A Founder Mutation in EHD1 Presents with Tubular Proteinuria and Deafness.

BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

Primary high-grade myoepithelial carcinoma of the lung: A study of three cases illustrating frequent SMARCB1-deficiency and review of the literature.

Primary myoepithelial carcinoma of the lung is exceptionally rare and, hence, remained poorly characterized. We present 3 tumors affecting 2 males and 1 female aged 60 to 84 years. Tumor size ranged from 4 to 10 cm. All presented as well circumscribed non-encapsulated peripheral solitary masses. One patient died postoperatively. The other two were lost to follow-up. Histologically, all tumors were high-grade with predominance of myxoid/chordoid (2) and rhabdoid (1) pattern. Immunohistochemistry (IHC) showed reactivity with vimentin, pankeratin, EMA and smooth muscle actin. Two tumors were SMARCB1-deficient (one with additional loss of SMARCA2 and PBRM1). RNA sequencing revealed no gene fusions. Review of reported cases (total: 16) showed that pulmonary myoepithelial carcinoma affects both sexes equally at a median age of 60 years (24-84), presents predominantly as peripheral masses (69%) in the lower lobes (66%) of smokers (70%) with a median size of 6 cm (1.5-13), and originates as high-grade de novo carcinoma. Forty percent of patients died of disease at a median of 12.5 months (0 to 62). Only 40% of patients were disease free at last follow-up (median, 9.5 months). Prominent lobulation and myxoid stroma were frequent histological features. Most tumors displayed variable combinations of epithelioid, spindle, plasmacytoid, clear, ovoid or round cells. Three of 6 tumors subjected to different RNA panels showed EWSR1 rearrangements (fused to PBX1, ZNF444 or to unknown partner). Two of 3 tumors lacking gene fusions were SMARCB1-deficient (both showed secondary EWSR1 FISH abnormalities due to 22q deletion). Primary pulmonary myoepithelial carcinoma is a rare aggressive malignancy that recapitulates its soft tissue and salivary counterpart. Exclusion of metastasis from other primaries is mandatory and can only be achieved by detailed clinical history and imaging.

A novel loss-of-function mutation in LACC1 underlies hereditary juvenile arthritis with extended intra-familial phenotypic heterogeneity.

OBJECTIVE: To investigate phenotypic and molecular characteristics of a consanguineous family with autosomal-recessive, polyarticular, juvenile isiopathic arthriris (JIA) with extra-articular manifestations, including renal amyloidosis and Crohn's disease, associated with a novel homozygous truncating variant in LACC1. METHODS: Whole exome sequencing (WES) or targeted Sanger verification were performed in 15 participants. LACC1 expression and cytokine array were analysed in patient-derived and CRISPR/Cas9-generated LACC1-knockout macrophages (Mϕ). RESULTS: A homozygous truncating variant (p.Glu348Ter) in LACC1 was identified in three affected and one asymptomatic family member, and predicted harmful by causing premature stop of the LACC1 protein sequences, and by absence from ethnically-matched controls and public variation databases. Expression studies in patient-derived macrophages (Mϕ) showed no endogenous p.Glu348Ter-LACC1 RNA transcription or protein expression, compatible with nonsense-mediated mRNA decay. WES analysis in the asymptomatic homozygous subject for p. Glu348Ter-LACC1 detected an exclusive heterozygous variant (p.Arg928Gln) in complement component C5. Further complement activity analysis suggested a protective role for the p.Arg928Gln-C5 variant as a phenotypic modifier of LACC1-associated disease. Finally, cytokine profile analysis indicated increased levels of pro-inflammatory cytokines in LACC1-disrupted as compared with wild-type Mϕ. CONCLUSIONS: Our findings reinforce the role of LACC1 disruption in autosomal-recessive JIA, extend the clinical spectrum and intra-familial heterogeneity of the disease-associated phenotype, indicate a modulatory effect of complement factor C5 on phenotypic severity, and suggest an inhibitory role for wild-type LACC1 on pro-inflammatory pathways.

SATB2 is not a reliable diagnostic marker for distinguishing between oral osteosarcoma and fibro-osseous lesions of the jaws.

