Publications by authors named "Peter M Ferguson"

30 Publications

  • Page 1 of 1

Transplant-associated penile Kaposi sarcoma managed with single agent paclitaxel chemotherapy: a case report.

BMC Urol 2021 Jun 7;21(1):87. Epub 2021 Jun 7.

Department of Renal Medicine and Renal Transplant, Royal Prince Alfred Hospital, Missenden Rd, Camperdown, NSW, Australia.

Background: Kaposi's sarcoma is an uncommon complication in renal transplant patients, and typically presents with cutaneous lesions on the lower extremities. Penile involvement has been reported only rarely. Management of cutaneous-limited disease is primarily reduction of immunosuppression and conversion to an mTOR-inhibitor, whereas the treatment of disseminated disease in transplant patients is more variable.

Case Presentation: A 75-year-old male, originally from Somalia, received a deceased-donor kidney transplant for diabetic and hypertensive nephropathy. Seven months post-transplant he presented with lower limb lesions, oedema and bilateral deep vein thromboses. He then developed a fast-growing painful lesion on his penile shaft. A biopsy of this lesion confirmed KS, and a PET scan demonstrated disseminated disease in the lower extremities, penis and thoracic lymph nodes. His tacrolimus was converted to sirolimus, and his other immunosuppression was reduced. He was treated with single agent paclitaxel chemotherapy in view of his rapidly progressing, widespread disease. The penile lesion completely resolved, and the lower extremity lesions regressed significantly. His kidney allograft function remained stable throughout treatment.

Conclusion: This case illustrates a rare presentation of an uncommon post-transplant complication and highlights the need for a high index of suspicion of KS in transplant patients presenting with atypical cutaneous lesions. It serves to demonstrate that the use of single agent paclitaxel chemotherapy, switch to an mTORi and reduction in immunosuppression where possible produces excellent short-term outcomes, adding to the body of evidence for this management strategy in disseminated Kaposi's sarcoma.
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http://dx.doi.org/10.1186/s12894-021-00855-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8186205PMC
June 2021

γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival.

Cancer Immunol Res 2021 Mar 5. Epub 2021 Mar 5.

Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia.

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4 or CD8 T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2 γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0817DOI Listing
March 2021

Programmed death ligand-1 (PD-L1) as a predictive marker for immunotherapy in solid tumours: a guide to immunohistochemistry implementation and interpretation.

Pathology 2021 Feb 30;53(2):141-156. Epub 2020 Dec 30.

Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital and NSW Health Pathology, Sydney, NSW, Australia; The University of Sydney, Sydney, NSW, Australia. Electronic address:

Immunotherapy with checkpoint inhibitors is well established as an effective treatment for non-small cell lung cancer and melanoma. The list of approved indications for treatment with PD-1/PD-L1 checkpoint inhibitors is growing rapidly as clinical trials continue to show their efficacy in patients with a wide range of solid tumours. Clinical trials have used a variety of PD-L1 immunohistochemical assays to evaluate PD-L1 expression on tumour cells, immune cells or both as a potential biomarker to predict response to immunotherapy. Requests to pathologists for PD-L1 testing to guide choice of therapy are rapidly becoming commonplace. Thus, pathologists need to be aware of the different PD-L1 assays, methods of evaluation in different tumour types and the impact of the results on therapeutic decisions. This review discusses the key practical issues relating to the implementation of PD-L1 testing for solid tumours in a pathology laboratory, including evidence for PD-L1 testing, different assay types, the potential interchangeability of PD-L1 antibody clones and staining platforms, scoring criteria for PD-L1, validation, quality assurance, and pitfalls in PD-L1 assessment. This review also explores PD-L1 IHC in solid tumours including non-small cell lung carcinoma, head and neck carcinoma, triple negative breast carcinoma, melanoma, renal cell carcinoma, urothelial carcinoma, gastric and gastroesophageal carcinoma, colorectal carcinoma, hepatocellular carcinoma, and endometrial carcinoma. The review aims to provide pathologists with a practical guide to the implementation and interpretation of PD-L1 testing by immunohistochemistry.
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http://dx.doi.org/10.1016/j.pathol.2020.10.007DOI Listing
February 2021

Molecular analysis of primary melanoma T cells identifies patients at risk for metastatic recurrence.

Nat Cancer 2020 Feb 20;1(2):197-209. Epub 2020 Jan 20.

Brigham and Women's Hospital, Department of Dermatology and Harvard Medical School, Boston, MA, USA.

