Discovery of methylated circulating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer.

OBJECTIVE: Mutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC). DESIGN: Genome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci (EYA4, GRIA4, ITGA4, MAP3K14-AS1, MSC). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy. RESULTS: Methylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for EYA4, 68.5% for GRIA4, 69.7% for ITGA4, 69.1% for MAP3K14-AS1% and 65.1% for MSC. Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival. CONCLUSION: This five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.

Antibodies are forever: a study using 12-26-year-old expired antibodies.

AIMS: The aim of this study was to investigate whether the shelf-life of diagnostic antibodies is longer than the expiry date on the label. METHODS AND RESULTS: Four independent laboratories tested a small number of diagnostic antibodies kept at +4°C for 12-26 years, and found them to work perfectly on routine histology sections. CONCLUSIONS: Diagnostic antibodies may have a workable half-life in excess of 10 years, and the emphasis on performance should shift to the preservation of antigenic targets in the tissue.

Pathology of ANCA-associated vasculitis.

According to the recent 2012 Chapel Hill Consensus Conference, ANCA-associated vasculitis is classified in the group of small vessel vasculitis. Pauci-immune necrotizing crescentic glomerulonephritis is the morphological hallmark of ANCA-associated vasculitis. Crescentic damage of the glomerular tuft is characterized by macrophage accumulation through vascular cell adhesion molecule-1 (VCAM-1) activation. Macrophages have a key role in chronic progression of renal damage due to production of substances involved in matrix remodelling [transforming growth factor-beta (TGFß)]. Diffuse interstitial infiltration of T and B lymphocytes and macrophages is another frequent morphological feature. Tubulitis is considered an important marker of worse prognosis. Unfortunately, the renal changes on long-term follow-up are largely unknown due to the small number of studies with repeat biopsy in these disorders. Although a standardized score for renal biopsies was developed previously, a final histopathologic classification is still lacking. The European Vasculitis Study Group (EUVAS) proposed a classification system based on glomerular pathology as assessed by light microscopy.

Extramedullary hemopoiesis and littoral cell angioma of the spleen: our experience and review.

Littoral cell angioma (LCA) is a rare primary vascular neoplasm of the spleen. A 54-year-old man was referred to our emergency department for abdominal pain. A CT scan showed multiple round hypodense lesions of various sizes throughout the spleen. The spleen was increased in volume. An MRI confirmed the lesion with a suspect of multiple angiomas vs. amartomas. The haematologists excluded any haematological disease. After a collegial discussion, we decided to perform laparotomic splenectomy. Histologically, the multiple lesions consisted in anastomosing vascular channels lined by plump cells. There was an increased number of dysmorphic megakaryocites inside the splenic parenchyma and along the tumour's border, known signs of extramedullary hemopoiesis, whose etiology in our patient was unexplained. To the best of our knowledge this is the third description of the association between littoral cell angioma and extramedullary hemopoiesis. LCA is a rare primary splenic vascular tumour that originates from the splenic littoral cells. The diagnosis of littoral cell angioma is confirmed histologically and on immunohistochemistry. This case report underlines the rarity of this type of benign splenic neoplams, but since the malignant potential of LCA, we recommend close clinical follow- up of patients with LCA of the spleen.

Small Bowel Intussusception Due to Rare Cardiac Intimal Sarcoma Metastasis: A Case Report.

Background: Intimal sarcomas are rare malignant mesenchymal tumors arising from the heart and large blood vessels. Their intraluminal growth leads to vascular obstructive symptoms and peripheral neoplastic embolization. Direct infiltration of the lungs or metastases to the pulmonary system, occur in 40% of cases and extrathoracic spread is frequent, also in presentation. Intussusception is an unusual event in adults, accounting for <5% of bowel obstructions. In most cases it is caused by a malignancy and requires surgical resection. Case Presentation: We describe a rare case of a 50-year-old man suffering of bowel obstruction due to intussusception sustained by a small bowel metastasis of a primary cardiac intimal sarcoma. One year and a half before the onset of abdominal symptoms, a grade II intimal sarcoma was removed from his left atrium and consequently he followed a chemotherapy protocol. Four months later a CT scan revealed local recurrence. Eighteen months after heart surgery he referred to the ER with abdominal pain. CT scan showed an ileal intussusception and the patient was scheduled for surgery. A tract of 10 cm ileus was removed containing an intramural polypoid solid mass. Histological analyses revealed a grade II intimal sarcoma consistent with his first diagnosis. Conclusion: Primary heart tumors are late found and often partially resected, therefore metastatic pathways are to be expected. Adult small bowel intussusception is a rare event and caused by a malignancy in one third of cases. Therefore, our recommendation is to always resect the tract involved in order to perform a proper diagnosis.