OBJECTIVE: Special AT-rich binding protein 2 (SATB2) is an immunohistochemical marker for osteoblast differentiation. Our aim was to investigate SATB2 expression in oral osteosarcoma and other bone-producing oral tumors/reactive lesions to evaluate its usefulness as a diagnostic marker. STUDY DESIGN: A total of 74 intraosseous and soft tissue bone-producing surgical samples and 10 samples of reactive bone tissue were stained with SATB2, including osteosarcoma/chondrosarcoma (n = 16), fibro-osseous lesions (n = 42), central giant cell granuloma (n = 6), osteoblastoma (n = 1), and gingival lesions (n = 9). Nuclear labeling of the stromal spindle cells and intensity of staining was scored and analyzed. RESULTS: The intraosseous (n = 65/65) and soft tissue samples (n = 9/9) diffusely expressed SATB2. The strongest expression was observed in juvenile aggressive ossifying fibroma (n = 2/2). Weak SATB2 expression was observed in the stromal spindle cells adjacent to reactive bone tissue (periosteal bone reaction). CONCLUSIONS: Our results indicate that SATB2 is not a reliable diagnostic marker for oral osteosarcoma but has practical use in detecting cells with osteoblast differentiation in histologic samples with scant bone production or in differentiating between a periosteal bone reaction and neoplastic bone induced by the tumor mesenchymal cells. Targeting SATB2 as an alternative therapy in oral osteosarcoma, fibro-osseous lesions, and central giant cell granuloma should be further investigated.

Clinicopathologic analysis of verrucous hyperplasia, verrucous carcinoma and squamous cell carcinoma as part of the clinicopathologic spectrum of oral proliferative verrucous leukoplakia: A literature review and analysis.

OBJECTIVE: Proliferative verrucous leukoplakia is classified as a potentially malignant disorder because of its high rate of malignant transformation. PVL progresses in a series of clinical stages where the early stage represents multiple, multifocal leukoplakias with a high recurrence rate. The intermediate and late stages are clinically exophytic lesion, diagnosed microscopically as verrucous hyperplasia that often progresses into verrucous carcinoma and/or squamous cell carcinoma. There is no single histologic definition and the diagnosis is retrospective following observed progression of the disorder. The goal of the current study was to conduct a literature review and analysis of PVL in the later stages to gain further knowledge on their clinicopathologic features. DATA SOURCES: Medline's PubMed and Google Scholar were searched for adequately documented cases from 1985 to 2018. References of published articles were searched for additional cases. REVIEW METHODS: Overall, 57 manuscripts were analyzed. 35/57 manuscripts provided adequate data on the clinicopathologic features in the premalignant and malignant stages. RESULTS: Malignant transformation rate was 50% (average of 57 months). Gingiva, palate and buccal mucosa were the most common locations. Clinicopathologic features included; well differentiated carcinoma (78%), perineural invasion (3%), lymph node metastasis (4%); distant metastasis (0%), average duration of illness (65 months), DOD-dead of disease (44%). Moderate dysplasia, severe dysplasia and carcinoma in situ were exceptionally uncommon in the premalignant stages (0.8%). CONCLUSION: Prognostic factors such as perineural invasion, lymph node metastasis and distant metastasis were uncommon occurrences which may have practical implications on treatment. Further studies are needed to substantiate our findings.

Three-step management of a newborn with a giant, highly vascularized, cervical teratoma: a case report.

BACKGROUND: A giant congenital cervical teratoma is often highly vascularized; thus, in addition to a life-threatening airway occlusion at birth it comprises a high risk for significant and lethal blood loss during resection. In the case presented, an endovascular embolization of the carotid artery that supplied a giant congenital cervical teratoma was done as part of a three-stage treatment soon after birth and contributed to an overall good outcome. Embolization in cases of cervical teratomas was not described previously. CASE PRESENTATION: We present a case of a preterm newborn from a Sephardic jewish origin with a giant, highly vascularized, congenital cervical teratoma that was managed successfully in three stages: (1) delivery by an ex utero intrapartum treatment procedure after extensive preoperative planning and followed by tracheostomy, (2) endovascular embolization of the carotid artery that supplied the tumor in order to decrease blood loss during resection, and (3) complete surgical resection. The parents were involved in all the ethical and medical decisions, starting just after the cervical mass was diagnosed prenatally. CONCLUSION: The management of giant congenital cervical teratoma is often challenging from both a medical and ethical prospective. Meticulous perinatal planning and parents' involvement is crucial. Endovascular embolization of the tumor feeding vessels can significantly improve the resection outcome and overall prognosis.

