A single-cell tumor immune atlas for precision oncology.

The tumor immune microenvironment is a main contributor to cancer progression and a promising therapeutic target for oncology. However, immune microenvironments vary profoundly between patients, and biomarkers for prognosis and treatment response lack precision. A comprehensive compendium of tumor immune cells is required to pinpoint predictive cellular states and their spatial localization. We generated a single-cell tumor immune atlas, jointly analyzing published data sets of >500,000 cells from 217 patients and 13 cancer types, providing the basis for a patient stratification based on immune cell compositions. Projecting immune cells from external tumors onto the atlas facilitated an automated cell annotation system. To enable in situ mapping of immune populations for digital pathology, we applied SPOTlight, combining single-cell and spatial transcriptomics data and identifying colocalization patterns of immune, stromal, and cancer cells in tumor sections. We expect the tumor immune cell atlas, together with our versatile toolbox for precision oncology, to advance currently applied stratification approaches for prognosis and immunotherapy.

Novel prognostic nomogram for predicting recurrence-free survival in medullary thyroid carcinoma.

AIMS: Recently, there have been attempts to improve prognostication and therefore better guide treatment for patients with medullary thyroid carcinoma (MTC). In 2022, the International MTC Grading System (IMTCGS) was developed and validated using a multi-institutional cohort of 327 patients. The aim of the current study was to build upon the findings of the IMTCGS to develop and validate a prognostic nomogram to predict recurrence-free survival (RFS) in MTC. METHODS AND RESULTS: Data from 300 patients with MTC from five centres across the USA, Europe, and Australia were used to develop a prognostic nomogram that included the following variables: age, sex, AJCC stage, tumour size, mitotic count, necrosis, Ki67 index, lymphovascular invasion, microscopic extrathyroidal extension, and margin status. A process of 10-fold cross-validation was used to optimize the model's performance. To assess discrimination and calibration, the area-under-the-curve (AUC) of a receiver operating characteristic (ROC) curve, concordance-index (C-index), and dissimilarity index (D-index) were calculated. Finally, the model was externally validated using a separate cohort of 87 MTC patients. The model demonstrated very strong performance, with an AUC of 0.94, a C-index of 0.876, and a D-index of 19.06. When applied to the external validation cohort, the model had an AUC of 0.9. CONCLUSIONS: Using well-established clinicopathological prognostic variables, we developed and externally validated a robust multivariate prediction model for RFS in patients with resected MTC. The model demonstrates excellent predictive capability and may help guide decisions on patient management. The nomogram is freely available online at https://nomograms.shinyapps.io/MTC_ML_DFS/.

Complement-mediated thrombotic microangiopathy on a background of Alport syndrome: A case report.

Here we present a case of complement-mediated thrombotic microangiopathy (TMA) in a patient who has a background of Stage 5 chronic kidney disease secondary to Alport syndrome. We explain our approach to the diagnosis of TMA, especially the reliance on non-renal manifestations of TMA and the role of kidney biopsy given there was a background of advanced kidney impairment at baseline.

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.

Ki67 proliferation index in medullary thyroid carcinoma: a comparative study of multiple counting methods and validation of image analysis and deep learning platforms.

AIMS: The International Medullary Thyroid Carcinoma Grading System, introduced in 2022, mandates evaluation of the Ki67 proliferation index to assign a histological grade for medullary thyroid carcinoma. However, manual counting remains a tedious and time-consuming task. METHODS AND RESULTS: We aimed to evaluate the performance of three other counting techniques for the Ki67 index, eyeballing by a trained experienced investigator, a machine learning-based deep learning algorithm (DeepLIIF) and an image analysis software with internal thresholding compared to the gold standard manual counting in a large cohort of 260 primarily resected medullary thyroid carcinoma. The Ki67 proliferation index generated by all three methods correlate near-perfectly with the manual Ki67 index, with kappa values ranging from 0.884 to 0.979 and interclass correlation coefficients ranging from 0.969 to 0.983. Discrepant Ki67 results were only observed in cases with borderline manual Ki67 readings, ranging from 3 to 7%. Medullary thyroid carcinomas with a high Ki67 index (≥ 5%) determined using any of the four methods were associated with significantly decreased disease-specific survival and distant metastasis-free survival. CONCLUSIONS: We herein validate a machine learning-based deep-learning platform and an image analysis software with internal thresholding to generate accurate automatic Ki67 proliferation indices in medullary thyroid carcinoma. Manual Ki67 count remains useful when facing a tumour with a borderline Ki67 proliferation index of 3-7%. In daily practice, validation of alternative evaluation methods for the Ki67 index in MTC is required prior to implementation.

