A Detailed Histologic and Molecular Assessment of the Diffuse Sclerosing Variant of Papillary Thyroid Carcinoma.

Diffuse sclerosing variant papillary thyroid carcinoma (DS-PTC) is characterized clinically by a predilection for children and young adults, bulky neck nodes, and pulmonary metastases. Previous studies have suggested infrequent BRAFV600E mutation but common RET gene rearrangements. Using strict criteria, we studied 43 DS-PTCs (1.9% of unselected PTCs in our unit). Seventy-nine percent harbored pathogenic gene rearrangements involving RET, NTRK3, NTRK1, ALK, or BRAF; with the remainder driven by BRAFV600E mutations. All 10 pediatric cases were all gene rearranged (P = .02). Compared with BRAFV600E-mutated tumors, gene rearrangement was characterized by psammoma bodies involving the entire lobe (P = .038), follicular predominant or mixed follicular architecture (P = .003), pulmonary metastases (24% vs none, P = .04), and absent classical, so-called "BRAF-like" atypia (P = .014). There was no correlation between the presence of gene rearrangement and recurrence-free survival. Features associated with persistent/recurrent disease included pediatric population (P = .030), gene-rearranged tumors (P = .020), microscopic extrathyroidal extension (P = .009), metastases at presentation (P = .007), and stage II disease (P = .015). We conclude that DS-PTC represents 1.9% of papillary thyroid carcinomas and that actionable gene rearrangements are extremely common in DS-PTC. DS-PTC can be divided into 2 distinct molecular subtypes and all BRAFV600E-negative tumors (1.5% of papillary thyroid carcinomas) are driven by potentially actionable oncogenic fusions.

Clear Cell Sarcoma of the Kidney (CCSK) With BCOR-CCNB3 Fusion: A Rare Case Report With a Brief Review of the Literature.

Pediatric renal tumors are a rare entity and majority of these tumors are accounted for by Wilms tumor. The second most common renal tumor is clear cell sarcoma of the kidney (CSSK). Most of the CSSK have either BCOR-internal tandem duplication (ITD) or YWHAE-NUTM2B/E fusion. The sarcomas with BCOR-CCNB3 fusion are well documented in soft tissue and bone tumors, but are extremely rare in the pediatric renal setting. We are reporting an extremely rare case of pediatric clear cell sarcoma of the kidney (CSSK) with BCOR-CCNB3 fusion, which was a diagnostic challenge on morphological grounds. A final diagnosis could only be reached after multiple reviews and NGS based RNA fusion testing. We have also performed a brief review of literature which revealed eight (8) other cases of this rare entity.

The EUS molecular evaluation of pancreatic cancer: A prospective multicenter cohort trial.

BACKGROUND AND OBJECTIVES: Patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (A-PDAC) are not candidates for surgical resection and are often offered palliative chemotherapy. The ready availability of a safe and effective tumor sampling technique to provide material for both diagnosis and comprehensive genetic profiling is critical for informing precision medicine in A-PDAC, thus potentially increasing survival. The aim of this study is to examine the feasibility and benefits of routine comprehensive genomic profiling (CGP) of A-PDAC using EUS-FNA material. METHODS: This is a prospective cohort study to test the clinical utility of fresh frozen or archival EUS-FNA samples in providing genetic material for CGP. The results of the CGP will be reviewed at a molecular tumor board. The proportion of participants that have a change in their treatment recommendations based on their individual genomic profiling will be assessed. Correlations between CGP and stage, prognosis, response to treatment and overall survival will also be investigated. This study will open to recruitment in 2020, with a target accrual of 150 A-PDAC patients within 36 months, with a 2-year follow-up. It is expected that the majority of participants will be those who have already consented for their tissue to be biobanked in the Victorian Pancreatic Cancer Biobank at the time of diagnostic EUS-FNA. Patients without archival or biobanked material that is suitable for CGP may be offered a EUS-FNA procedure for the purposes of obtaining fresh frozen material. DISCUSSION: This trial is expected to provide crucial data regarding the feasibility of routine CGP of A-PDAC using EUS-FNA material. It will also provide important information about the impact of this methodology on patients' survival.

