Graft-derived cfDNA Monitoring in Plasma and Bile During Normothermic Machine Perfusion in Liver Transplantation Is Feasible and a Potential Tool for Assessing Graft Viability.

BACKGROUND: Ex vivo normothermic machine perfusion (NMP) is an organ preservation technique that enables an extended assessment of graft suitability before liver transplantation (LT). Established monitoring protocols used during NMP vary significantly in their assessment of transplant suitability when applied to the same grafts. Graft-derived cell-free DNA (gdcfDNA) analysis is an emerging tool for monitoring graft health post-transplantation. We investigated the feasibility of monitoring gdcfDNA during NMP for LT in a proof-of-concept, observational study. METHODS: Serial plasma and bile samples were collected during NMP for 10 consecutive grafts, at 15 min post-machine reperfusion and then 2-h intervals. Digital polymerase chain reaction was used to quantify gdcfDNA at each time point. RESULTS: Five grafts were suitable for LT, there were no cases of primary nonfunction or death in the recipients. gdcfDNA was quantified in all bile and plasma samples (n > 100). In plasma, gdcfDNA concentrations climbed post-machine reperfusion until 4.25 h (median 2.25 h = 15.98 × 10 6 copies/mL, 4.25 h = 40.21 × 10 6 copies/mL). gdcfDNA levels then diverged significantly when comparing the viable and non-viable graft groups (6.25 h, median viable: 117.15 × 10 6 copies/mL versus non-viable: 16.72 × 10 6 copies/mL, P = 0.01). These opposing trends correlated in each graft and in all cases with the viable/non-viable outcome. There was a trend of gradual decline in bile gdcfDNA from viable grafts post-machine reperfusion; discarded grafts showed more variable patterns of release. CONCLUSIONS: gdcfDNA analysis during NMP is a feasible and potential tool to inform viability assessment during NMP for LT. Bile gdcfDNA monitoring offers the prospect of an objective means to assess the degree of biliary injury associated with organ procurement.

Point-of-care detection of fibrosis in liver transplant surgery using near-infrared spectroscopy and machine learning.

Introduction: Visual assessment and imaging of the donor liver are inaccurate in predicting fibrosis and remain surrogates for histopathology. We demonstrate that 3-s scans using a handheld near-infrared-spectroscopy (NIRS) instrument can identify and quantify fibrosis in fresh human liver samples. Methods: We undertook NIRS scans on 107 samples from 27 patients, 88 from 23 patients with liver disease, and 19 from four organ donors. Results: Liver disease patients had a median immature fibrosis of 40% (interquartile range [IQR] 20-60) and mature fibrosis of 30% (10%-50%) on histopathology. The organ donor livers had a median fibrosis (both mature and immature) of 10% (IQR 5%-15%). Using machine learning, this study detected presence of cirrhosis and METAVIR grade of fibrosis with a classification accuracy of 96.3% and 97.2%, precision of 96.3% and 97.0%, recall of 96.3% and 97.2%, specificity of 95.4% and 98.0% and area under receiver operator curve of 0.977 and 0.999, respectively. Using partial-least square regression machine learning, this study predicted the percentage of both immature (R 2 = 0.842) and mature (R 2 = 0.837) with a low margin of error (root mean square of error of 9.76% and 7.96%, respectively). Conclusion: This study demonstrates that a point-of-care NIRS instrument can accurately detect, quantify and classify liver fibrosis using machine learning.

Looking beyond workforce parity: addressing gender inequity in pathology.

While women pathologists have made up over one-third of pathologists in the Australian workforce for over 15 years and at least 50% since 2019, they are under-represented in senior leadership roles, scientific publications, grant recipients, editorial boards, key presentations, and professional awards. This is not unique to pathology and is seen in the broader medical and academic community. Barriers to gender equity and equality in pathology, medicine and academia include gender stereotypes, gender-based discrimination, structural and organisational barriers as well as broader social and cultural barriers. A diverse leadership reflective of the whole professional body and the broader community is important for optimal health outcomes. It is the responsibility and moral duty of individuals and organisations to address any gender disparities, inequities, and inequalities by monitoring, identifying, and acting on gender biases and systemic barriers that hinder appropriate levels of representation by women.

Implementing structured pathology reporting protocol for non-melanocytic skin cancers: practical considerations.