Metastatic solid tumours to the penis: a clinicopathologic evaluation of 109 cases from an international collaboration.

AIMS: To elucidate the spectrum of metastatic tumours to the penis and their clinicopathologic features. METHODS: The databases and files of 22 pathology departments from eight countries on three continents were queried to identify metastatic solid tumours of the penis and to characterize their clinical and pathologic features. RESULTS: We compiled a series of 109 cases of metastatic solid tumours that secondarily involved the penis. The mean patient age at diagnosis was 71 years (range, 7-94 years). Clinical presentation commonly included a penile nodule/mass (48/95; 51%) and localised pain (14/95; 15%). A prior history of malignancy was known in 92/104 (89%) patients. Diagnosis was made mainly on biopsy (82/109; 75%), or penectomy (21/109; 19%) specimens. The most common penile locations were the glans (45/98; 46%) and corpus cavernosum (39/98; 39%). The most frequent histologic type was adenocarcinoma (56%). Most primary carcinomas originated in the genitourinary (76/108; 70%) and gastrointestinal (20/108; 18%) tracts, including prostate (38/108; 35%), urinary bladder (27/108; 25%), and colon/rectum (18/108; 17%). Concurrent or prior extrapenile metastases were identified in 50/78 (64%) patients. Clinical follow-up (mean 22 months, range 0-171 months) was available for 87/109 (80%) patients, of whom 46 (53%) died of disease. CONCLUSION: This is the largest study to date of metastatic solid tumours secondarily involving the penis. The most frequent primaries originated from the genitourinary and gastrointestinal tracts. Metastatic penile tumours usually presented with penile nodules/masses and pain, and they often occurred in the setting of advanced metastatic disease, portending poor clinical outcomes.

Survival, symptoms and hospitalization of older patients with advanced chronic kidney disease managed without dialysis.

BACKGROUND: Shared decision-making (SDM) is important when considering whether an older patient with advanced chronic kidney disease (CKD) should be managed with dialysis or conservative kidney management (CKM). Physicians may find these conversations difficult because of the relative paucity of data on patients managed without dialysis. METHODS: This prospective observational study was conducted in a unit supported by a multidisciplinary Kidney Supportive Care (KSC) programme, in a cohort of 510 patients (280 CKM and 230 dialysis) ≥65 years of age with CKD stages 4 and 5. Survival was evaluated using logistic regression and Cox proportional hazards models. Linear mixed models were utilized to assess symptoms over time. RESULTS: CKM patients were older (mean 84 versus 74 years; P < .001) and almost 2-fold more likely to have three or more comorbidities (P < .001). The median survival of CKM patients was lower compared with dialysis from all time points: 14 months [interquartile range (IQR) 6-32] versus 53 (IQR 28-103) from decision of treatment modality or dialysis start date (P < .001); 15 months (IQR 7-34) versus 64 (IQR 30-103) from the time the estimated glomerular filtration rate (eGFR) was ≤15 mL/min/1.73 m2 (P < .001); and 8 months (IQR 3-18) versus 49 (19-101) from eGFR ≤10 mL/min/1.73 m2. A total of 59% of CKM patients reported an improvement in symptoms by their third KSC clinic visit (P < .001). The rate of unplanned hospitalization was 2-fold higher in the dialysis cohort. CONCLUSIONS: CKM patients survive a median of 14 months from the time of modality choice and have a lower rate of hospitalization than dialysis patients. Although the symptom burden in advanced CKD is high, most elderly CKM patients managed through an integrated KSC programme and can achieve improvement in their symptoms over time. These data might help with SDM.

Expanding the clinicopathological spectrum of succinate dehydrogenase-deficient renal cell carcinoma with a focus on variant morphologies: a study of 62 new tumors in 59 patients.