Genomic and Clinical Significance of Multiple Primary Lung Cancers as Determined by Next-Generation Sequencing.

INTRODUCTION: Marked variations in survival rates have brought into question whether standard clinicopathologic classification should be applied to patients presenting with multiple primary lung cancers (MPLCs). This study investigated the genetic profiles of MPLCs in a cohort of patients using next-generation sequencing and correlated results to clinicopathologic data and patient outcome. METHODS: Patients treated surgically with curative intent for two putative primaries of similar histopathology from January 2000 to December 2019 at St Vincent's Hospital Melbourne. DNA and RNA was extracted from formalin-fixed, paraffin-embedded tumor tissue and sequenced on an Ion Torrent Personal Genome Machine system. Patient outcome was determined by overall survival and disease-free survival. RESULTS: A total of 40 cases fulfilled the inclusion criteria. Mutational profiling was concordant with clinicopathologic diagnosis in most cases; however, seven cases (17.5%) revealed shared mutations suggesting metastatic disease and this was associated with a substantial reduction in overall survival (p < 0.05). CONCLUSIONS: Our results suggest that gene sequencing technologies are potentially a more accurate diagnostic and prognostic tool compared with traditional histopathologic evaluation in patients presenting with suspected MPLCs, which could better guide management and predict outcomes.

ALK-Rearranged Non-Small Cell Lung Cancer in 2020: Real-World Triumphs in an Era of Multigeneration ALK-Inhibitor Sequencing Informed by Drug Resistance Profiling.

Since its discovery in 2007, we have seen the lives of patients diagnosed with advanced anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancers (NSCLC) transform with the advent of molecular therapies with first-, second-, and third-generation ALK inhibitors now available in the clinic. Despite great gains in patient survival now measured in years and preserved quality of life with targeted therapies, drug resistance is unfortunately inevitably encountered in this rare and unique molecular subset of lung cancer, and patients will eventually succumb to the disease. As these patients are often young, fit, and never smokers, the clinical and scientific communities have aligned to expedite drug development and access. Drug resistance profiling and further strategies are being explored through clinical trials, including the evaluation of specific drug sequencing and combinations to overcome such resistance and promote patient longevity. The cases of this report focus on precision medicine and aim to portray the pertinent aspects to consider when treating ALK-rearranged NSCLC in 2020, an ever-shifting space. By way of case examples, this report offers valuable information to the treating clinician, including the evolution of systemic treatments and the management of oligo-progression and multisite drug resistance. With the maturation of real-world data, we are fortunate to be experiencing quality and length of life for patients with this disease surpassing prior expectations in advanced lung cancer. KEY POINTS: This report focuses on the importance of genetic analysis of serial biopsies to capture the dynamic therapeutic vulnerabilities of a patient's tumor, providing a perspective on the complexity of ALK tyrosine kinase inhibitor (ALKi) treatment sequencing. These case examples contribute to the literature on ALK-rearranged and oncogene addicted non-small cell lung cancer (NSCLC), providing a framework for care in the clinic. In oligo-progressive disease, local ablative therapy and continuation of ALKi postprogression should be considered with potential for sustained disease control. ALK G1202R kinase domain mutations (KDM), highly prevalent at resistance to second-generation ALKi resistances, may emerge in non-EML4-ALK variant 3 cases and is sensitive to third-generation lorlatinib. When in compound with one or more ALK KDMs, resistance to lorlatinib is expected. In the case of rampantly progressive disease, rebiopsy and redefining biology in a timely manner may be informative.

Slim electrodes for improved targeting in deep brain stimulation.