Non-melanocytic skin cancers (NMSCs) account for five times the incidence of all other cancers combined and cost US $6 billion annually. These are the most frequent specimens encountered in community pathology practice in many Western countries. Lack of standardised structured pathology reporting protocols (SPRPs) can result in omission of critical information or miscommunication leading to suboptimal patient management. The lack of standardised data has significant downstream public health implications, including insufficient data for reliable development of prognostic tools and health-economy planning. The Royal College of Pathologists of Australasia has developed an NMSC SPRP. A multidisciplinary expert committee including pathologists, surgeons, dermatologists, and radiation and medical oncologists from high volume cancer centres was convened. A systematic literature review was performed to identify evidence for including elements as mandatory standards or best practice guidelines. The SPRP and accompanying commentary of evidence, definitions and criteria was peer reviewed by external stakeholders. Finally, the protocol was revised following feedback and trialled in multiple centres prior to implementation. Some parameters utilised clinically for determining management and prognosis including tumour depth, lymphovascular invasion or distance to the margins lack high level evidence in NMSC. Dermatologists, surgeons, and radiation oncologists welcomed the SPRP. Pathologists indicated that the variety of NMSC specimens ranging from curettes to radical resections as well as significant differences in the biological behaviour of different tumours covered by the NMSC umbrella made use of a single protocol difficult. The feedback included that using a SPRP for low risk NMSC was neither clinically justified nor compensated adequately by the Australian Medicare Reimbursement Schedule. Following stakeholder feedback, the SPRP implementation was restricted to excision specimens of head and neck NMSC; and low-risk NMSC, such as superficial basal cell carcinoma, were excluded. Implementing NMSC SPRP fulfils an unmet clinical need. Unlike other cancers, NMSCs generate a range of specimen types and are reported in a wide range of pathology practices. Limiting use of SPRP to NMSC at higher risk of progression and providing formatted templates for easy incorporation into laboratory information systems were essential to successful deployment. In the future, further consideration should be given to implementing the SPRP to include all relevant specimens, including non-head and neck and low-risk NMSC specimens.

Altered endosomal-lysosomal biogenesis in melanoma.

Cutaneous melanoma is the deadliest form of skin neoplasm and its high mortality rates could be averted by early accurate detection. While the detection of melanoma is currently reliant upon melanin visualisation, research into melanosome biogenesis, as a key driver of pathogenesis, has not yielded technology that can reliably distinguish between atypical benign, amelanotic and melanotic lesions. The endosomal-lysosomal system has important regulatory roles in cancer cell biology, including a specific functional role in melanosome biogenesis. Herein, the involvement of the endosomal-lysosomal system in melanoma was examined by pooled secondary analysis of existing gene expression datasets. A set of differentially expressed endosomal-lysosomal genes was identified in melanoma, which were interconnected by biological function. To illustrate the protein expression of the dysregulated genes, immunohistochemistry was performed on samples from patients with cutaneous melanoma to reveal candidate markers. This study demonstrated the dysregulation of Syntenin-1, Sortilin and Rab25 may provide a differentiating feature between cutaneous melanoma and squamous cell carcinoma, while IGF2R may indicate malignant propensity in these skin cancers.

Clinical and financial impacts of abnormal liver biochemistry after liver transplantation.

OBJECTIVE: After liver transplant (LT), many investigations are needed to evaluate abnormal liver function test (LFT), which has poor specificity for graft function and complication. A single center retrospective audit of all adult single organ LT from 1/1/2015 to 31/12/2017 was performed. Demographic, clinical and investigation data from the LT database and electronic medical records and cost data from the hospital's Business Intelligence Unit were analyzed. Patients were classified into uncomplicated or complicated LFT by 2 independent investigators and the number, type, and cost of investigations in the first 30 post-operative days were analyzed. Investigations prior to liver biopsy was sub-analyzed. RESULTS: There was 170 LT with 87 cases of uncomplicated LFT (51.2%) and 83 cases of complicated LFT (48.8%). Most patients with complicated LFT had additional investigations (97.6%), most commonly cholangiogram (55.4%) and liver biopsy (LBx) (50.6%). The additional investigations cost was $1863.3 (95% CI 1289.0-2437.6). Although most LBx (73.8%) showed evidence of rejection, LBx was often not the initial investigation of choice. Current LFT based post-transplant monitoring is inefficient. It remains difficult to determine which patient will benefit from an early invasive procedure like LBx, using LFT alone without further imaging investigations.

Pitfalls in Cutaneous Melanoma Diagnosis and the Need for New Reliable Markers.