The Iron-Klotho-VDR Axis Is a Major Determinant of Proximal Convoluted Tubule Injury in Haptoglobin 2-2 Genotype Diabetic Nephropathy Patients and Mice.

The haptoglobin (Hp) genotype (1-1 and 2-2) is a major determinant of nephropathy progression in diabetes mellitus patients. Hp 2-2 diabetic mice have impaired Hb clearance and increased iron deposits and oxidative stress in the proximal tubules (PCT), leading to increased renal injury. However, the precise mechanism of the PCT injury in diabetic nephropathy (DN) remains elusive. In the kidney, 1,25(OH)2D3 suppresses the inflammatory response to renal tubular injury and requires normal renal expression of the α-klotho protein. In this study, we set out to test the hypothesis that the increased renal iron deposits in the PCT of Hp 2-2 DN affect the α-klotho-vitamin D receptor (VDR) axis and thereby exacerbates the PCT injury generated by the iron deposits. Immunohistochemical analysis of human and mouse kidney biopsies along with western blot analysis showed that the increased iron deposits in the PCT of the Hp 2-2 genotype were accompanied with significantly decreased α-klotho and VDR renal expression but significantly increased 1-α-hydroxylase renal expression. In conclusion, the iron-klotho-VDR axis is a major player in the mechanism contributing to iron-mediated PCT injury in diabetic Hp 2-2 mice and patients. Targeting this axis may open the way for new ideas regarding the pathogenesis and treatment of DN.

Characterization of Factors Affecting the Detection Limit of EGFR p.T790M in Circulating Tumor DNA.

OBJECTIVE: Circulating tumor DNA is a promising noninvasive tool for cancer monitoring. One of the challenges in applying this tool is the detection of low-frequency mutations. The detection limit of these mutations varies between different molecular methods. The aim of this study is to characterize the factors affecting the limit of detection for epidermal growth factor receptor p.T790M mutation in circulating tumor DNA of patients with lung adenocarcinoma. METHODS: DNA was extracted from plasma samples of 102 patients. For sequencing the DNA, we used 2 different next-generation sequencing-based platforms: Ion Torrent Personal Genome Machine (56 cases) and Roche/454 (46 cases). Serially diluted synthetic DNA samples carrying the p.T790M mutation were sequenced using the Ion Torrent Personal Genome Machine for validation. Limit of detection was determined through the analysis of non-hot-spot nonreference reads, which were regarded as sequencing artifacts. RESULTS: The frequency of the non-hot-spot nonreference reads was higher in Ion Torrent Personal Genome Machine compared to Roche/454 (0.07% ± 0.08% and 0.03% ± 0.06%, respectively, P < .001). We found that different base type substitutions occur with different frequency. Since the base substitution leading to p.T790M mutation is C>T transition, its frequency was used to determine the limit of detection for the assay. Based on the C>T non-hot-spot nonreference allele frequency, we found that the limit of detection is 0.18% in Ion Torrent Personal Genome Machine and 0.1% in Roche/454. Based on these values, 48% and 56% of the cases were positive for T790M mutation in Ion Torrent Personal Genome Machine and Roche/454 groups, respectively. Agreement between duplicates was 76% in Ion Torrent Personal Genome Machine and 72% in Roche/454. Using serially diluted synthetic DNA samples carrying the p.T790M mutation, we could identify mutations with allele frequency of 0.18% or more using the Ion Torrent Personal Genome Machine, supporting our approach to determine the detection limit. CONCLUSION: Both the sequencing platform and the specific nucleotide change affect the limit of detection and should therefore be determined in the validation process of new assays.

Mutant KRAS Circulating Tumor DNA Is an Accurate Tool for Pancreatic Cancer Monitoring.