Primary melanomas >1 mm thickness are potentially curable by resection, but can recur metastatically. We assessed the prognostic value of T cell fraction (TCFr) and repertoire T cell clonality, measured by high-throughput-sequencing of the T cell receptor beta chain (TRB) in T2-T4 primary melanomas (n=199). TCFr accurately predicted progression-free survival (PFS) and was independent of thickness, ulceration, mitotic rate, or age. TCFr was second only to tumor thickness in its predictive value, using a gradient boosted model. For accurate PFS prediction, adding TCFr to tumor thickness was superior to adding any other histopathological variable. Furthermore, a TCFr >20% was protective regardless of tumor ulceration status, mitotic rate or presence of nodal disease. TCFr is a quantitative molecular assessment that predicts metastatic recurrence in primary melanoma patients whose disease has been resected surgically. This study suggests that a successful T cell-mediated antitumor response can be present in primary melanomas.
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http://dx.doi.org/10.1038/s43018-019-0019-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725220PMC
February 2020

Intranuclear inclusions are a distinguishing morphological feature of renal cell carcinoma with leiomyomatous stroma.

Pathology 2021 Jun 4;53(4):543-545. Epub 2020 Dec 4.

Department of Tissue Pathology and Diagnostic Oncology, New South Wales Health Pathology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia; Central Clinical School, The University of Sydney, Sydney, NSW, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.pathol.2020.08.026DOI Listing
June 2021

Whole-genome sequencing of acral melanoma reveals genomic complexity and diversity.

Nat Commun 2020 10 16;11(1):5259. Epub 2020 Oct 16.

Center for Rare Melanomas, University of Colorado Cancer Center, Aurora, Colorado, USA.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed. Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature. Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1. Mutations and amplification of KIT are also common. Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common. Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.
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http://dx.doi.org/10.1038/s41467-020-18988-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567804PMC
October 2020

The tumour immune landscape and its implications in cutaneous melanoma.

Pigment Cell Melanoma Res 2021 May 1;34(3):529-549. Epub 2020 Oct 1.

Melanoma Institute Australia, The University of Sydney, Sydney, Australia.

The field of tumour immunology has rapidly advanced in the last decade, leading to the advent of effective immunotherapies for patients with advanced cancers. This highlights the critical role of the immune system in determining tumour development and outcome. The tumour immune microenvironment (TIME) is highly heterogeneous, and the interactions between tumours and the immune system are vastly complex. Studying immune cell function in the TIME will provide an improved understanding of the mechanisms underpinning these interactions. This review examines the role of immune cell populations in the TIME based on their phenotype, function and localisation, as well as contextualising their position in the dynamic relationship between tumours and the immune system. We discuss the function of immune cell populations, examine their impact on patient outcome and highlight gaps in current understanding of their roles in the TIME, both in cancers in general and specifically in melanoma. Studying the TIME by evaluating both pro-tumour and anti-tumour effects may elucidate the conditions which lead to tumour growth and metastasis or immune-mediated tumour regression. Moreover, an in-depth understanding of these conditions could contribute to improved prognostication, more effective use of current immunotherapies and guide the development of novel treatment strategies and therapies.
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http://dx.doi.org/10.1111/pcmr.12926DOI Listing
May 2021

Tumor Mutation Burden and Structural Chromosomal Aberrations Are Not Associated with T-cell Density or Patient Survival in Acral, Mucosal, and Cutaneous Melanomas.

Cancer Immunol Res 2020 11 11;8(11):1346-1353. Epub 2020 Sep 11.

Melanoma Institute Australia, The University of Sydney, North Sydney, New South Wales, Australia.

Tumor mutation burden (TMB) has been proposed as a key determinant of immunogenicity in several cancers, including melanoma. The evidence presented thus far, however, is often contradictory and based mostly on RNA-sequencing data for the quantification of immune cell phenotypes. Few studies have investigated TMB across acral, mucosal, and cutaneous melanoma subtypes, which are known to have different TMB. It is also unknown whether chromosomal structural mutations [structural variant (SV) mutations] contribute to the immunogenicity in acral and mucosal melanomas where such aberrations are common. We stained 151 cutaneous and 35 acral and mucosal melanoma patient samples using quantitative IHC and correlated immune infiltrate phenotypes with TMB and other genomic profiles. TMB and SVs did not correlate with the densities of CD8 lymphocytes, CD103 tumor-resident T cells (Trm), CD45RO cells, and other innate and adaptive immune cell subsets in cutaneous and acral/mucosal melanoma tumors, respectively, including in analyses restricted to the site of disease and in a validation cohort. In 43 patients with stage III treatment-naïve cutaneous melanoma, we found that the density of immune cells, particularly Trm, was significantly associated with patient survival, but not with TMB. Overall, TMB and chromosomal structural aberrations are not associated with protective antitumor immunity in treatment-naïve melanoma.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0835DOI Listing
November 2020

Primary dermal melanoma: clinical behaviour, prognosis and treatment.

Eur J Surg Oncol 2020 11 1;46(11):2131-2139. Epub 2020 May 1.

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia; Royal Prince Alfred Hospital, Camperdown, NSW, Australia; Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia. Electronic address:

Purpose: Primary dermal melanoma (PDM) is a subtype of cutaneous melanoma, confined to the dermis, which poses a challenging clinical dilemma. It may represent a true primary melanoma or a dermal cutaneous metastasis. This study aimed to delineate the histopathological characteristics and prognosis of PDM in a large patient cohort to guide appropriate treatment strategies.