Most succinate dehydrogenase (SDH)-deficient renal cell carcinomas (RCCs) demonstrate stereotypical morphology characterized by bland eosinophilic cells with frequent intracytoplasmic inclusions. However, variant morphologic features have been increasingly recognized. We therefore sought to investigate the incidence and characteristics of SDH-deficient RCC with variant morphologies. We studied a multi-institutional cohort of 62 new SDH-deficient RCCs from 59 patients. The median age at presentation was 39 years (range 19-80), with a slight male predominance (M:F = 1.6:1). A relevant family history was reported in 9 patients (15%). Multifocal or bilateral tumors were identified radiologically in 5 patients (8%). Typical morphology was present at least focally in 59 tumors (95%). Variant morphologies were seen in 13 (21%) and included high-grade nuclear features and various combinations of papillary, solid, and tubular architecture. Necrosis was present in 13 tumors, 7 of which showed variant morphology. All 62 tumors demonstrated loss of SDHB expression by immunohistochemistry. None showed loss of SDHA expression. Germline SDH mutations were reported in all 18 patients for whom the results of testing were known. Among patients for whom follow-up data was available, metastatic disease was reported in 9 cases, 8 of whom had necrosis and/or variant morphology in their primary tumor. Three patients died of disease. In conclusion, variant morphologies and high-grade nuclear features occur in a subset of SDH-deficient RCCs and are associated with more aggressive behavior. We therefore recommend grading all SDH-deficient RCCs and emphasize the need for a low threshold for performing SDHB immunohistochemistry in any difficult to classify renal tumor, particularly if occurring at a younger age.

Histopathological tumour response scoring in resected pancreatic cancer following neoadjuvant therapy: international interobserver study (ISGPP-1).

BACKGROUND: Most tumour response scoring systems for resected pancreatic cancer after neoadjuvant therapy score tumour regression. However, whether treatment-induced changes, including tumour regression, can be identified reliably on haematoxylin and eosin-stained slides remains unclear. Moreover, no large study of the interobserver agreement of current tumour response scoring systems for pancreatic cancer exists. This study aimed to investigate whether gastrointestinal/pancreatic pathologists can reliably identify treatment effect on tumour by histology, and to determine the interobserver agreement for current tumour response scoring systems. METHODS: Overall, 23 gastrointestinal/pancreatic pathologists reviewed digital haematoxylin and eosin-stained slides of pancreatic cancer or treated tumour bed. The accuracy in identifying the treatment effect was investigated in 60 patients (30 treatment-naive, 30 after neoadjuvant therapy (NAT)). The interobserver agreement for the College of American Pathologists (CAP) and MD Anderson Cancer Center (MDACC) tumour response scoring systems was assessed in 50 patients using intraclass correlation coefficients (ICCs). An ICC value below 0.50 indicated poor reliability, 0.50 or more and less than 0.75 indicated moderate reliability, 0.75 or more and below 0.90 indicated good reliability, and above 0.90 indicated excellent reliability. RESULTS: The sensitivity and specificity for identifying NAT effect were 76.2 and 49.0 per cent respectively. After NAT in 50 patients, ICC values for both tumour response scoring systems were moderate: 0.66 for CAP and 0.71 for MDACC. CONCLUSION: Identification of the effect of NAT in resected pancreatic cancer proved unreliable, and interobserver agreement for the current tumour response scoring systems was suboptimal. These findings support the recently published International Study Group of Pancreatic Pathologists recommendations to score residual tumour burden rather than tumour regression after NAT.

Switch/sucrose-non-fermentable (SWI/SNF) complex (SMARCA4, SMARCA2, INI1/SMARCB1)-deficient colorectal carcinomas are strongly associated with microsatellite instability: an incidence study in 4508 colorectal carcinomas.