OBJECTIVE: The efficacy of deep brain stimulation can be limited by factors including poor selectivity of stimulation, targeting error, and complications related to implant reliability and stability. We aimed to improve surgical outcomes by evaluating electrode leads with smaller diameter electrode and microelectrodes incorporated which can be used for assisting targeting. APPROACH: Electrode arrays were constructed with two different diameters of 0.65 mm and the standard 1.3 mm. Micro-electrodes were incorporated into the slim electrode arrays for recording spiking neural activity. Arrays were bilaterally implanted into the medial geniculate body (MGB) in nine anaesthetised cats for 24-40 h using stereotactic techniques. Recordings of auditory evoked field potentials and multi-unit activity were obtained at 1 mm intervals along the electrode insertion track. Insertion trauma was evaluated histologically. MAIN RESULTS: Evoked auditory field potentials were recorded from ring and micro-electrodes in the vicinity of the medial geniculate body. Spiking activity was recorded from 81% of the microelectrodes approaching the MGB. Histological examination showed localized surgical trauma along the implant. The extent of haemorrhage surrounding the track was measured and found to be significantly reduced with the slim electrodes (541 ± 455 µm vs. 827 ± 647 µm; P < 0.001). Scoring of the trauma, focusing on tissue disruption, haemorrhage, oedema of glial parenchyma and pyknosis, revealed a significantly lower trauma score for the slim electrodes (P < 0.0001). SIGNIFICANCE: The slim electrodes reduced the extent of acute trauma, while still providing adequate electrode impedance for both stimulating and recording, and providing the option to target stimulate smaller volumes of tissue. The incorporation of microelectrodes into the electrode array may allow for a simplified, single-step surgical approach where confirmatory micro-targeting is done with the same lead used for permanent implantation.

RET gene rearrangements occur in a subset of pancreatic acinar cell carcinomas.

Pancreatic acinar cell carcinoma is relatively rare (1 to 2% of pancreatic malignancies) but may be under-recognized. In contrast to pancreatic ductal adenocarcinoma, most acinar cell carcinomas lack mutations in KRAS, DPC, CDKN2A or TP53, but appear to have a high incidence of gene rearrangements, with up to 20% reported to be driven by BRAF fusions. With the development of a new class of RET-specific tyrosine kinase inhibitors, which appear to have particularly strong activity against RET gene rearranged tumours, there is now considerable interest in identifying RET gene rearrangements across a wide range of cancers. RET rearrangements have been reported to occur at a very low incidence (<1%) in all pancreatic carcinomas. We postulated that given its unique molecular profile, RET gene rearrangements may be common in acinar cell carcinomas. We performed fluorescent in-situ hybridization (FISH) studies on a cohort of 40 acinar cell spectrum tumours comprising 36 pure acinar cell carcinomas, three pancreatoblastomas and one mixed acinar-pancreatic neuroendocrine tumour. RET gene rearrangements were identified in 3 (7.5%) cases and BRAF gene rearrangements in 5 (12.5%). All gene rearranged tumours were pure acinar cell carcinomas. Our findings indicate that amongst all pancreatic carcinomas, acinar carcinomas are highly enriched for potentially actionable gene rearrangements in RET or BRAF. FISH testing is inexpensive and readily available in the routine clinical setting and may have a role in the assessment of all acinar cell carcinomas-at this stage to recruit patients for clinical trials of new targeted therapies, but perhaps in the near future as part of routine care.

Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS-mutant lung adenocarcinoma.

The KRAS oncoprotein, a critical driver in 33% of lung adenocarcinoma (LUAD), has remained an elusive clinical target due to its perceived undruggable nature. The identification of dependencies borne through common co-occurring mutations are sought to more effectively target KRAS-mutant lung cancer. Approximately 20% of KRAS-mutant LUAD carry loss-of-function mutations in KEAP1, a negative regulator of the antioxidant response transcription factor NFE2L2/NRF2. We demonstrate that Keap1-deficient KrasG12D lung tumors arise from a bronchiolar cell-of-origin, lacking pro-tumorigenic macrophages observed in tumors originating from alveolar cells. Keap1 loss activates the pentose phosphate pathway, inhibition of which, using 6-AN, abrogated tumor growth. These studies highlight alternative therapeutic approaches to specifically target this unique subset of KRAS-mutant LUAD cancers.

FGFR3-TACC3 is an oncogenic fusion protein in respiratory epithelium.