Methods: A search of the Melanoma Research Database at Melanoma Institute Australia was conducted to identify all possible PDM patients at our institution diagnosed from 1978 to 2013. Overall, melanoma-specific and disease-free survival outcomes of the PDM group were compared to those of similar cohorts of Stage I-II and Stage IV M1a melanoma patients based on propensity score matching.

Results: Sixty-two PDM patients were identified from the MRD with a median follow-up of 6.3 years. Five-year survival was 87.1% and overall survival was 74.2%. PDMs had a significantly improved overall survival (p = 0.0002) and melanoma-specific survival (p = 0.001) compared to Stage I-II controls, however there was no difference in disease-free survival (p = 0.08). PDMs also demonstrated improved overall survival (p < 0.0001), melanoma-specific survival (p < 0.0001) and disease-free survival (p < 0.0001) compared to Stage IV M1a controls.

Conclusion: These findings demonstrate that PDMs have a more favorable prognosis compared to stage I-II cutaneous melanomas and suggest that these are in fact true primary lesions. This study thus provides evidence to justify a treatment approach, by way of a wide local excision and possibly sentinel lymph node biopsy, as for early stage primary cutaneous melanomas.
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http://dx.doi.org/10.1016/j.ejso.2020.04.043DOI Listing
November 2020

Vaccines adjuvanted with an NKT cell agonist induce effective T-cell responses in models of CNS lymphoma.

Immunotherapy 2020 04 22;12(6):395-406. Epub 2020 Apr 22.

Malaghan Institute of Medical Research, P.O. Box 7060, Wellington 6242, New Zealand.

The efficacy of anti-lymphoma vaccines that exploit the cellular adjuvant properties of activated natural killer T (NKT) cells were examined in mouse models of CNS lymphoma. Vaccines were prepared by either loading the NKT cell agonist, α-galactosylceramide onto irradiated and heat-shocked B- and T-lymphoma cells, or chemically conjugating α-galactosylceramide to MHC-binding peptides from a lymphoma-associated antigen. Vaccine efficacy was analyzed in mice bearing intracranial tumors. Both forms of vaccine proved to be effective in preventing lymphoma engraftment through activity of T cells that accessed the CNS. Established lymphoma was harder to treat with responses constrained by Tregs, but this could be overcome by depleting Tregs prior to vaccination. Simply designed NKT cell-activating vaccines enhance T-cell responses and have the potential to protect against CNS lymphoma development or prevent CNS relapse. To be effective against established CNS lymphoma, vaccines need to be combined with Treg suppression.
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http://dx.doi.org/10.2217/imt-2019-0134DOI Listing
April 2020

Diagnostic accuracy of pigmented labial macules by in vivo reflectance confocal microscopy and correlation among techniques.

J Am Acad Dermatol 2020 Mar 5. Epub 2020 Mar 5.

Department of Dermatology, Hospital del Mar-Parc de Salut Mar, Departament de Medicina de la UAB, Universitat Autònoma de Barcelona, Barcelona, Spain; Institut Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain.

Background: Pigmented labial macules (PLMs) are clinical, dermoscopic, and histopathologic challenges.

Objective: To describe and evaluate the utility of reflectance confocal microscopy (RCM) in PLMs and to establish a correlation between dermoscopy, RCM, histopathology, and immunohistochemistry.

Methods: Prospective study of PLMs from 4 tertiary referral dermatology centers. The study included 51 biopsy specimen-proven PLMs. Dermoscopic, RCM images, and histopathologic preparations were evaluated for malignant criteria. Diagnostic accuracy of RCM for melanoma diagnosis, RCM Lip Score previously reported, and κ values between techniques were calculated.

Results: Included were 5 melanomas and 46 benign PLMs. Dermoscopically, melanomas exhibited more frequently ≥3 colors and ≥3 structures. With RCM, pagetoid spreading, epithelial disarray, continuous proliferation of atypical cells around papillae, nonhomogeneously distributed papillae, marked cellular atypia, and a higher number of dendritic cells per papillae were more frequent in melanomas. The RCM Lip Score was significantly higher in malignant lesions. Good κ values were observed in most of the evaluated features. A perfect sensitivity and specificity was obtained combining dermoscopy and RCM.

Limitations: A low number of melanomas were obtained.

Conclusions: RCM improves lip melanoma diagnosis, and the RCM Lip Score represents a useful tool for the evaluation of a PLM.
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http://dx.doi.org/10.1016/j.jaad.2020.02.067DOI Listing
March 2020

Melanoma pathology reporting and staging.

Mod Pathol 2020 01 22;33(Suppl 1):15-24. Epub 2019 Nov 22.

University of Texas, MD Anderson Cancer Center, Houston, TX, 77030, USA.

