Utility of multigene panel next-generation sequencing in routine clinical practice for identifying genomic alterations in newly diagnosed metastatic nonsmall cell lung cancer.

BACKGROUND: The standard of care in newly diagnosed metastatic non-small cell lung cancer (NSCLC) is to test for aberrations in three genes for driver mutations - ALK, ROS1 and epidermal growth factor receptor (EGFR) - and also for immunohistochemistry to be performed for programmed death-ligand 1 expression level. Next-generation sequencing (NGS), with or without RNA fusion testing, is increasingly used in standard clinical practice to identify patients with potentially actionable mutations. Stratification of NGS mutation tiers is currently based on the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT) Tiers I-V and X. AIM: Our aim was to analyse NSCLC tumour samples for the prevalence of Tiers I-V mutations to establish guidance for current and novel treatments in patients with metastatic disease. METHODS: NGS was performed employing the Oncomine Precision Assay (without RNA fusion testing) that interrogates DNA hotspot variants across 45 genes to screen 210 NSCLC tissue samples obtained across six Sydney hospitals between June 2021 and March 2022. RESULTS: In our cohort, 161 of 210 (77%) had at least one gene mutation identified, with 41 of 210 (20%) having two or more concurrent mutations. Tier I mutations included 42 of 210 (20%) EGFR mutations (EIA) and five of 210 (3%) MET exon 14 skipping mutations (EIB). Non-Tier I variants included 22 of 210 (11%) KRAS G12C hotspot mutations (EIIB), with a further 47 of 210 (22%) having non-G12C KRAS (EX) mutations. NGS testing revealed an additional 15% of cases with Tier II ESCAT mutations in NSCLC. Forty-six percent of patients also demonstrated potential Tier III and IV mutations that are currently under investigation in early-phase clinical trials. CONCLUSIONS: In addition to identifying patients with genomic alterations suitable for clinically proven standard-of-care therapeutic options, the 45-gene NGS panel has significant potential in identifying potentially actionable non-Tier 1 mutations that may become future standard clinical practice in NSCLC.

KRAS and BRAF Mutation Rates and Survival Outcomes in Colorectal Cancer in an Ethnically Diverse Patient Cohort.

KRAS and BRAF mutation rates in colorectal cancer (CRC) reported from various mono-ethnic studies vary amongst different ethnic groups. However, these differences in mutation rates may not be statistically significant or may be due to differences in environmental and/or laboratory factors across countries rather than racial genetic differences. Here, we compare the KRAS/BRAF mutation rates and survival outcomes in CRC between ethnic groups at a single institution. We also investigate the contributions of genetic, environmental, and laboratory factors to the variations in KRAS/BRAF mutation rates reported from different countries. Clinicopathological data from 453 ethnically diverse patients with CRC were retrospectively analyzed at Liverpool Hospital, NSW Australia (2014-2016). KRAS/BRAF mutations were detected using real-time PCR (Therascreen kits from Qiagen). Mismatch repair (MMR) status was determined using immunohistochemical staining. Four ethnic groups were analyzed: Caucasian, Middle Eastern, Asian, and South American. Overall survival data were available for 406 patients. There was no significant difference in KRAS mutation rates between Caucasians (41.1%), Middle Easterners (47.9%), Asians (44.8%), and South Americans (25%) (p = 0.34). BRAF mutation rates differed significantly between races (p = 0.025), with Caucasians having the highest rates (13.5%) and Middle Easterners the lowest (0%). A secondary analysis in which Caucasians were divided into three subgroups showed that ethnic grouping correlated significantly with KRAS mutation rate (p = 0.009), with central and eastern Europeans having the highest rates (58.3%). There were no significant differences in overall survival (OS) or disease-free survival (DFS) between the four races. The similarity in KRAS mutation rates across races raises the possibility that the differences in KRAS mutation rates reported from various countries may either not be statistically significant or may be due to environmental and/or laboratory factors rather than underlying racial genetic differences. In contrast, we verified that BRAF mutation rates differ significantly between races, suggesting racial genetic differences may be responsible for the discrepant BRAF mutation rates reported from different countries.

Aberrant Expression of RAD52, Its Prognostic Impact in Rectal Cancer and Association with Poor Survival of Patients.