AIMS: Loss of expression of mammalian switch/sucrose-non-fermentable (SWI/SNF) [BRG1/BRM-associated factor (BAF)] complex subunits, including SMARCA4, SMARCA2 and INI1/SMARCB1 (termed SWI/SNF complex deficiency), has been reported in colorectal carcinomas (CRCs) but its frequency and clinical significance are uncertain. METHODS AND RESULTS: We performed immunohistochemistry for SMARCA4, SMARCA2 and SMARCB1 on 4508 consecutive resected CRCs. Loss of SMARCA4 expression was found in 13 cases (0.3%), loss of SMARCA2 expression was found in 59 cases (1.3%), and loss of SMARCB1 expression was found in 21 cases (0.4%). Some CRCs showed loss of expression of more than one subunit, so that 84 CRCs (1.7%) were deficient for at least one component. SWI/SNF complex deficiency was associated with higher grade, a right-sided location, mismatch repair deficiency, and BRAF V600E mutation (P < 0.05); 5.8% of mismatch repair-deficient (MMRd) cases and 5.4% of BRAF V600E-mutant cases were SWI/SNF complex-deficient, as compared with 0.9% and 0.4% of mismatch repair-proficient and BRAF-wild-type cases (P < 0.001). Any loss of SMARCB1 expression and global loss of SMARCA2 expression were associated with statistically significant worse overall survival, whereas SMARCA4-deficient cases showed a trend only towards poor overall survival (P = 0.121). In multivariate analysis, any loss of SMARCA4 expression and global loss of SMARCA2 expression were associated with worse survival [odds ratio (OR) 3.33, P = 0.019; and OR 3.39, P < 0.001]. Of particular note, among the subgroup of cases that were MMRd and BRAF V600E-mutated (otherwise considered to be a good prognostic group), loss of SMARCA4 expression was associated with much worse median survival (10.5 months versus 110.9 months; P = 0.003). CONCLUSIONS: SWI/SNF complex deficiency is rare in CRC but is enriched in MMRd cases. Identifying these cases has morphological associations and prognostic significance, and in the future may have potential therapeutic implications.

Genomic analyses identify molecular subtypes of pancreatic cancer.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

DNA damage-inducible transcript 3 immunohistochemistry is highly sensitive for the diagnosis of myxoid liposarcoma but care is required in interpreting the significance of focal expression.

AIMS: Myxoid liposarcoma (MLPS) is characterised by DNA damage-inducible transcript 3 (DDIT3) gene rearrangements, confirmation of which is commonly used diagnostically. Recently, DDIT3 immunohistochemistry (IHC) has been reported to be highly sensitive and, when strict criteria are employed, specific for the diagnosis of MLPS. The aim of this study was to independently investigate DDIT3 IHC as a diagnostic marker for MLPS. METHODS AND RESULTS: DDIT3 IHC was performed on 52 MLPS and on 152 mimics on whole sections, and on 515 non-MLPS sarcomas in tissue microarray format. Only one MLPS (which had undergone acid-based decalcification) was completely negative. With inclusion of this case if any nuclear expression is considered to indicate positivity, the overall sensitivity of DDIT3 is 98% (51 of 52 cases) and the specificity is 94% (633 of 667 non-MLPS cases are negative). If a cut-off of >10% of neoplastic cells is required for positivity, then the sensitivity remains 98% (51/52) and the specificity is 98.5% (657 of 667 non-MLPS cases are negative). If a cut-off of >50% of cells is required for positivity, then the sensitivity is 96% (50 of 52 cases) but the specificity improves to 100%. CONCLUSIONS: Diffuse nuclear DDIT3 expression occurs in the overwhelming majority of MLPSs, and can be used to confirm the diagnosis in most cases without the need for molecular testing. A complete absence of expression argues strongly against MLPS, and almost completely excludes this diagnosis, particularly if there is consideration of technical factors such as decalcification. The significance of focal DDIT3 expression should be interpreted in the morphological and clinical context, although most tumours showing only focal expression are not MLPS.

Whole genomes redefine the mutational landscape of pancreatic cancer.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Precision Oncology in Surgery: Patient Selection for Operable Pancreatic Cancer.

OBJECTIVE: We aimed to define preoperative clinical and molecular characteristics that would allow better patient selection for operative resection. BACKGROUND: Although we use molecular selection methods for systemic targeted therapies, these principles are not applied to surgical oncology. Improving patient selection is of vital importance for the operative treatment of pancreatic cancer (pancreatic ductal adenocarcinoma). Although surgery is the only chance of long-term survival, 80% still succumb to the disease and approximately 30% die within 1 year, often sooner than those that have unresected local disease. METHOD: In 3 independent pancreatic ductal adenocarcinoma cohorts (total participants = 1184) the relationship between aberrant expression of prometastatic proteins S100A2 and S100A4 and survival was assessed. A preoperative nomogram based on clinical variables available before surgery and expression of these proteins was constructed and compared to traditional measures, and a postoperative nomogram. RESULTS: High expression of either S100A2 or S100A4 was independent poor prognostic factors in a training cohort of 518 participants. These results were validated in 2 independent patient cohorts (Glasgow, n = 198; Germany, n = 468). Aberrant biomarker expression stratified the cohorts into 3 distinct prognostic groups. A preoperative nomogram incorporating S100A2 and S100A4 expression predicted survival and nomograms derived using postoperative clinicopathological variables. CONCLUSIONS: Of those patients with a poor preoperative nomogram score, approximately 50% of patients died within a year of resection. Nomograms have the potential to improve selection for surgery and neoadjuvant therapy, avoiding surgery in aggressive disease, and justifying more extensive resections in biologically favorable disease.