BACKGROUND: Many new pancreatic cancer treatment combinations have been discovered in recent years, yet the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains grim. The advent of new treatments highlights the need for better monitoring tools for treatment response, to allow a timely switch between different therapeutic regimens. Circulating tumor DNA (ctDNA) is a tool for cancer detection and characterization with growing clinical use. However, currently, ctDNA is not used for monitoring treatment response. The high prevalence of KRAS hotspot mutations in PDAC suggests that mutant KRAS can be an efficient ctDNA marker for PDAC monitoring. SUBJECTS, MATERIALS, AND METHODS: Seventeen metastatic PDAC patients were recruited and serial plasma samples were collected. CtDNA was extracted from the plasma, and KRAS mutation analysis was performed using next-generation sequencing and correlated with serum CA19-9 levels, imaging, and survival. RESULTS: Plasma KRAS mutations were detected in 5/17 (29.4%) patients. KRAS ctDNA detection was associated with shorter survival (8 vs. 37.5 months). Our results show that, in ctDNA positive patients, ctDNA is at least comparable to CA19-9 as a marker for monitoring treatment response. Furthermore, the rate of ctDNA change was inversely correlated with survival. CONCLUSION: Our results confirm that mutant KRAS ctDNA detection in metastatic PDAC patients is a poor prognostic marker. Additionally, we were able to show that mutant KRAS ctDNA analysis can be used to monitor treatment response in PDAC patients and that ctDNA dynamics is associated with survival. We suggest that ctDNA analysis in metastatic PDAC patients is a readily available tool for disease monitoring. IMPLICATIONS FOR PRACTICE: Avoiding futile chemotherapy in metastatic pancreatic ductal adenocarcinoma (PDAC) patients by monitoring response to treatment is of utmost importance. A novel biomarker for monitoring treatment response in PDAC, using mutant KRAS circulating tumor DNA (ctDNA), is proposed. Results, although limited by small sample numbers, suggest that ctDNA can be an effective marker for disease monitoring and that ctDNA level over time is a better predictor of survival than the dynamics of the commonly used biomarker CA19-9. Therefore, ctDNA analysis can be a useful tool for monitoring PDAC treatment response. These results should be further validated in larger sample numbers.

The Role of Histopathology and Immunohistochemistry in the Diagnosis of Cutaneous Leishmaniasis Without "Discernible" Leishman-Donovan Bodies.

BACKGROUND: Histopathology plays an important role in the diagnosis of cutaneous leishmaniasis (CL) but Leishman-Donovan (LD) bodies may not always be discernible. Recently, anti-CD1a antibody (Ab), clone MTB1, was found to decorate LD bodies immunohistochemically. OBJECTIVE: Can histopathology without discernible LD bodies be used to diagnose CL, and can immunohistochemistry using anti-CD1a Ab, clone MTB1, detect LD bodies in these cases. METHODS: Suspected CL lesions were studied histopathologically and immunohistochemically, and the patients' clinical files were reviewed. RESULTS: Of the 196 patients with suspected CL, direct smear demonstrated LD bodies in 50 (25.5%). Of the remaining 146 patients, 118 underwent biopsy. In 56 (47.5%) patients, the hematoxylin-eosin-stained sections revealed LD bodies. In 47 (39.8%) patients, LD bodies were not discerned but the histopathology demonstrated histiocytic infiltrates with varying numbers of plasma cells along with other inflammatory cells, and negative Ziehl-Neelsen and periodic acid-Schiff stains. This pattern was termed "histopathology consistent with leishmaniasis." The history, clinical findings, and response to anti-leishmania therapy supported the diagnosis of CL in all of them, and immunostains for CD1a, clone MTB1, detected LD bodies in 11 (23.4%) of these 47 patients. CONCLUSIONS: "Histopathology consistent with CL" along with appropriate clinical findings supports the diagnosis of CL in an endemic area, and immunostains with CD1a Ab, clone MTB1, may help in the minority of the cases.

Clinically significant sub-clonality for common drivers can be detected in 26% of KRAS/EGFR mutated lung adenocarcinomas.