The DNA damage response enables cells to survive and maintain genome integrity. RAD52 is a DNA-binding protein involved in the homologous recombination in DNA repair, and is important for the maintenance of tumour genome integrity. We investigated possible correlations between RAD52 expression and cancer survival and response to preoperative radiotherapy. RAD52 expression was examined in tumour samples from 179 patients who underwent surgery for rectal cancer, including a sub-cohort of 40 patients who were treated with neoadjuvant therapy. A high score for RAD52 expression in the tumour centre was significantly associated with worse disease-free survival (DFS; p = 0.045). In contrast, reduced RAD52 expression in tumour centre samples from patients treated with neoadjuvant therapy (n = 40) significantly correlated with poor DFS (p = 0.025) and overall survival (OS; p = 0.048). Our results suggested that RAD52 may have clinical value as a prognostic marker of tumour response to neoadjuvant radiation and both disease-free status and overall survival in patients with rectal cancer.

Pathological prognostication of paediatric adrenocortical tumours: Is a gold standard emerging?

BACKGROUND: Criteria for the pathological classification of adult adrenocortical tumours (ACTs) have been found to overestimate the malignant potential of childhood ACTs. We sought to evaluate the accuracy and utility of criteria developed for paediatric ACT compared to current criteria for adults. METHODS: ACTs treated between January 2006 and December 2016 in two paediatric institutions were evaluated. Patients classified clinically as malignant (CM) had locally invasive disease at surgery requiring extensive en bloc resection to achieve clear margins, had local recurrence or distant metastasis. Slides were reviewed by pathologists blinded to the clinical outcome. A grade was assigned to each tumor according to the Weiss, Aubert, Wieneke and Dehner-Hill criteria. The pathological grade was compared to the clinical outcome. RESULTS: The median follow-up was 60 months (interquartile range 25-80 months). Based on clinical criteria, of 22 patients 14 (64%) had a benign course and eight (34%) behaved malignant. The malignant potential was overestimated by Weiss criteria in 23% and Aubert criteria in 27%. Wieneke and Dehner-Hill criteria showed good clinicopathological correlation; no child who had a benign course was classified as malignant. The Dehner-Hill criteria, however, classified five (23%) children as intermediate risk of which three had a clinically benign and two a CM course. CONCLUSION: The Wieneke criteria accurately predicts the clinical course in childhood ACTs and could be considered the gold standard in their pathological characterization.

Overexpression of the MRE11-RAD50-NBS1 (MRN) complex in rectal cancer correlates with poor response to neoadjuvant radiotherapy and prognosis.

BACKGROUND: The MRE11/RAD50/NBS1 (MRN) complex plays an essential role in detecting and repairing double-stranded breaks, and thus the potential roles of MRE11, RAD50 and NBS1 proteins in the pathogenesis of various cancers is the subject of investigation. This study was aimed at assessing the three-protein panel of MRN complex subunits as a potential radiosensitivity marker and evaluating the prognostic and clinicopathological implications of MRN expression in rectal cancer. METHODS: Samples from 265 rectal cancer patients treated with surgery and adjuvant chemoradiotherapy, including samples from 55 patients who were treated with neoadjuvant radiotherapy between 2000 and 2011, were analyzed. Expression of MRN complex proteins in tissue samples was determined by immunohistochemistry. Univariate and multivariate analyses were carried out to identify clinicopathological characteristics that are associated with the MRN three-protein panel expression in rectal cancer samples. RESULTS: In Kaplan-Meier survival analyses, we found that high level expression of MRN complex proteins in postoperative samples was associated with poor disease-free (p = 0.021) and overall (P = 0.002) survival. Interestingly, high MRN expression also correlated with poor disease-free (P = 0.047) and overall (P = 0.024) survival in the neoadjuvant radiotherapy subgroup. In multivariate analysis, combined MRN expression (hazard ratio = 2.114, 95% confidence interval 1.096-4.078, P = 0.026) and perineural invasion (hazard ratio = 2.160, 95% confidence interval 1.209-3.859, P = 0.009) were significantly associated with a worse disease-free survival. CONCLUSIONS: Expression levels of MRN complex proteins significantly predict disease-free survival in rectal cancer patients, including those treated with neoadjuvant radiotherapy, and may have value in the management of these patients.

Microsatellite Instability Testing and Prognostic Implications in Colorectal Cancer.