Cutaneous melanoma is one of the most aggressive forms of skin cancer, with the development of advanced stage disease resulting in a high rate of patient mortality. Accurate diagnosis of melanoma at an early stage is essential to improve patient outcomes, as this enables treatment before the cancer has metastasised. Histopathologic analysis is the current gold standard for melanoma diagnosis, but this can be subjective due to discordance in interpreting the morphological heterogeneity in melanoma and other skin lesions. Immunohistochemistry (IHC) is sometimes employed as an adjunct to conventional histology, but it remains occasionally difficult to distinguish some benign melanocytic lesions and melanoma. Importantly, the complex morphology and lack of specific biomarkers that identify key elements of melanoma pathogenesis can make an accurate confirmation of diagnosis challenging. We review the diagnostic constraints of melanoma heterogeneity and discuss issues with interpreting routine histology and problems with current melanoma markers. Innovative approaches are required to find effective biomarkers to enhance patient management.

Intraductal tubulopapillary neoplasm.

A man aged above 70 years old with a medical history of ulcerative colitis presented with unintentional weight loss. A pancreatic mass associated with pancreatic duct dilatation was detected on imaging procedures. Initial investigations including fine needle aspiration and cytology examination were inconclusive. A diagnosis of intraductal tubulopapillary neoplasm (ITPN) was made with histopathology and immunohistochemistry examination on a surgically resected specimen. Two years after surgery, the patient remained well with no radiological evidence of recurrence.ITPN is a rare pancreatic duct tumour with limited case reports in medical literature. Risk factors are not well established. We report the first case of ITPN occurring in a patient with ulcerative colitis. A typical presentation of this rare tumour is reported to encourage clinicians to consider ITPN in the differential diagnoses of a pancreatic mass.

Multiple eruptive squamoproliferative lesions during anti-PD1 immunotherapy for metastatic melanoma: Pathogenesis, immunohistochemical analysis and treatment.

Treatment with anti-PD1 inhibitors may enhance the risk for developing low grade squamoproliferative skin tumors. Immunohistochemical (IHC) analysis of the immune tumor microenvironment (TME) allows exploration of the pathogenesis and relationship with the PD1/PDL1 axis. Patients with eruptive keratoacanthoma (KA)-like lesions were recruited from the Melanoma Institute Australia, a tertiary referral specialist melanoma treatment center from January 2015 to August 2017. Clinicopathologic evaluation and IHC features of tumor cells (PDL1 expression) and peritumoral microenvironment (PD1, FOXP3, PDL1, CD4:CD8 expressing cells) in 12 eruptive KA-like lesions, were compared with solitary KAs in age and sex matched non-anti-PD1 treated controls. Four patients with repeated episodes of eruptive KA-like and lichenoid lesions developing 2-7 months after commencing pembrolizumab for AJCC stage IV melanoma, were recruited. Eruptive KA-like squamoproliferative lesions occurred in sun exposed sites and in areas of resolving, concomitant or delayed lichenoid reactions. Histologically, the lesions were well-differentiated squamoproliferative lesions resembling infundibulocystic squamous cell carcinoma or KA. IHC of cases and controls revealed low PDL1 expression of both squamous tumor cells and the TME immune cells. The numbers of immunosuppressive FOXP3 positive Tregs and PD1-expressing T-cells were higher in the cases than the controls but the CD4:CD8 ratio (2:1) was similar. The patients best responded to acitretin and were managed surgically if they demonstrated neoplastic features. Accelerated squamoproliferative growth in actinically damaged keratinocytes associated with lichenoid eruptions may be unmasked in patients treated with anti-PD1 immunotherapy potentially contributed to by a local cutaneous immunosuppressed TME.

Residual melanoma in wide local excision specimens after 'complete' excision of primary cutaneous in situ and invasive melanomas.