The pathological diagnosis of melanoma can be challenging. The provision of an appropriate biopsy and pertinent history can assist in establishing an accurate diagnosis and reliable estimate of prognosis. In their reports, pathologists should document both the criteria on which the diagnosis was based as well as important prognostic parameters. For melanoma, such prognostic parameters include tumor thickness, ulceration, mitotic rate, lymphovascular invasion, neurotropism, and tumor-infiltrating lymphocytes. Disease staging is important for risk stratifying melanoma patients into prognostic groups and patient management recommendations are often stage based. The 8th edition American Joint Committee on Cancer (AJCC) Melanoma Staging System was implemented in 2018 and several important changes were made. Tumor thickness and ulceration remain the key T category criteria. T1b melanomas were redefined as either ulcerated melanomas <1.0 mm thick or nonulcerated melanomas 0.8-1.0 mm thick. Although mitotic rate was removed as a T category criterion in the 8th edition, it remains a very important prognostic factor and should continue to be documented in primary melanoma pathology reports. It was also recommended in the 8th edition that tumor thickness be recorded to the nearest 0.1 mm (rather than the nearest 0.01 mm). In the future, incorporation of additional prognostic parameters beyond those utilized in the current version of the staging system into (web based) prognostic models/clinical tools will likely facilitate more personalized prognostic estimates. Evaluation of molecular markers of prognosis is an active area of current research; however, additional data are needed before it would be appropriate to recommend use of such tests in routine clinical practice.
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http://dx.doi.org/10.1038/s41379-019-0402-xDOI Listing
January 2020

Molecular Profiling of Noncoding Mutations Distinguishes Nevoid Melanomas From Mitotically Active Nevi in Pregnancy.

Am J Surg Pathol 2020 03;44(3):357-367

Royal Prince Alfred Hospital.

The accurate recognition of subtle melanomas and their distinction from benign mimics is an oft-recurring diagnostic problem, critical for patient management. Melanomas that bear resemblance to benign nevi (so-called nevoid melanomas, NMs) and benign mitotically active nevi in pregnancy (MANP) are 2 lesions particularly prone to error. Molecular data, including analysis of noncoding regions, in MANP and NM are very limited. This study sought to identify differences in clinical, pathologic, and molecular characteristics between MANP and NMs to facilitate correct diagnosis and reduce the risk of overtreatment or undertreatment. Clinicopathologic characteristics of NM (n=18) and MANP (n=30) were evaluated, and mutation data were analyzed using next-generation sequencing for available cases in each group (NM, n=8; MANP, n=12). All MANP showed innocent histopathologic characteristics apart from increased mitotic activity, frequently in both superficial and deep parts of the lesion (median dermal mitotic rate: 2/mm, range: 1 to 7/mm). All cases of NM demonstrated a characteristic nevoid silhouette, subtle atypical architectural and cytologic features, and variable mitoses (median mitotic rate: 3/mm, range: 1 to 5/mm). Median NM tumor thickness was 1.4 mm. Four of 10 NM patients with follow-up had metastatic disease, including 3 patients who developed widespread metastases, with 1 disease-related death. No other recurrences have been identified (follow-up period: 24 to 60 mo). None of the 15 MANP patients with available follow-up had a recurrence. Most NMs harbored hotspot mutations in NRAS (6/8, 75%). Noncoding mutations were significantly more common in NMs than in MANP (median: 4 vs. 0, P=0.0014). Copy number alterations were infrequent but, when present, were seen in NMs (3/8 NMs vs. 0/12 MANP). All NMs but only 1 of 12 MANP had >1 abnormality in the noncoding regions. Similar to conventional common acquired nevi, MANP mostly harbored driver BRAF mutations, while activating NRAS mutations, noncoding mutations, and copy number alterations were rare. NM and MANP have subtle but recognizable distinguishing histopathologic characteristics that are underpinned by molecular differences. Mutation analysis of targeted noncoding mutations may assist in the diagnosis of difficult lesions.
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http://dx.doi.org/10.1097/PAS.0000000000001406DOI Listing
March 2020

Whole-genome landscape of mucosal melanoma reveals diverse drivers and therapeutic targets.

Nat Commun 2019 07 18;10(1):3163. Epub 2019 Jul 18.

Department of Pathology, University of California, San Francisco, CA, 94143, USA.

Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type. To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors. Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2. Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8. SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas. TERT aberrations and ATRX mutations are associated with alterations in telomere length. Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.
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http://dx.doi.org/10.1038/s41467-019-11107-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639323PMC
July 2019

Improving diagnostic accuracy for suspicious melanocytic skin lesions: New Australian melanoma clinical practice guidelines stress the importance of clinician/pathologist communication.

Aust J Gen Pract 2019 06;48(6):357-362

MD, Chairman, Australian Melanoma Clinical Practice Guidelines Working Party; Professor of Melanoma and Surgical Oncology, University of Sydney, Sydney, NSW; Surgeon, Royal Prince Alfred Hospital and Melanoma Institute Australia, Sydney, NSW.

Background: Incorrect or delayed diagnosis of melanoma may lead to inappropriate treatment, poor clinical outcomes, increased cost and medicolegal consequences. The provision of pertinent clinical information is essential for accurate pathological diagnosis of cutaneous melanocytic tumours. Failure to provide this information may contribute to poor outcomes.