Given the crucial predictive implications of microsatellite instability (MSI) in colorectal cancer (CRC), MSI screening is commonly performed in those with and at risk for CRC. Here, we compared results from immunohistochemistry (IHC) and the droplet digital PCR (ddPCR) MSI assay on formalin-fixed paraffin-embedded tumor samples from 48 patients who underwent surgery for colon and rectal cancer by calculating Cohen's kappa measurement (k), revealing high agreement between the methods (k = 0.915). We performed Kaplan-Meier survival analyses and univariate and multivariate Cox regression to assess the prognostic significance of ddPCR-based MSI and to identify clinicopathological features associated with CRC outcome. Patients with MSI-high had better overall survival (OS; p = 0.038) and disease-free survival (DFS; p = 0.049) than those with microsatellite stability (MSS). When stratified by primary tumor location, right-sided CRC patients with MSI-high showed improved DFS, relative to those with MSS (p < 0.001), but left-sided CRC patients did not. In multivariate analyses, MSI-high was associated with improved OS (hazard ratio (HR) = 0.221, 95% confidence interval (CI): 0.026-0.870, p = 0.042), whereas the loss of DNA mismatch repair protein MutL homolog 1 (MLH1) expression was associated with worse OS (HR = 0.133, 95% CI: 0.001-1.152, p = 0.049). Our results suggest ddPCR is a promising tool for MSI detection. Given the opposing effects of MSI-high and MLH1 loss on OS, both ddPCR and IHC may be complementary for the prognostic assessment of CRC.

Multigene panel next generation sequencing in metastatic colorectal cancer in an Australian population.

BACKGROUND: Next generation sequencing (NGS) is increasingly used in standard clinical practice to identify patients with potentially actionable mutations. Stratification of NGS mutation tiers is currently based on the European Society of Medical Oncology (ESMO) Scale for Clinical Actionability of Molecular Targets (ESCAT[E]) Tier I-V & X. Allele frequency is also increasingly recognised as an important prognostic tool in advanced cancer. The aim of this study was to determine the genomic mutations in metastatic colorectal cancer (CRC) in an Australian multicultural population and their influence on survival outcomes. METHODS: Next generation sequencing with the 50-gene panel Oncomine Precision Assay™ was used on 180 CRC tissue samples obtained across six Sydney hospitals between June 2021 and March 2022. RESULTS: From 180 samples, 147 (82%) had at least one gene mutation identified with 68 (38%) having two or more concurrent mutations. Tier I variants included RAS wild-type [EI] in 73 (41%) and BRAF V600E [EIA] in 27 (15%). Non-tier I variants include 2 (1%) ERBB2 amplification [EIIB], 26 (15%) PIK3CA hotspot mutations [EIIIA] and 9 (5%) MET focal amplifications [EIIIA]. NGS testing revealed an additional 22% of cases with Tier II & III mutations. 43% of patients also presented with potentially actionable Tier III & IV mutations. Patients with concurrent TP53 and RAS mutations had significantly reduced overall survival (6.1 months versus 21.1 months, p <0.01). High KRAS allele frequency, as defined by those with over 20% variant allele frequency (VAF), also demonstrated reduced overall survival (12.1 months versus 42.9 months, p = 0.04). CONCLUSIONS: In addition to identifying patients with genomic alterations suitable for clinically proven standard of care therapeutic options, the 50 gene NGS panel has significant potential in identifying potentially actionable non-tier 1 mutations and therefore may become future standard clinical practice.

Prognostic Significance of MRE11 Overexpression in Colorectal Cancer Patients.

Meiotic recombination 11 (MRE11) plays a critical role in the DNA damage response and maintenance of genome stability and is associated with the prognosis for numerous malignancies. Here, we explored the clinicopathological significance and prognostic value of MRE11 expression in colorectal cancer (CRC), a leading cause of cancer-related deaths worldwide. Samples from 408 patients who underwent surgery for colon and rectal cancer between 2006 and 2011, including a sub-cohort of 127 (31%) patients treated with adjuvant therapy, were analyzed. In Kaplan-Meier survival analyses, we found that high MRE11 expression in the tumor center (TC) was significantly associated with poor disease-free survival (DFS; p = 0.045) and overall survival (OS; p = 0.039). Intriguingly, high MRE11 expression in the TC was also significantly correlated with reduced DFS (p = 0.005) and OS (p = 0.010) in the subgroup with right-sided primary CRC. In multivariate analyses, high MRE11 expression (hazard ratio [HR] = 1.697, 95% confidence interval [CI]: 1.034-2.785; p = 0.036) and lymphovascular/perineural invasion (LVI/PNI; HR = 1.922, 95% CI 1.122-3.293; p = 0.017) showed significant association with worse OS in patients with right-sided tumors but not those with left-sided tumors. Moreover, in patients with right-sided tumors, high MRE11 was associated with worse OS for those with lymph node involvement (p = 0.006) and LVI/PNI (p = 0.049). Collectively, our results suggest that MRE11 may serve as an independent prognostic marker in those with right-sided severe CRC, with clinical value in the management of these patients.

Necrotising granulomatous mass of the foreskin with inguinal lymphadenopathy: a rare presentation of syphilis.