Structural rearrangements of the genome can drive lung tumorigenesis through the generation of fusion genes with oncogenic properties. Advanced genomic approaches have identified the presence of a genetic fusion between fibroblast growth factor receptor 3 (FGFR3) and transforming acidic coiled-coil 3 (TACC3) in non-small cell lung cancer (NSCLC), providing a novel target for FGFR inhibition. To interrogate the functional consequences of the FGFR3-TACC3 fusion in the transformation of lung epithelial cells, we generated a novel transgenic mouse model that expresses FGFR3-TACC3 concomitant with loss of the p53 tumor suppressor gene. Intranasal delivery of an Ad5-CMV-Cre virus promoted seromucinous glandular transformation of olfactory cells lining the nasal cavities of FGFR3-TACC3 (LSL-F3T3) mice, which was further accelerated upon loss of p53 (LSL-F3T3/p53). Surprisingly, lung tumors failed to develop in intranasally infected LSL-F3T3 and LSL-F3T3/p53 mice. In line with these observations, we demonstrated that intranasal delivery of Ad5-CMV-Cre induces widespread Cre-mediated recombination in the olfactory epithelium. Intra-tracheal delivery of Ad5-CMV-Cre into the lungs of LSL-F3T3 and LSL-F3T3/p53 mice, however, resulted in the development of lung adenocarcinomas. Taken together, these findings provide in vivo evidence for an oncogenic function of FGFR3-TACC3 in respiratory epithelium.

Synergy between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small-Cell Lung Cancer with an Altered Immune Microenvironment.

The lung presents a highly oxidative environment, which is tolerated through engagement of tightly controlled stress response pathways. A critical stress response mediator is the transcription factor nuclear factor erythroid-2-related factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1). Alterations in the KEAP1/NRF2 pathway have been identified in 23% of lung adenocarcinomas, suggesting that deregulation of the pathway is a major cancer driver. We demonstrate that inactivation of Keap1 and Pten in the mouse lung promotes adenocarcinoma formation. Notably, metabolites identified in the plasma of Keap1f/f/Ptenf/f tumor-bearing mice indicate that tumorigenesis is associated with reprogramming of the pentose phosphate pathway. Furthermore, the immune milieu was dramatically changed by Keap1 and Pten deletion, and tumor regression was achieved utilizing immune checkpoint inhibition. Thus, our study highlights the ability to exploit both metabolic and immune characteristics in the detection and treatment of lung tumors harboring KEAP1/NRF2 pathway alterations.

Adenoid Cystic Carcinoma Presenting as Abducens Nerve Palsy Secondary to Cavernous Sinus Lesion.

BACKGROUND: We report a 61-year-old who presented with a right-sided abducens nerve palsy secondary to a middle cranial fossa adenoid cystic carcinoma (ACC) extending into the cavernous sinus. PURPOSE: This case represents a unique presentation of intracranial ACC with a large middle cranial fossa mass and only a small extracranial component. METHODS: Review of the literature was undertaken to identify cases of intracranial ACC and their range of presentations. RESULTS: Our results show that this is the first reported case of an ACC presenting mostly as an intracranial mass with an isolated cranial nerve lesion. CONCLUSION: Our case highlights the importance of a broad differential diagnosis, particularly in circumstances where there are atypical features of lesions on radiographic imaging.

Correlation between molecular analysis, diagnosis according to the 2015 WHO classification of unresected lung tumours and TTF1 expression in small biopsies and cytology specimens from 344 non-small cell lung carcinoma patients.

We investigated correlations between diagnosis according to the 2015 World Health Organization (WHO) classification of unresected lung tumours, molecular analysis and TTF1 expression in small biopsy and cytology specimens from 344 non-small cell lung carcinoma (NSCLC) patients. One case failed testing for EGFR, KRAS and ALK abnormalities and six had insufficient tumour for ALK testing. Overall mutation rate in 343 cases was 48% for the genes tested, with 19% EGFR, 33% KRAS and 4% BRAF mutations, and 5% ALK rearrangements detected. More EGFR-mutant (78%) and ALK-rearranged (75%) tumours had morphologic adenocarcinoma than KRAS-mutant (56%) tumours. Despite no significant difference in the overall rate of any molecular abnormality between morphologic adenocarcinoma (52%) and NSCLC, favour adenocarcinoma (47%) (p = 0.18), KRAS mutations were detected more frequently in the latter group. No significant difference in the overall rate of any molecular abnormality between TTF1 positive (49%) and TTF1 negative tumours (44%) (p = 0.92) was detected, but more EGFR-mutant (97%) and ALK-rearranged tumours (92%) were TTF1 positive than KRAS-mutant tumours (68%). Rates of EGFR, KRAS and BRAF mutations and ALK rearrangements in this Australian NSCLC patient population are consistent with the published international literature. Our findings suggest that 2015 WHO classification of unresected tumours may assist in identifying molecular subsets of advanced NSCLC.