Objective: The aim of this article is to highlight important clinical information that clinicians can provide to pathologists to facilitate accurate diagnosis of melanocytic tumours.

Discussion: Pertinent clinical information includes patient age, sex, tumour site, specimen orientation (if appropriate), history of the lesion, presence of any clinically or dermoscopically suspicious areas within the lesion (including apparent regression), access to any relevant clinical and/or dermoscopic photographs and prior pathology reports, melanoma history and risk factors, and history of concurrent or recent pregnancy. If the clinical features are not concordant with the pathology findings, the clinician and pathologist should discuss the case to identify the reason for incongruence.
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http://dx.doi.org/10.31128/AJGP-11-18-4759DOI Listing
June 2019

Neoadjuvant dabrafenib combined with trametinib for resectable, stage IIIB-C, BRAF mutation-positive melanoma (NeoCombi): a single-arm, open-label, single-centre, phase 2 trial.

Lancet Oncol 2019 07 3;20(7):961-971. Epub 2019 Jun 3.

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia; Royal North Shore Hospital, St Leonards, Sydney, NSW, Australia.

Background: Adjuvant dabrafenib plus trametinib therapy improves relapse-free survival in patients with resected stage III melanoma. We aimed to ascertain the proportion of patients who would have a pathological response and a response according to Response Evaluation Criteria in Solid Tumors (RECIST) after neoadjuvant dabrafenib plus trametinib therapy for resectable clinical stage III melanoma.

Methods: NeoCombi was a single-arm, open-label, single-centre, phase 2 study done at Melanoma Institute Australia (Sydney, NSW, Australia). Eligible patients were adults (aged ≥18 years) with histologically confirmed, resectable, RECIST-measurable, clinical stage IIIB-C (American Joint Committee on Cancer [AJCC] 7th edition), BRAF-mutant melanoma, and had an Eastern Cooperative Oncology Group performance status of 1 or lower. Patients received 150 mg dabrafenib orally, twice daily, plus 2 mg trametinib orally, once daily, for 52 weeks (12 weeks of neoadjuvant therapy before complete resection of the pre-therapy tumour bed, and 40 weeks of adjuvant therapy thereafter). CT and PET scans were done at baseline and before resection. The primary outcomes were the proportion of patients achieving a complete pathological response and the proportion of patients achieving a response according to RECIST at week 12, analysed as per protocol. This trial is registered with ClinicalTrials.gov, NCT01972347, and follow-up of patients is ongoing.

Findings: Between Aug 20, 2014, and April 19, 2017, 40 patients were screened, of whom 35 eligible patients were enrolled, received neoadjuvant dabrafenib plus trametinib, and underwent resection. At the data cutoff (Sept 24, 2018), median follow-up was 27 months (IQR 21-36). At resection, 30 (86%) patients achieved a RECIST response; 16 (46%; 95% CI 29-63) had a complete response and 14 (40%; 24-58) had a partial response. Five patients (14%; 95% CI 5-30) had stable disease, and no patients progressed. After resection and pathological evaluation, all 35 patients achieved a pathological response, of whom 17 (49%; 95% CI 31-66) patients had a complete pathological response and 18 (51%; 95% CI 34-69) had a non-complete pathological response. Treatment-related serious adverse events occurred in six (17%) of 35 patients and grade 3-4 adverse events occurred in ten (29%) patients. No treatment-related deaths were reported.

Interpretation: Neoadjuvant dabrafenib plus trametinib therapy could be considered in the management of RECIST-measurable resectable stage III melanoma as it led to a high proportion of patients achieving a complete response according to RECIST and a high proportion of patients achieving a complete pathological response, with no progression during neoadjuvant therapy.

Funding: GlaxoSmithKline; Novartis; National Health and Medical Research Council, Australia; and Melanoma Institute Australia.
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http://dx.doi.org/10.1016/S1470-2045(19)30331-6DOI Listing
July 2019

Recurrent hotspot SF3B1 mutations at codon 625 in vulvovaginal mucosal melanoma identified in a study of 27 Australian mucosal melanomas.

Oncotarget 2019 Jan 29;10(9):930-941. Epub 2019 Jan 29.

Melanoma Institute Australia, The University of Sydney, Sydney, Australia.

Introduction: Clinical outcomes for mucosal melanomas are often poor due to a lack of effective systemic drug therapies. Identifying driver genes in mucosal melanoma may enhance the understanding of disease pathogenesis and provide novel opportunities to develop effective therapies.

