A number of common driver mutations have been identified in melanoma, but other genetic or epigenetic aberrations are also likely to play a role in the pathogenesis of melanoma and present potential therapeutic targets. Translocations of the anaplastic lymphoma kinase (ALK), for example, have been reported in spitzoid melanocytic neoplasms leading to kinase-fusion proteins that result in immunohistochemically detectable ALK expression. In this study, we sought to determine whether ALK was also expressed in nonspitzoid primary and metastatic cutaneous melanomas. ALK immunohistochemistry was performed on 603 melanomas (303 primary and 300 metastatic tumors) from 600 patients. ALK immunohistochemistry expression was identified in 7 primary and 9 metastatic tumors. In 5 of 7 primary tumors and in 6 of 9 metastatic lesions, the majority of tumor cells were immunoreactive for ALK. In the other 2 primary and 3 metastatic lesions, positive staining was identified in less than half of the tumor cells. ALK positivity was found in the presence or absence of BRAF or NRAS mutations. In contrast to prior observations with ALK-positive Spitz tumors, none of the ALK-positive melanomas harbored a translocation. Instead, the ALK-positive melanomas predominantly expressed the recently described ALK isoform, ALK, which lacks the extracellular and transmembrane domains of wild-type ALK, consists primarily of the intracellular tyrosine kinase domain, and originates from an alternative transcriptional initiation site within the ALK gene. The findings are clinically relevant as patients with metastatic melanoma who have ALK expression may potentially benefit from treatment with ALK kinase inhibitors.

Histological pattern of Merkel cell carcinoma sentinel lymph node metastasis improves stratification of Stage III patients.

Sentinel lymph node biopsy is used to stage Merkel cell carcinoma, but its prognostic value has been questioned. Furthermore, predictors of outcome in sentinel lymph node positive Merkel cell carcinoma patients are poorly defined. In breast carcinoma, isolated immunohistochemically positive tumor cells have no impact, but in melanoma they are considered significant. The significance of sentinel lymph node metastasis tumor burden (including isolated tumor cells) and pattern of involvement in Merkel cell carcinoma are unknown. In this study, 64 Merkel cell carcinomas involving sentinel lymph nodes and corresponding immunohistochemical stains were reviewed and clinicopathological predictors of outcome were sought. Five metastatic patterns were identified: (1) sheet-like (n=38, 59%); (2) non-solid parafollicular (n=4, 6%); (3) sinusoidal, (n=11, 17%); (4) perivascular hilar (n=1, 2%); and (5) rare scattered parenchymal cells (n=10, 16%). At the time of follow-up, 30/63 (48%) patients had died with 21 (33%) attributable to Merkel cell carcinoma. Patients with pattern 1 metastases had poorer overall survival compared with patients with patterns 2-5 metastases (P=0.03), with 22/30 (73%) deaths occurring in pattern 1 patients. Three (10%) deaths occurred in patients showing pattern 5, all of whom were immunosuppressed. Four (13%) deaths occurred in pattern 3 patients and 1 (3%) death occurred in a pattern 2 patient. In multivariable analysis, the number of positive sentinel lymph nodes (1 or 2 versus >2, P<0.0001), age (<70 versus ≥70, P=0.01), sentinel lymph node metastasis pattern (patterns 2-5 versus 1, P=0.02), and immune status (immunocompetent versus suppressed, P=0.03) were independent predictors of outcome, and could be used to stratify Stage III patients into three groups with markedly different outcomes. In Merkel cell carcinoma, the pattern of sentinel lymph node involvement provides important prognostic information and utilizing this data with other clinicopathological features facilitates risk stratification of Merkel cell carcinoma patients who may have management implications.