Genetic sub-clonality has been described in multiple malignancies, however the presence of sub-clonality for major drivers in lung adenocarcinoma and its clinical significance is a subject under debate. Using molecular and morphometric approach, 347 lung adenocarcinoma samples were analyzed for KRAS and EGFR sub-clonality, which was further correlated with clinical and pathological variables.KRAS and EGFR mutations were identified in 100 (29%) and 82 (23%) cases, respectively. One hundred and forty four KRAS or EGFR positive cases were also available for morphometric analysis, among which 37 (26%) were defined as sub-clonal. The presence of sub-clonality was associated with shorter survival time (p=0.02). Interestingly, cases with sub-clonality were also associated with earlier disease stage (89% vs 66% stage I disease in sub-clonal vs clonal cases, respectively, p=0.01) and less lymph node involvement (8% vs 25% in sub-clonal vs clonal cases, respectively, p=0.02). Our findings demonstrate the presence of sub-clonality for mutations in common drivers in lung adenocarcinoma and link it both to earlier disease stage and to poor survival. These findings are in line with the different evolutionary models that can present with genetic sub-clonality.

Risk for molecular contamination of tissue samples evaluated for targeted anti-cancer therapy.

With the increasing usage of sensitive PCR technology for pharmacogenetics, cross contamination becomes a significant concern. Researchers employed techniques which basically include replacing laboratory equipment after each sample preparation; however, there are no recommended guidelines. In the present work we wanted to evaluate the risk of cross contamination during tissue processing using the routine precaution measures. Twenty-one surgical samples of lung adenocarcinoma were used, of which 7 contained EGFR exon 19 mutation, 7 contained EGFR exon 21 mutation (p.L858R) and 7 were EGFR wild-type. The samples were ordered by alternating the mutation group to maximize the potential for cross contamination and underwent tissue sectioning and de-paraffinization. The entire process was performed using the same tools. Following DNA extraction all samples underwent PCR amplification and were scrutinized for small fractions of EGFR mutation using deep sequencing with the Ion torrent PGM technology. Twenty samples yielded results. The fraction of mutated copies was 41 ± 23% (range 11-66) for the cases with known exon 19 mutation and 48±24% (range 0-65) for the cases with known exon 21 mutations. No in-frame exon 19 deletion mutations were identified in the wild-type (WT) and exon 21 groups. The fraction of EGFR exon 21 (codon 858) mutations was 0.018±0.014% (range 0-0.05%) in the WT and exon 19 groups, which was not statistically different than the background sequencing artifact noise for the same base-pair alteration (p = 0.21). Our results suggest that standard precautions are sufficient for molecular pathology diagnosis of surgical samples and are not associated with increased risk of cross contamination.

Computerized cytometry and wavelet analysis of follicular lesions for detecting malignancy: A pilot study in thyroid cytology.

BACKGROUND: The cytologic diagnosis of indeterminate lesions of the thyroid involves much uncertainty, and the final diagnosis often requires operative resection. Computerized cytomorphometry and wavelets analysis were examined to evaluate their ability to better discriminate between benign and malignant lesions based on cytology slides. METHODS: Cytologic reports from patients who underwent thyroid operation in a single, tertiary referral center were retrieved. Patients with Bethesda III and IV lesions were divided according to their final histopathology. Cytomorphometry and wavelet analysis were performed on the digitized images of the cytology slides. RESULTS: Cytology slides of 40 patients were analyzed. Seven patients had a histologic diagnosis of follicular malignancy, 13 had follicular adenomas, and 20 had a benign goiter. Computerized cytomorphometry with a combination of descriptors of nuclear size, shape, and texture was able to predict quantitatively adenoma versus malignancy within the indeterminate group with 95% accuracy. An automated wavelets analysis with a neural network algorithm reached an accuracy of 96% in identifying correctly malignant vs. benign lesions based on cytology. CONCLUSION: Computerized analysis of cytology slides seems to be more accurate in defining indeterminate thyroid lesions compared with conventional cytologic analysis, which is based on visual characteristics on cytology as well as the expertise of the cytologist. This pilot study needs to be validated with a greater number of samples. Providing a successful validation, we believe that such methods carry promise for better patient treatment.