Results: Somatic variant analysis identified (6 of 27: 22%) as the most commonly mutated gene, followed by (3 of 27: 11%). Other less frequently mutated genes (4% otherwise stated) included (7%), (7%), , , , , , , and . Recurrent SF3B1 p.R625 hotspot mutations were exclusively detected in vulvovaginal (5 of 19: 26%) and anorectal melanomas (3 of 5:60%). The only other SF3B1 mutation was a p.C1123Y mutation that occurred in a conjunctival mucosal melanoma.-mutated patients were associated with shorter overall survival (OS; 34.9 months) and progression-free survival (PFS; 16.9 months) compared to non--mutated patients (OS: 79.7 months, log-rank = 0.1172; PFS: 35.7 months, log-rank = 0.0963).

Conclusion: Molecular subgroups of mucosal melanoma with mutations occurred predominantly in the vulvovaginal region. mutations may have a negative prognostic impact.

Methods: Formalin-fixed biopsies were collected from 27 pathologically-confirmed mucosal melanomas. Genomic DNA was isolated from the tumor tissue and sequenced using a novel dual-strand amplicon sequencing technique to determine the frequency and types of mutations across 45 target genes.
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http://dx.doi.org/10.18632/oncotarget.26584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398173PMC
January 2019

Integrated molecular and immunophenotypic analysis of NK cells in anti-PD-1 treated metastatic melanoma patients.

Oncoimmunology 2019;8(2):e1537581. Epub 2018 Oct 31.

Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia.

: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma. : Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line). : Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (< 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells' cytotoxicity. : A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.
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http://dx.doi.org/10.1080/2162402X.2018.1537581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343795PMC
October 2018

Enhancing T cell responses and tumour immunity by vaccination with peptides conjugated to a weak NKT cell agonist.

Org Biomol Chem 2019 01;17(5):1225-1237

The Ferrier Research Institute, Victoria University of Wellington, Lower Hutt, New Zealand.

Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.
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http://dx.doi.org/10.1039/c8ob02982bDOI Listing
January 2019

Staging of Cutaneous Melanoma: Is There Room for Further Improvement?

JAMA Netw Open 2018 05 18;1(1):e180086. Epub 2018 May 18.

Melanoma Institute Australia, The University of Sydney, Sydney, Australia.

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http://dx.doi.org/10.1001/jamanetworkopen.2018.0086DOI Listing
May 2018

Inter- and intrapatient heterogeneity of indoleamine 2,3-dioxygenase expression in primary and metastatic melanoma cells and the tumour microenvironment.

Histopathology 2019 May;74(6):817-828

Melanoma Institute Australia, The University of Sydney, Sydney, Australia.

Aims: Indoleamine 2,3-dioxygenase (IDO), an immunomodulatory enzyme, facilitates immune escape by tumours and promotes tumour progression. IDO inhibitors with and without additional anti-PD-1 therapy have been evaluated in recent and ongoing melanoma clinical trials, but IDO expression in melanoma tumours, and therefore its potential role as a predictive biomarker remains unknown. This study sought to evaluate IDO expression in immunotherapy-naive metastatic melanoma patients in order to determine patterns of expression in corresponding primary melanomas, locoregional metastases and distant metastases.

Methods And Results: Here, we evaluated IDO expression using immunohistochemistry in 99 melanoma tumour samples from 43 immunotherapy-naive patients with metastatic melanoma to determine patterns of expression in primary melanomas (n = 29), locoregional metastases (n = 36) and distant metastases (n = 34). Thirty-seven per cent of patients demonstrated tumour IDO expression in at least one specimen. Twelve of 35 patients (34%) with longitudinal specimens (i.e. two or more separate specimens from different disease stages in the same patient) displayed heterogeneous IDO staining between samples. Tumour IDO expression positively correlated with tumour-infiltrating lymphocyte (TIL) score as well as the number of IDO-expressing mononuclear cells in the primary melanoma (P < 0.0001 and P = 0.0011, respectively) and nodal metastases (P = 0.049 and P = 0.037, respectively), but not in distant metastases. Furthermore, tumour IDO expression correlated positively with PD-L1 expression by melanoma cells among all specimens (P = 0.0073).

Conclusions: Therefore, while assessment of tumour IDO expression warrants evaluation in melanoma patient cohorts treated with IDO inhibitors dosed at levels proven to inhibit the target by pharmacodynamic assessment, its utility as a biomarker may be limited by intertumoral heterogeneity.
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http://dx.doi.org/10.1111/his.13814DOI Listing
May 2019

Correlation Between Surgical and Histologic Margins in Melanoma Wide Excision Specimens.

Ann Surg Oncol 2019 Jan 16;26(1):25-32. Epub 2018 Oct 16.

Melanoma Institute Australia, The University of Sydney, North Sydney, NSW, Australia.

Introduction: Wide surgical excision is the standard treatment for localized primary cutaneous melanomas, with a narrow histologic margin associated with an increased risk of local recurrence. The correlation between surgical and histologic margins is poorly documented in the literature.

Methods: An audit was performed to (1) document the shrinkage of formalin-fixed specimens, and (2) use a precisely measured surgical margin in vivo to predict the histologic margin. For patients presenting for wide excision of melanomas and other malignant skin tumors, measured surgical margin, in vivo and ex vivo specimen width, and histologic margins after formalin fixation were recorded. The effects of clinicopathologic characteristics, including age, sex, body mass index (BMI), tumor type, anatomic site, and presence of visible tumor in predicting specimen shrinkage and histologic margin were assessed.