Wide local excision (WLE) to achieve adequate clearance margins is the standard initial definitive treatment for patients with biopsy-proven primary cutaneous melanoma. Residual melanoma in WLE specimens after prior complete excision-biopsy (CEB) is reported in 0-6.3% of cases. However, studies evaluating the prevalence, clinicopathological features and relevance of persistent disease in WLE specimens are limited. This study sought to determine the frequency of and clinicopathological characteristics associated with residual melanoma in WLE specimens performed after a CEB of primary cutaneous or acral melanoma (in situ or invasive) with clinically and histologically tumour-free margins, and assess its relevance. A review of the research database and pathology archives of a large Australian tertiary referral melanoma treatment centre was performed. Eligible patients were those for whom a definitive WLE was performed after CEB of a primary melanoma (in situ or invasive) with negative clinical and histological margins, between May 2013 and May 2015. All partial biopsies were excluded. Of 640 eligible patients, 510 (79.7%) had invasive melanoma and 130 (20.3%) had melanoma in situ. Residual disease was identified in 20 cases (20/640, 3.1%), of which three (15%) were melanoma in situ on CEB and 17 (85%) were invasive melanoma. On univariate analysis, the presence of residual disease in WLE specimens was associated with lentigo maligna (LM)/LM melanoma (LMM) subtype [odds ratio (OR) 10.33; 95% confidence interval (CI) 2.84-37.54; p=0.004], nodular melanoma (NM) subtype (OR 4.92; 95% CI 1.53-15.85; p=0.0076) and, for invasive tumours, higher tumour mitotic rate (mean 7.7, SD 7.51 vs 3.4, SD 4.83; OR 1.11; 95% CI 1.04-1.18; p=0.0014). Breslow thickness >4 mm was associated with a higher risk of residual disease (OR 7.30; 95% CI 1.88, 28.26; p=0.004). Cases with residual disease had primary tumours with a significantly larger diameter (median 14 mm, range 4-25) than those without residual disease (median 9 mm, range 2-60), (OR 1.07; 95% CI 1.03-1.11; p≤0.001) and were also more likely to be amelanotic (38% vs 14%), (OR 3.69; 95% CI 1.17, 11.60; p=0.026). Residual disease was associated with assessment of >3 slides of tissue (OR 6.98; 95% CI 1.54-31.62; p=0.0118) and complete blocking of the scar (OR 31.69; 95% CI 3.98-252.21; p=0.0011). Residual melanoma in WLE specimens is an infrequent occurrence. Risk factors for residual disease are LM/LMM and NM melanoma subtypes, higher mitotic rate, larger lesion diameter and amelanosis. Tumours with these features warrant more extensive pathological sampling. WLE after CEB for melanoma remains an important procedure to reduce local recurrence; however, limited pathological sampling of the WLE scar is probably appropriate for cases lacking high risk features.

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 Levels of Hepatocyte-Specific Methylation in Cell-Free DNA Are a Strong Negative Predictor for Acute T Cell-Mediated Rejection Requiring Treatment Following Liver Transplantation.

Graft-derived cell-free DNA (gdcfDNA) quantification is a promising, minimally invasive tool for detecting acute T cell-mediated rejection (ATCMR) following liver transplantation (LT). We investigated the utility of measuring hepatocyte-specific methylation in cfDNA (HS-cfDNA) to quantify gdcfDNA, examining its accuracy in detecting ATCMR in a prospective, cross-sectional study. Blood was collected from LT recipients immediately prior to graft biopsy for suspected rejection. HS-cfDNA was quantified using droplet-digital polymerase chain reaction. Prebiopsy liver function tests (LFTs) and HS-cfDNA levels were correlated with biopsy results and the primary outcome of treated biopsy-proven acute rejection (tBPAR). A total of 51 patients were recruited; 37 had evidence of rejection on biopsy and 20 required treatment. As much as 11 patients needed inpatient treatment for rejection. HS-cfDNA significantly outperformed LFTs in identifying patients with tBPAR, particularly those needing inpatient treatment (area under the curve, 73.0%; 95% confidence interval, 55.4%-90.6%; P = 0.01). At a threshold of <33.5% of the total cfDNA fraction, HS-cfDNA had a specificity of 97%, correctly excluding tBPAR in 30/31 patients. Quantifying graft-specific methylation in cfDNA has a major advantage over previous gdcfDNA techniques: it does not require genotyping/sequencing, lending it greater feasibility for translation into transplantation care. Low levels of HS-cfDNA were a strong negative predictor for tBPAR (negative predictive value, 86%) and may have a future role in triaging patients prior to invasive graft biopsies.

Combination immunotherapy with nivolumab and ipilimumab in patients with rare gynecological malignancies: results of the CA209-538 clinical trial.