Cancer-associated fibroblasts are an infrequent finding in the microenvironment of proliferative verrucous leukoplakia-associated squamous cell carcinoma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are generally associated with negative prognostic factors. This study compares the clinicopathologic impact of CAFs in oral squamous cell carcinoma in patients with a history of proliferative verrucous leukoplakia (p-scca) and patients with conventional squamous cell carcinoma of the buccal mucosa, gingiva, and palate (c-scca). METHODS: A retrospective clinicopathologic and immunohistochemical analysis of 97 tumor specimens from 78 patients (13 patients with proliferative verrucous leukoplakia-associated squamous cell carcinoma (n = 32) and conventional squamous cell carcinoma from the buccal mucosa, gingiva, and palate (n = 65) was conducted. Immunostaining with anti-alpha-smooth muscle actin (α-SMA) antibody was used to evaluate the presence of CAFs. RESULTS: α-SMA expression was an infrequent finding in p-scca and seen in only 6% of p-scca compared to 40% of c-scca (P < 0.0004). In the c-scca subgroup, α-SMA significantly correlated with tumor size (T) (P = 0.009), tumor thickness (P < 0.0009), perineural invasion (P = 0.009), and microscopic grade (P = 0.018). CONCLUSIONS: The presence of CAFs was an infrequent finding in our p-scca cohort which may contribute to its seemingly slower growing and less invasive growth pattern. In the cohort of c-scca patients, higher levels of CAFs correlated with microscopic invasiveness, tumor size, and perineural invasion. Practically, these are important observations as targeting strategies are being developed to combat carcinoma types where CAFs significance has been validated.

The prognostic significance of heparanase expression in metastatic melanoma.

BACKGROUND: Heparanase expression is induced in many types of cancers, including melanoma, and promotes tumor growth, angiogenesis and metastasis. However, there is insufficient data regarding heparanase expression in the metastatic lesions that are the prime target for anti-cancer therapeutics. To that end, we examined heparanase expression in metastatic melanoma and its correlation with clinical parameters. RESULTS: Heparanase staining was detected in 88% of the samples, and was strong in 46%. For the entire cohort of metastatic melanoma patients, no apparent correlation was found between heparanase staining intensity and survival. However, in a sub group of 46 patients diagnosed as stage IVc melanoma, strong heparanase staining was associated with reduced survival rates [hazard ratio=2.1; 95%CI 1.1-4.1, p=0.025]. MATERIAL AND METHODS: Paraffin sections from 69 metastatic melanomas were subjected to immunohistochemical analysis, applying anti-heparanase antibody. The clinical and pathological data, together with heparanase staining intensity, were evaluated in a logistic regression model for site of metastasis and survival. Slides were also stained for the heparanase-homolog, heparanase-2 (Hpa2). CONCLUSIONS: Heparanase is highly expressed in metastatic melanoma and predicts poor survival of stage IVc melanoma patients, justifying the development and implementation of heparanase inhibitors as anti-cancer therapeutics.

Angiosarcoma arising in association with vascular Dacron grafts and orthopedic joint prostheses: clinicopathologic, immunohistochemical, and molecular study.

Angiosarcoma may rarely arise near an inert foreign body material including vascular grafts and metal joint prostheses. Sixteen such cases have been reported since 1972 but mostly in the radiologic or surgical literature without detailed histologic or molecular analyses. We herein describe the clinicopathologic and molecular features of 2 new cases and reanalyzed 3 previously reported cases of angiosarcoma that developed in association with Dacron grafts for vascular repair (n=3) or related to orthopedic metal prostheses for joint replacement (n=2). All patients were men aged 50 to 84 years (median, 71 years). Mean time to development of angiosarcoma was 9 years (range, 4.6-17 years). Symptoms were recurrent bleeding/loosening of prosthesis for suspected infection (in the joint prosthesis cases) and fatigue, weight loss, and abdominal symptoms in the Dacron-associated cases. Four patients died of disease within 1 to 24 months (mean, 8 months). One patient was alive after radical surgery, radiochemotherapy, and embolization of pulmonary metastases (17 months). Histologically, all tumors were high-grade epithelioid neoplasms with a predominant solid growth pattern and variable vasoformation. All tumors expressed CD31, ERG, FLI-1, and variably pancytokeratin (diffuse in 3 cases), but none expressed D2-40, MDM2, or CDK4. Fluorescence in situ hybridization analysis revealed no MDM2 or CDK4 alterations. MYC was expressed in all cases, but only 1 case was MYC amplified by fluorescence in situ hybridization. Angiosarcomas are exceedingly rare fatal complications of long-standing metal and Dacron prostheses. Awareness of their morphology and frequent cytokeratin expression is necessary to avoid misdiagnosis as metastatic carcinoma. Limited awareness of their existence explains delayed clinical diagnosis in most of cases. Absence of MDM2/CDK4 alterations underlines their distinction from intimal-type sarcomas.