We report the case of a 24-year-old man who presented with a 5-week history of painful right inguinal lymphadenopathy, weight loss and non-ulcerative foreskin mass. The patient's symptoms progressed despite initial antibiotic therapy. The foreskin mass was clinically suspicious for squamous cell carcinoma; however, histopathology of both the foreskin mass and inguinal lymph node showed necrotising granulomatous inflammation. Extensive immunohistochemistry testing was inconclusive and could not identify a causative microorganism. Ultimately, serology was positive for Treponema pallidum and he was treated with intramuscular benzathine penicillin. This is an unusual case, which highlights the importance of extensive investigation for differential diagnoses of penile mass and exemplifies the resurgence of syphilis in developed countries.

A Diagnostic Dilemma of White Matter Lesions and Cerebral Oedema without Identifiable Cause-A Neurological Conundrum.

INTRODUCTION: This paper describes a case of bi-frontal vasogenic oedema associated with bilateral frontal lobe and left parietal lobe white matter lesions where extensive investigations, including brain biopsy, failed to establish a diagnosis. CASE REPORT: A 67-year-old female presented with three weeks' history of memory loss, fatigue, insomnia, nausea, and occasional dysphasia. Physical examination was unremarkable, yet cerebral CT and MRI showed bilateral frontal lobe vasogenic oedema. Extensive investigations, including: biochemical; radiological; immunological; microbiological; haematological; histopathological; and cytological, failed to establish a confirmed diagnosis. A multidisciplinary team could not achieve a consensus for this atypical presentation. Brain biopsy was unusual, showing destructive inflammatory and subtly granulomatous disease, but an exhaustive list of auxiliary tests could not confirm a cause, and consensus favoured glial fibrillary acidic protein (GFAP) autoimmune encephalopathy. DISCUSSION: A definitive diagnosis could not be established for this patient despite a gamut of investigations. Although some of the presenting features were consistent with GFAP astrocytopathy, initial staining of the patient's CSF for neuronal antibodies was negative. Her symptoms and radiological changes of brain imaging improved without any corticosteroid therapy. CONCLUSIONS: Through this case report, the aim is to add to the repository of neurological sciences in the hope that future similar presentations could potentially lead to discovery of a new aetiology or contribute towards better understanding of an existing disease process.

Early Postoperative Low Expression of RAD50 in Rectal Cancer Patients Associates with Disease-Free Survival.

BACKGROUND: Molecular biomarkers have the potential to predict response to the treatment of rectal cancer. In this study, we aimed to evaluate the prognostic and clinicopathological implication of RAD50 (DNA repair protein RAD50 homolog) expression in rectal cancer. METHODS: A total of 266 rectal cancer patients who underwent surgery and received chemo- and radiotherapy between 2000 and 2011 were involved in the study. Postoperative RAD50 expression was determined by immunohistochemistry in surgical samples (n = 266). RESULTS: Using Kaplan-Meier survival analysis, we found that low RAD50 expression in postoperative samples was associated with worse disease free survival (p = 0.001) and overall survival (p < 0.001) in early stage/low-grade tumors. In a comparison of patients with low vs. high RAD50 expression, we found that low levels of postoperative RAD50 expression in rectal cancer tissues were significantly associated with perineural invasion (p = 0.002). CONCLUSION: Expression of RAD50 in rectal cancer may serve as a prognostic biomarker for long-term survival of patients with perineural invasion-positive tumors and for potential use in early stage and low-grade rectal cancer assessment.

Tubular carcinoma of the breast: axillary involvement and prognostic factors.

BACKGROUND: Tubular carcinoma (TC) of the breast has a very favourable prognosis. The role for axillary staging in small TC was questioned. This study investigated the frequency of axillary metastases and prognostic factors in pure TC of the breast. It involved a retrospective review of prospectively collected data. METHODS: A consecutive series of patients presenting to The Strathfield Breast Centre (TSBC) between 1988 and 2011 were reviewed. Only pure TC was included. Information collected included demographics, surgery, pathology, adjuvant therapy and survival. RESULTS: Pure TC accounted for 146 out of 6110 cases of operable breast cancer. Ninety-five per cent were node negative (micrometastases and isolated tumour cells excluded). Ninety-eight per cent of those with known oestrogen receptor status were oestrogen receptor positive. Median tumour size was 10 mm (range 1-52 mm). Ten-year survival was 97%. Twelve per cent of patients had more than one tumour (either ipsilateral or contralateral). Eight patients had recurrent disease. All were node negative. Three of these patients died of their disease. CONCLUSION: Axillary metastases are uncommon in pure TC. Recurrent disease is not readily predicted by tumour size or node status.