NTRK gene rearrangements are highly enriched in MLH1/PMS2 deficient, BRAF wild-type colorectal carcinomas-a study of 4569 cases.

NTRK gene rearrangements are important to identify as predictors of response to targeted therapy in many malignancies. Only 0.16-0.3% of colorectal carcinomas (CRCs) harbor these fusions making universal screening difficult. We therefore investigated whether pan-Trk immunohistochemistry (IHC), mismatch repair deficiency (MMRd), and BRAFV600E mutation status could be used to triage molecular testing for NTRK gene rearrangements in CRC. CRCs from 4569 unselected patients underwent IHC in TMA format with two different anti-pan-Trk rabbit monoclonal antibodies. All positive cases were confirmed on whole sections and underwent RNA-sequencing. Pan-Trk IHC was positive in 0.2% of CRCs (9/4569). Both antibodies demonstrated similar staining characteristics with diffuse positive staining in all neoplastic cells. Of note 8/9 (89%) IHC positive cases were both MMRd (all showing MLH1/PMS2 loss) and lacked BRAFV600E mutation. That is, IHC was positive in 5.3% (8/152) MLH1/PMS2/BRAFV600E triple negative CRCs, but only 0.02% (1/4417) not showing this phenotype. All nine IHC positive CRCs demonstrated gene rearrangements (LMNA-NTRK1 in 5 CRCs, TPR-NTRK1, STRM-NTRK1, MUC2-NTRK2, and NTRK1 with an unknown partner in one each), suggesting close to 100% specificity for IHC in this sub-population. NTRK fusions were associated with right sided (p = 0.02), larger tumors (p = 0.029) with infiltrative growth (p = 0.021). As a part of universal Lynch syndrome screening many institutions routinely test all CRCs for MMRd, and then proceed to reflex BRAFV600E mutation testing in MLH1/PMS2 negative CRCs. We conclude that performing pan-Trk IHC on this preselected subgroup of MLH1/PMS2/BRAFV600E triple negative CRCs (only 3.3% of all CRC patients) is a resource effective approach to identify the overwhelming majority of CRC patients with NTRK gene fusions.

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.

RAF1 rearrangements are common in pancreatic acinar cell carcinomas.

There is now evidence that gene fusions activating the MAPK pathway are relatively common in pancreatic acinar cell carcinoma with potentially actionable BRAF or RET fusions being found in ~30%. We sought to investigate the incidence of RAF1 fusions in pancreatic malignancies with acinar cell differentiation. FISH testing for RAF1 was undertaken on 30 tumors comprising 25 'pure' acinar cell carcinomas, 2 mixed pancreatic acinar-neuroendocrine carcinomas, 1 mixed acinar cell-low grade neuroendocrine tumor and 2 pancreatoblastomas. RAF1 rearrangements were identified in 5 cases and confirmed by DNA and RNA sequencing to represent oncogenic fusions (GATM-RAF1, GOLGA4-RAF1, PDZRN3-RAF1, HERPUD1-RAF1 and TRIM33-RAF1) and to be mutually exclusive with BRAF and RET fusions, as well as KRAS mutations. Large genome-wide copy number changes were common and included 1q gain and/or 1p loss in all five RAF1 FISH-positive acinar cell carcinomas. RAF1 expression by immunohistochemistry was found in 3 of 5 (60%) of fusion-positive cases and no FISH-negative cases. Phospho-ERK1/2 expression was found in 4 of 5 RAF1-fusion-positive cases. Expression of both RAF1 and phospho-ERK1/2 was heterogeneous and often only detected at the tumor-stroma interface, thus limiting their clinical utility. We conclude that RAF1 gene rearrangements are relatively common in pancreatic acinar cell carcinomas (14.3% to 18.5% of cases) and can be effectively identified by FISH with follow up molecular testing. The combined results of several studies now indicate that BRAF, RET or RAF1 fusions occur in between one third and one-half of these tumors but are extremely rare in other pancreatic malignancies. As these fusions are potentially actionable with currently available therapies, a strong argument can be made to perform FISH or molecular testing on all pancreatic acinar cell carcinomas.