Variation in KRAS driver substitution distributions between tumor types is determined by both mutation and natural selection.

Different tumor types vary greatly in their distribution of driver substitutions. Here, we analyzed how mutation and natural selection contribute to differences in the distribution of KRAS driver substitutions between lung, colon and pancreatic adenocarcinomas. We were able to demonstrate that both differences in mutation and differences in selection drive variation in the distribution of KRAS driver substitutions between tumor types. By accounting for the effects of mutation on the distribution of KRAS driver substitutions, we could identify specific KRAS driver substitutions that are more favored by selection in specific tumor types. Such driver substitutions likely improve fitness most when they occur within the context of the tumor type in which they are preferentially favored. Fitting with this, we found that driver substitutions that are more favored by natural selection in a specific type of tumor tend to associate with worse clinical outcomes specifically in that type of tumor.

Subclonality for BRAF Mutation in Papillary Thyroid Carcinoma Is Associated With Earlier Disease Stage.

CONTEXT: The presence of driver mutations only in a subset of tumor cells within a single lesion, defined as subclonality, is being appreciated as a clinically significant factor. BRAF mutation is the most common driver mutation in papillary thyroid carcinoma (PTC). There are conflicting data in the literature regarding the presence of BRAF mutation subclonality in PTC and its clinical significance. OBJECTIVE: The purpose of the present study was to use a molecular and morphometric approach to determine BRAF clonality status and its clinical-pathological correlates. DESIGN: Fifty-nine cases of PTC were studied. DNA extracted from the tumors underwent deep sequencing to determine the percentage of BRAF mutant allele copies. Additionally, we used computerized morphometry to determine the fraction of tumor cells in each sample. Using both variables, we were able to determine the presence or absence of subclonality for BRAF mutation, which was further correlated with clinical, pathological, and prognostic data. RESULTS: BRAF mutation was found in 49 (83%) cases. The average percentage of tumor cells and of BRAF mutant alleles in the samples were 68.1 ± 9.8 and 26 ± 6.7, respectively. Based on the molecular and morphometric analysis, 11 (24%) cases were found to be subclonal for BRAF mutation. Tumors with subclonal BRAF mutations were significantly smaller compared to tumors with clonal mutation (0.82 ± 0.38 cm vs 1.37 ± 0.57 cm, P = .005) and were less likely to have lymph node metastasis (0% vs 32%, P = .03). CONCLUSIONS: In PTC, subclonality for BRAF mutation is associated with earlier stage. Molecular-morphometric analysis of PTC can provide clonality information with potential clinical significance.

Intratumor Heterogeneity of KRAS Mutation Status in Pancreatic Ductal Adenocarcinoma Is Associated With Smaller Lesions.

OBJECTIVES: Recent studies have demonstrated intratumor heterogeneity (ITH) for genetic mutations in various tumors and suggest that ITH might have significant clinical impact. Because KRAS is the most commonly mutated oncogene in pancreatic ductal adenocarcinoma and has an important role in pancreatic carcinogenesis, the purpose of this study was to evaluate ITH for KRAS gene mutation and its clinical significance. METHODS: Deep sequencing of 47 pancreatic ductal adenocarcinoma cases was used to determine the fraction of KRAS mutant alleles. In addition, computerized morphometry was used to calculate the fraction of tumor cells. After analysis of both results, cases were classified as ITH or as having amplification of mutant KRAS. The presence of ITH was correlated with clinical and pathological factors. RESULTS: KRAS mutation was found in 38 (81%) cases, of which 12 (32%) showed ITH and 9 (23%) were found to have KRAS mutant allele amplification. The presence of ITH was associated with smaller tumors, whereas amplification was associated with higher tumor diameter. CONCLUSIONS: In pancreatic ductal adenocarcinoma, ITH for KRAS gene mutation was associated with smaller tumors. It is possible that, as the tumor progresses, more cells carry this mutation, which leads to more aggressive tumor features.