Comparative Study of Clinical, Pathological, Radiological, and Genetic Features of Patients With Adult Ocular Adnexal Xanthogranulomatous Disease, Erdheim-Chester Disease, and IgG4-Related Disease of the Orbit/Ocular Adnexa.

PURPOSE: To compare and contrast the clinical, radiologic, pathologic, and genetic features of patients with ocular adnexal IgG4-related disease (IgG4-RD) and patients with adult ocular adnexal xanthogranulomatous disease (XG). METHODS: This retrospective review study identified patients with histological evidence of either disease from records of the pathology department of our hospital from 1996 to 2014. Clinical, imaging, and a variety of histopathologic features were collected for 23 patients with IgG4-RD and 13 patients with XG. Next generation sequencing with a 50-gene cancer screening panel was performed on biopsy tissues from 10 patients in each group. RESULTS: Statistical differences between the 2 groups include eyelid (67%; p = 0.0002) and anterior orbital (75%; p = 0.0352) predilection for XG except for Erdheim-Chester disease subgroup which was more posterior and diffuse. Eyelid involvement was rare (4%) for IgG4-RD. Involvement of orbital nerves was seen in 30% of IgG4-RD and 0% in XG (p = 0.0695). Five patients with IgG4-RD developed malignancy (4 lymphoma, 1 leiomyosarcoma), but none of XG patients. Discriminating pathological features were the presence of any IgG4+ plasma cells (p = 0.0121) and the ratio of IgG4+/IgG+ plasma cells (p =0.0294) for IgG4-RD. Five of 12 (42%) patients with XG had sufficient numbers of IgG4+ plasma cells/high power field to fulfill published diagnostic criteria for IgG4-RD, and 5 (42%) had a ratio of IgG4+/IgG+ plasma cells over 40%, but the numbers overall were less than seen in the IgG4-RD patients. The only genetic difference between the 2 groups was that BRAF V600E mutation was found in 1 of the 2 Erdheim-Chester disease patients, which form a subgroup of XG. CONCLUSIONS: IgG4-RD and XG share clinical, imaging, and histopathological features including IgG4+ plasma cells. Significant differences were the eyelid involvement in XG, orbital nerve involvement, and an elevated IgG4+/IgG+ ratio in IgG4-RD and the only genetic abnormality found was BRAF V600E mutation in the Erdheim-Chester disease subgroup of XG.

Clinical validation of the 50 gene AmpliSeq Cancer Panel V2 for use on a next generation sequencing platform using formalin fixed, paraffin embedded and fine needle aspiration tumour specimens.

The advent of massively parallel sequencing has caused a paradigm shift in the ways cancer is treated, as personalised therapy becomes a reality. More and more laboratories are looking to introduce next generation sequencing (NGS) as a tool for mutational analysis, as this technology has many advantages compared to conventional platforms like Sanger sequencing. In Australia all massively parallel sequencing platforms are still considered in-house in vitro diagnostic tools by the National Association of Testing Authorities (NATA) and a comprehensive analytical validation of all assays, and not just mere verification, is a strict requirement before accreditation can be granted for clinical testing on these platforms. Analytical validation of assays on NGS platforms can prove to be extremely challenging for pathology laboratories. Although there are many affordable and easily accessible NGS instruments available, there are no standardised guidelines as yet for clinical validation of NGS assays. We present an accreditation development procedure that was both comprehensive and applicable in a setting of hospital laboratory for NGS services. This approach may also be applied to other NGS applications in service laboratories.