Radiologic and Genomic Evolution of Individual Metastases during HER2 Blockade in Colorectal Cancer.

Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated.

Cytomorphologic and molecular features of hobnail variant of papillary thyroid carcinoma: case series and literature review.

Recent reports indicate that hobnail papillary thyroid carcinoma (HPTC) is a rare, but very aggressive variant of papillary thyroid carcinoma. The authors describe the cytological features of five HPTC on fine-needle aspiration biopsies (FNAB). Moreover, their immunophenotype and the presence of B-RAF mutation by pyrosequencing were investigated. The patients' (three females and two males) age ranged from 27 to 86 (mean 65) years. Tumor size ranged from 2 to 9 cm (mean 4.2 cm). FNAB were highly cellular with a bloody background and scant colloid. The cells were arranged in papillary-like clusters or in micropapillary groups. The cell population consisted of medium-sized cells with "tear-drop" cytoplasm, apically placed nuclei that produced a surface bulge leading to a hobnail appearance. At higher magnification, nuclei showed variable degrees of atypia, occasional pink intranuclear pseudoinclusions, and grooves. Nuclear stratification and atypical mitotic figures were usually present. Immunocytochemistry revealed positive staining for thyroglobulin, thyroid transcriptor factor-1, Hector Battifora Mesothelial Antigen-1, partial loss of E-cadherin expression, and nuclear expression of p53 protein. B-RAF mutation was present in three out of five cytological cases. Immunohistochemical and molecular results were confirmed on histological sections. Recognizing the unique cytological features of HPTC should help to avoid misdiagnosis of this rare variant.

[Pathology of the ANCA-associated vasculitides]. / Patologia delle vasculiti ANCA-associate.

The international guidelines emphasize the crucial role of renal biopsy in the diagnosis of Anti-Neutrophil Cytoplasmic Autoantibody (ANCA)- associated glomerulonephritis (Microscopic Polyangiitis, Granulomatosis with Polyangiitis and Eosinophilic Granulomatosis with Polyangiitis). 1) According to the recent 2012 Chapel Hill Consensus Conference, ANCA- associated vasculitides are classified in the group of small vessel vasculitis. 2) Pauci-immune necrotizing crescentic glomerulonephritis is the morphological hallmark of ANCA-associated vasculitis. The lesion can vary widely in severity and extent of the damage, from segmental tuft necrosis to massive circumferential crescents and frequent peri-glomerular granulomatous reaction. 3) The ANCA test is highly specific for these types of autoimmune vasculitides but renal biopsy still remains the gold standard for final diagnosis, prognostic evaluation and therapeutic intervention, although discrepancy between clinical and morphological features is frequently found. 4) Crescentic damage of the glomerular tuft is characterized by monocyte.- macrophage accumulation through Vascular Cell Adhesion Molecule-1 (VCAM-1) activation. Activated macrophages are considered to have a key role in the chronic progression of renal damage due to their ability to produce substances directly involved in matrix remodelling (Tumor Growth Factor b). 5) Diffuse and marked interstitial infiltration of T, B lymphocytes and monocyte- macrophages is another frequent morphological feature with aspects of tubulitis recently being considered important markers for a worse prognosis. 6) Unfortunately, repeat biopsies are infrequently performed in these disorders and long-term renal changes have remained largely unidentified. 7) Despite the formulation of standardized scoring for renal biopsies, definitive histopathologic classification is still lacking. The European Vasculitis Study (EUVAS) group has proposed a classification system based on glomerular pathology as assessed by light microscopy which is, in turn, divided into four categories: focal, crescentic, sclerotic and mixed. A preliminary correlation with clinical features in 100 cases of ANCA-associated vasculitis has demonstrated that renal biopsy categories were the only independent predictors for the estimated glomerular flow rate (eGFR). The international study currently way is being evaluated by EUVAS for possible confirmation of the classification.