BACKGROUND: Patients with rare cancers represent 55% of all gynecological malignancies and have poor survival outcomes due to limited treatment options. Combination immunotherapy with the anti-programmed cell death protein 1 (anti-PD-1) antibody nivolumab and the anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) antibody ipilimumab has demonstrated significant clinical efficacy across a range of common malignancies, justifying evaluation of this combination in rare gynecological cancers. METHODS: This multicenter phase II study enrolled 43 patients with advanced rare gynecological cancers. Patients received induction treatment with nivolumab and ipilimumab at a dose of 3 mg/kg and 1 mg/kg, respectively, every 3 weeks for four doses. Treatment was continued with nivolumab monotherapy at 3 mg/kg every 2 weeks until disease progression or a maximum of 2 years. The primary endpoint was the proportion of patients with disease control at week 12 (complete response, partial response or stable disease (SD) by Response Evaluation Criteria In Solid Tumor V.1.1). Exploratory evaluations correlated clinical outcomes with tumor programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB). RESULTS: The objective response rate in the radiologically evaluable population was 36% (12/33 patients) and in the intention-to-treat population was 28% (12/43 patients), with additional 7 patients obtaining SD leading to a disease control rate of 58% and 44%, respectively. Durable responses were seen across a range of tumor histologies. Thirty-one (72%) patients experienced an immune-related adverse event (irAE) with a grade 3/4 irAE observed in seven (16%) patients. Response rate was higher among those patients with baseline PD-L1 expression (≥1% on tumor cells) but was independent of TMB. CONCLUSIONS: Ipilimumab and nivolumab combination treatment has significant clinical activity with a favorable safety profile across a range of advanced rare gynecological malignancies and warrants further investigation in these tumor types.

Evaluation of Crizotinib Treatment in a Patient With Unresectable GOPC-ROS1 Fusion Agminated Spitz Nevi.

IMPORTANCE: Spitz nevi are benign melanocytic neoplasms that classically present in childhood. Isolated Spitz nevi have been associated with oncogenic gene fusions in approximately 50% of cases. The rare agminated variant of Spitz nevi, thought to arise from cutaneous genetic mosaicism, is characterized by development of clusters of multiple lesions in a segmental distribution, which can complicate surgical removal. Somatic single-nucleotide variants in the HRAS oncogene have been described in agminated Spitz nevi, most of which were associated with an underlying nevus spilus. The use of targeted medical therapy for agminated Spitz nevi is not well understood. OBSERVATIONS: A girl aged 30 months presented with facial agminated Spitz nevi that recurred rapidly and extensively after surgery. Owing to the morbidity of further surgery, referral was made to a molecular tumor board. The patient's archival nevus tissue was submitted for extended immunohistochemical analysis and genetic sequencing. Strong ROS1 protein expression was identified by immunohistochemistry. Consistent with this, analysis of whole-genome sequencing data revealed GOPC-ROS1 fusions. These results indicated likely benefit from the oral tyrosine kinase inhibitor crizotinib, which was administered at a dosage of 280 mg/m2 twice daily. An excellent response was observed in all lesions within 5 weeks, with complete flattening after 20 weeks. CONCLUSIONS AND RELEVANCE: Given the response following crizotinib treatment observed in this case, the kinase fusion was believed to be functionally consequential in the patient's agminated Spitz nevi and likely the driver mutational event for growth of her nevi. The repurposing of crizotinib for GOPC-ROS1 Spitz nevi defines a new treatment option for these lesions, particularly in cases for which surgery is relatively contraindicated.

Combination immunotherapy with ipilimumab and nivolumab in patients with advanced adrenocortical carcinoma: a subgroup analysis of CA209-538.

Background: Adrenocortical carcinoma is a rare malignancy, with poor prognosis and limited treatment options for patients with advanced disease. Chemotherapy is the current standard first-line treatment, providing only a modest survival benefit. There is only limited treatment experience with immunotherapy using single-agent anti-PD-1/PD-L1 therapy. To date no clinical trials have been reported using combination immunotherapy with anti-CTLA-4 and anti-PD-1 blockade in this patient population. Methods: CA209-538 is a prospective multicentre clinical trial in patients with advanced rare cancers. Participants received the anti-PD-1 antibody nivolumab (3 mg/kg IV) and the anti-CTLA-4 antibody ipilimumab (1 mg/kg IV) every three weeks for four doses, followed by nivolumab (3 mg/kg IV) every two weeks and continued for up to 96 weeks, until disease progression or unacceptable toxicity. Response was assessed every 12 weeks by RECIST version 1.1. Primary endpoint was clinical benefit rate (complete response, partial response, stable disease at 12 weeks). Results: Six patients with adrenocortical carcinoma were enrolled and received treatment. Two patients (33%) have an ongoing partial response (10 and 25 months +) and two patients (33%) stable disease leading to a disease control rate of 66%. Both responders had tumors with a microsatellite instable phenotype. One patient rapidly progressed shortly after enrollment into the trial and did not undergo restaging. Immunotherapy-related toxicity was reported in all patients, with four patients (67%) experiencing grade 3/4 hepatitis leading to discontinuation of treatment. Conclusions: This is the first treatment experience using ipilimumab and nivolumab combination immunotherapy in patients with advanced adrenocortical carcinoma. Durable responses have been observed in a subset of patients suggesting that this treatment regimen should be further investigated in this patient population.