When used together SS18-SSX fusion-specific and SSX C-terminus immunohistochemistry are highly specific and sensitive for the diagnosis of synovial sarcoma and can replace FISH or molecular testing in most cases.

AIMS: Synovial sarcoma is defined by recurrent t(X;18)(p11;q11) translocations creating SS18-SSX1, SS18-SSX2 or SS18-SSX4 fusions. Recently, a novel rabbit monoclonal antibody designed to identify these fusions (SS18-SSX, clone E9X9V) was proposed to be highly specific (100%), but not completely sensitive (95%) for this diagnosis. Another antibody designed to identify the C-terminal end of SSX (SSX_CT, clone E5A2C) was proposed to be highly sensitive (100%), but not completely specific (96%). We sought to validate these antibodies in an independent cohort. METHODS AND RESULTS: We performed immunohistochemistry for SS18-SSX and SSX_CT on 39 synovial sarcoma samples from 25 patients with confirmed gene rearrangements. Thirty-four (87%) and 36 (92%) were positive for SS18-SSX and SSX_CT, respectively. False-negative staining was associated with suboptimally handled small biopsies and decalcified specimens, even when staining was diffuse and strong in subsequent optimally processed excisions and non-decalcified areas. None of 580 non-synovial sarcoma tumours (76 whole sections, 504 TMA samples) were positive for SS18-SSX (100% specificity), whereas 39 (93% specificity) were positive for SSX_CT. CONCLUSIONS: SS18-SSX fusion-specific IHC is 87-95% sensitive for the diagnosis of synovial sarcoma and highly (perhaps perfectly) specific. Therefore, positive SS18-SSX staining definitively confirms the diagnosis of synovial sarcoma. SSX_CT is less specific (93-96%) but highly sensitive (92%, but approaching 100% when suboptimally processed biopsies and decalcified specimens are excluded). Negative SSX_CT staining may therefore have an ancillary role as a rule-out test for synovial sarcoma. We caution that both antibodies are prone to false-negative staining in decalcified specimens.

Tailored first-line and second-line CDK4-targeting treatment combinations in mouse models of pancreatic cancer.

OBJECTIVE: Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4. DESIGN: Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens). RESULTS: Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach. CONCLUSION: This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.

The RING finger domain E3 ubiquitin ligases BRCA1 and the RNF20/RNF40 complex in global loss of the chromatin mark histone H2B monoubiquitination (H2Bub1) in cell line models and primary high-grade serous ovarian cancer.

Enzymatic factors driving cancer-associated chromatin remodelling are of increasing interest as the role of the cancer epigenome in gene expression and DNA repair processes becomes elucidated. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone modification that functions in histone cross-talk, transcriptional elongation, DNA repair, maintaining centromeric chromatin and replication-dependent histone mRNA 3'-end processing, as well as being required for the differentiation of stem cells. The loss of global H2Bub1 is seen in a number of aggressive malignancies and has been linked to tumour progression and/or a poorer prognosis in some cancers. Here, we analyse a large cohort of high-grade serous ovarian cancers (HGSOC) and show loss of global H2Bub1 in 77% (313 of 407) of tumours. Loss of H2Bub1 was seen at all stages (I-IV) of HGSOC, indicating it is a relatively early epigenomic event in this aggressive malignancy. Manipulation of key H2Bub1 E3 ubiquitin ligases, RNF20, RNF40 and BRCA1, in ovarian cancer cell line models modulated H2Bub1 levels, indicative of the role of these RING finger ligases in monoubiquitination of H2Bub1 in vitro. However, in primary HGSOC, loss of RNF20 protein expression was identified in just 6% of tumours (26 of 424) and did not correlate with global H2Bub1 loss. Similarly, germline mutation of BRCA1 did not show a correlation with the global H2Bub1 loss. We conclude that the regulation of tumour-associated H2Bub1 levels is complex. Aberrant expression of alternative histone-associated 'writer' or 'eraser' enzymes are likely responsible for the global loss of H2Bub1 seen in HGSOC.