Mapping of actionable mutations to histological subtype domains in lung adenocarcinoma: implications for precision medicine.

Precision medicine depends on the accurate identification of actionable mutations in a tumor sample. It is unknown how heterogeneous the distribution of such mutations can be in a tumor. Morphological (i.e. histopathological) heterogeneity is well described in lung adenocarcinoma and has been specifically recognized in the most recent official clinico-pathological classification. The most predominant subtype present is now used to classify each lung adenocarcinoma. No molecular profile exists to explain the intratumoral differences in lung adenocarcinoma morphology, despite the consistently observed association between specific predominant subtypes and poorer survival. Given a recent proposal stratifying lung adenocarcinoma into subtypes of differing metastatic potential, we questioned the assumption that major mutations are present uniformly throughout tumors; especially those showing discrete different subtypes. We selected formalin-fixed paraffin embedded lung adenocarcinoma specimens that showed discrete areas of different subtypes, extracted subtype DNA samples from those areas and screened for mutations in hotspot regions of the EGFR, KRAS and BRAF genes using high resolution melting. Sanger sequencing was used to confirm all identified mutations. Chromogenic in situ hybridization (CISH) was used to identify mutant allele specific imbalances in tumors with EGFR mutations. Interestingly, we found that KRAS and BRAF mutations could be confined to morphological domains of higher grade. On the other hand, EGFR mutations were found through all histological subtypes in each tumor consistent with the driver status of this mutation. Intratumoral heterogeneity has major implications for tumorigenesis, chemoresistance and the role of histopathology in molecular screening for precision medicine. This study not only confirms that intratumoral mutational heterogeneity does occur, but also that it is associated with morphologically distinct regions in some tumors. From a practical perspective, small biopsies may not adequately represent a tumor's full mutational profile, particularly for later arising but prognostically important mutations such as those in the KRAS and BRAF genes.

National Working Group Meeting on ALK diagnostics in lung cancer.

The global landscape of molecular testing is rapidly changing, with the recent publication of the International Association for the Study of Lung Cancer (IASLC)/College of American Pathologists (CAP) guidelines and the ALK Atlas. The IASLC/CAP guidelines recommend that tumors from patients with non-small cell lung cancer (NSCLC) be tested for ALK rearrangements in addition to epidermal growth factor receptor (EGFR) mutations. The spur for this recommendation is the availability of novel therapies that target these rearrangements. This article is based on coverage of a Pfizer-sponsored National Working Group Meeting on ALK Diagnostics in Lung Cancer, held around the 15th World Lung Cancer Conference, in Sydney on October 31, 2013. It is based on the presentations given by the authors at the meeting and the discussion that ensued. The content for this article was discussed and agreed on by the authors.

Well-differentiated papillary mesothelioma in association with endometrial carcinoma: a case report.

BACKGROUND: Well-differentiated papillary mesothelioma (WDPM) is an uncommon tumor usually arising in the peritoneum and mostly an incidental finding during abdominal and pelvic surgery. Its natural history and association with other neoplasms is not clearly understood. We present a rare case of WDPM in association with high-grade endometrial carcinoma. To our knowledge, there are only two previously reported cases in the English literature of WDPM in association with endometrial carcinoma. CASE: A 62-year-old woman underwent pelvic surgery for a high-grade endometrial adenocarcinoma. At laparotomy an extensive peritoneal nodular fibrotic reaction was present, raising the clinical possibility of metastatic disease; however, intraoperative frozen section reported this as a mesothelial reaction. Cytologic examination of peritoneal washings revealed cohesive clusters of reactive-appearing mesothelial cells, some with papillary morphology, and no evidence of adenocarcinoma. The peritoneal biopsies showed no metastatic carcinoma. The endometrial tumor was an endometrioid adenocarcinoma. CONCLUSION: The cytologic diagnosis of WDPM may be difficult because it is an uncommon entity and there are overlapping features with other neoplastic and nonneoplastic lesions of the female genital tract and peritoneum. Compounding this, WDPM may occur in association with other neoplasms. We highlight the potential for surgical and pathologic misinterpretation of this entity.