Results: In total, 252 specimens were evaluated. When compared with measured width in vivo, the formalin-fixed specimens showed a mean shrinkage of 14% (R = 0.98), regardless of patient age, sex, BMI, or site of the lesion. The measured surgical margin was not a strong predictor of the histologic margin, with a high degree of variability (R = 0.55) not explained by patient factors, tumor subtype, or presence of visible tumor at the time of excision (p > 0.05).

Conclusions: A consistent 14% shrinkage rate of wide excision specimens was found across all patients and excision sites, and we propose a clinically useful 15% correction factor that will account for fixation and shrinkage of cutaneous excision specimens. Excision margins measured by the surgeon were a poor predictor of the histologic margins.
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http://dx.doi.org/10.1245/s10434-018-6858-yDOI Listing
January 2019

When is surgery for metastatic melanoma still the most appropriate treatment option?

Expert Rev Anticancer Ther 2018 10 9;18(10):943-945. Epub 2018 Aug 9.

a Melanoma Institute Australia , The University of Sydney , North Sydney , Australia.

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http://dx.doi.org/10.1080/14737140.2018.1508346DOI Listing
October 2018

Melanoma protective antitumor immunity activated by catalytic DNA.

Oncogene 2018 09 29;37(37):5115-5126. Epub 2018 May 29.

Vascular Biology and Translational Research, School of Medical Sciences & UNSW Medicine, The University of New South Wales, Sydney, NSW, 2052, Australia.

Melanoma incidence is increasing worldwide, and although drugs such as BRAF/MEK small-molecule inhibitors and immune checkpoint antibodies improve patient outcomes, most patients ultimately fail these therapies and alternative treatment strategies are urgently needed. DNAzymes have recently undergone clinical trials with signs of efficacy and no serious adverse events attributable to the DNAzyme. Here we investigated c-Jun expression in human primary and metastatic melanoma. We also explored the role of T cell immunity in DNAzyme inhibition of primary melanoma growth and the prevention of growth in non-treated tumors after the cessation of treatment in a mouse model. c-Jun was expressed in 80% of melanoma cells in human primary melanomas (n = 17) and in 83% of metastatic melanoma cells (n = 38). In contrast, c-Jun was expressed in only 11% of melanocytes in benign nevi (n = 24). Dz13, a DNAzyme targeting c-Jun/AP-1, suppressed both Dz13-injected and untreated B16F10 melanoma growth in the same mice, an abscopal effect relieved in each case by administration of anti-CD4/anti-CD8 antibodies. Dz13 increased levels of cleaved caspase-3 within the tumors. New, untreated melanomas grew poorly in mice previously treated with Dz13. Administration of anti-CD4/anti-CD8 antibodies ablated this inhibitory effect and the tumors grew rapidly. Dz13 inhibited c-Jun expression, reduced intratumoral vascularity (vascular lumina area defined by CD31 staining), and increased CD4 cells within the tumors. This study provides the first demonstration of an abscopal effect of a DNAzyme on tumor growth and shows that Dz13 treatment prevents growth of subsequent new tumors in the same animal. Dz13 may be useful clinically as a therapeutic antitumor agent by preventing tumor relapse through adaptive immunity.
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http://dx.doi.org/10.1038/s41388-018-0306-0DOI Listing
September 2018

Blocking CTLA-4 while priming with a whole cell vaccine reshapes the oligoclonal T cell infiltrate and eradicates tumors in an orthotopic glioma model.

Oncoimmunology 2017;7(1):e1376154. Epub 2017 Sep 27.

Malaghan Institute of Medical Research, Wellington, New Zealand.

Vaccine-mediated cancer treatment is unlikely to induce long-term survival unless suppressive mechanisms are overcome. Given the success of antibody-mediated immune checkpoint blockade in relieving regulation of endogenous anti-tumor T cell responses in tumor-burdened hosts, we investigated whether checkpoint blockade could improve the efficacy of responses induced with a whole tumor-cell vaccine. We show that administration of a single dose of blocking antibody was sufficient to significantly enhance antitumor activity of the vaccine, inducing complete radiological regression of established intracranial tumors. The antibody or vaccine alone were ineffective in this setting. The antibody had to be administered before, or close to, vaccine administration, suggesting CTLA-4 blockade had an impact on early priming events. The combined treatment resulted in enhanced trapping of leukocytes in the lymphoid tissues, including T cells that had undergone significant proliferation. There were no obvious changes in the stimulatory function of antigen-presenting cells or the number and function of regulatory T cells, suggesting T cells were the targets of the checkpoint blockade. While tumors regressing under combined treatment were highly infiltrated with a variety of leukocytes, tumor eradication was dependent on CD4 T cells. Analysis of the TCR repertoire showed that the addition of anti-CTLA-4 at priming reshaped the repertoire of tumor infiltrating T cells. In particular, the oligoclonal populations became greater in magnitude and more diverse in specificity. Using anti-CTLA-4 in a restricted way to promote the priming phase of an anti-cancer vaccine may offer a useful way of harnessing clinical benefit from this powerful agent.
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http://dx.doi.org/10.1080/2162402X.2017.1376154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739554PMC
September 2017

HDAC inhibitors restore BRAF-inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma.

Int J Cancer 2018 05 20;142(9):1926-1937. Epub 2017 Dec 20.

The Centenary Institute, University of Sydney, Newtown, NSW, Australia.

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.
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http://dx.doi.org/10.1002/ijc.31199DOI Listing
May 2018

A self-adjuvanting vaccine induces cytotoxic T lymphocytes that suppress allergy.

Nat Chem Biol 2014 Nov 5;10(11):943-9. Epub 2014 Oct 5.

1] Malaghan Institute of Medical Research, Wellington, New Zealand. [2] School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.

Epitope-based peptide vaccines encompass minimal immunogenic regions of protein antigens to allow stimulation of precisely targeted adaptive immune responses. However, because efficacy is largely determined by the functional status of antigen-presenting cells (APCs) that acquire and present peptides to cells of the adaptive immune system, adjuvant compounds are needed to enhance immunogenicity. We present here a vaccine consisting of an allergen-derived peptide conjugated to a prodrug of the natural killer-like T (NKT) cell agonist α-galactosylceramide, which is highly effective in reducing inflammation in a mouse model of allergic airway inflammation. Unlike other peptide-adjuvant conjugates that directly activate APCs through pattern recognition pathways, this vaccine encourages third-party interactions with NKT cells to enhance APC function. Therapeutic efficacy was correlated with marked increases in the number and functional activity of allergen-specific cytotoxic T lymphocytes (CTLs), leading to suppression of immune infiltration into the lungs after allergen challenge in sensitized hosts.
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http://dx.doi.org/10.1038/nchembio.1640DOI Listing
November 2014

Using magnetic resonance imaging to evaluate dendritic cell-based vaccination.

PLoS One 2013 29;8(5):e65318. Epub 2013 May 29.

Malaghan Institute of Medical Research, Wellington, New Zealand.

Cancer immunotherapy with antigen-loaded dendritic cell-based vaccines can induce clinical responses in some patients, but further optimization is required to unlock the full potential of this strategy in the clinic. Optimization is dependent on being able to monitor the cellular events that take place once the dendritic cells have been injected in vivo, and to establish whether antigen-specific immune responses to the tumour have been induced. Here we describe the use of magnetic resonance imaging (MRI) as a simple, non-invasive approach to evaluate vaccine success. By loading the dendritic cells with highly magnetic iron nanoparticles it is possible to assess whether the injected cells drain to the lymph nodes. It is also possible to establish whether an antigen-specific response is initiated by assessing migration of successive rounds of antigen-loaded dendritic cells; in the face of a successfully primed cytotoxic response, the bulk of antigen-loaded cells are eradicated on-route to the node, whereas cells without antigen can reach the node unchecked. It is also possible to verify the induction of a vaccine-induced response by simply monitoring increases in draining lymph node size as a consequence of vaccine-induced lymphocyte trapping, which is an antigen-specific response that becomes more pronounced with repeated vaccination. Overall, these MRI techniques can provide useful early feedback on vaccination strategies, and could also be used in decision making to select responders from non-responders early in therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065318PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667033PMC
January 2014

Strongly magnetic iron nanoparticles improve the diagnosis of small tumours in the reticuloendothelial system by magnetic resonance imaging.

PLoS One 2013 20;8(2):e56572. Epub 2013 Feb 20.

Malaghan Institute of Medical Research and School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.

Despite advances in non-invasive medical imaging, accurate nodal staging of malignancy continues to rely on surgery. Superparamagnetic iron oxide nanoparticles (IONP) with lymphotropic qualities have shown some promise as contrast agents for MRI of the lymph nodes, but recent large-scale studies failed to show consistent detection of tumours below 5 mm. Herein we compare imaging of splenic and lymph node tissue using iron/iron oxide core/shell nanoparticles (Fe NP) that have superior magnetic qualities to IONP, to determine whether improved negative contrast in T(2)-weighted MRI can enhance the diagnosis of small tumours in the reticuloendothelial system. To provide an in vivo pre-clinical model of human lymph node micrometastases, breast cancer cells were injected into the spleens of mice, providing localised areas of tumour growth. MR images of groups of tumour-bearing and sham-treated animals were generated using a 1.5 T imaging system and analysed by two independent, blinded radiologists. Fe NP improved the sensitivity and specificity of MRI when compared to IONP, enabling accurate detection of tumours as small as 1-3 mm. The use of Fe NP as contrast agents have the potential to improve the diagnostic accuracy of MRI in cancer patients, leading to more rapid and effective treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056572PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3577855PMC
September 2013