One-instrument, objective microsatellite instability analysis using high-resolution melt.

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers. The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type. The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status. The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance. Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors. Novel technologies have been applied and concepts such as tumor mutation burden have been introduced. However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator. In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue. The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles. The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study. The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

Cancer and Pregnancy: Estimates in Italy from Record-Linkage Procedures between Cancer Registries and the Hospital Discharge Database.

The aim of this study is to describe the frequency and trend of pregnancy-associated cancer (PAC) in Italy, an increasingly relevant phenomenon due to postponing age at childbirth. To this purpose, a population-based retrospective longitudinal study design based on cohorts of women aged 15-49 diagnosed with cancer and concomitant pregnancy is proposed. The study uses 19 population-based Cancer Registries, covering about 22% of Italy, and linked at an individual level with Hospital Discharge Records. A total of 2,861,437 pregnancies and 3559 PAC are identified from 74,165 women of the cohort with a rate of 1.24 PAC per 1000 pregnancies. The most frequent cancer site is breast (24.3%), followed by thyroid (23.9%) and melanoma (14.3%). The most frequent outcome is delivery (53.1%), followed by voluntary termination of pregnancy and spontaneous abortion (both 12.0%). The trend of PAC increased from 2003 to 2015, especially when the outcome is delivery, thus confirming a new attitude of clinicians to manage cancer throughout pregnancy. This represents the first attempt in Italy to describe PAC from Cancer Registries data; the methodology is applicable to other areas with the same data availability. Evidence from this study is addressed to clinicians for improving clinical management of women with PAC.

Detection of BRAF mutations in malignant melanoma and colorectal cancer by SensiScreen® FFPE BRAF qPCR assay.

Mutations in BRAF exon 15 lead to conformational changes in its activation loops, resulting in constitutively active BRAF proteins which are implicated in the development of several human cancer types. Different BRAF inhibitors have been developed and introduced in clinical practice. Identification of BRAF mutations influences the clinical evaluation, treatment, progression and for that reason a sensitive and specific identification of BRAF mutations is on request from the clinic. Here we present the SensiScreen® FFPE BRAF qPCR Assay that uses a novel real-time PCR-based method for BRAF mutation detection based on PentaBases proprietary DNA analogue technology designed to work on standard real-time PCR instruments. The SensiScreen® FFPE BRAF qPCR Assay displays high sensitivity, specificity, fast and easy-to-use. The SensiScreen® FFPE BRAF qPCR Assay was validated on two different FFPE tumour biopsy cohorts, one cohort included malignant melanoma patients previously analyzed by the Cobas® 4800 BRAF V600 Mutation Test, and one cohort from colorectal cancer patients previously analyzed by mutant-enriched PCR and direct sequencing. All BRAF mutant malignant melanoma patients were confirmed with the SensiScreen® FFPE BRAF qPCR Assay and additional four new mutations in the malignant melanoma cohort were identified. All the previously identified BRAF mutations in the colorectal cancer patients were confirmed, and additional three new mutations not identified with direct sequencing were detected. Also, one new BRAF mutation not previously identified with ME-PCR was found. Furthermore, the SensiScreen® FFPE BRAF qPCR Assay identified the specific change in the amino acid. The SensiScreen® FFPE BRAF qPCR Assay will contribute to a more specific, time and cost saving approach to better identify and characterize mutations in patients affected by cancer, and consequently permits a better BRAF characterization that is fundamental for therapy decision.

ABCB4 variants in adult patients with cholestatic disease are frequent and underdiagnosed.

BACKGROUND: Heterozygous ABCB4 variants are not routinely tested in adults with cholestasis because of their supposed rarity and high costs. METHODS: Nineteen adult patients presenting with unexplained cholestasis, and/or recurrent gallstones were included; genotyping was not done in five due to lack of health insurance approval. RESULTS: heterozygous ABCB4 variants were identified in seven patients, followed by cascade testing of 12 family members: one patient underwent liver transplantation at age 40 for end-stage liver disease; one had compensated cirrhosis; all symptomatic adults had gallstones, including four with low phospholipid-associated cholelithiasis; four had intrahepatic cholestasis of pregnancy; all children and one 54-year old female were asymptomatic. Genotype: Families A and C: c.2211G>A (p.Ala737=) combined with c.959C>T (p.Ser320Phe) in one subject; Family B: c.1130T>C (p.Ile377Thr); Family D: large deletion removing ABCB4 exons 1-4 plus ABCB1, RUNDC3B, SLC25A40, DBF4, ADAM22 exons 1-3; Family E: c.1565T>C (p.Phe522Ser) ; Family F: c.1356+2T>C combined with c.217C>G (p.Leu73Val). All patients responded to ursodeoxycholic acid. CONCLUSIONS: We found ABCB4 variants in half of the adults with unexplained cholestasis and/or recurrent gallstones presenting at our center, suggesting that this condition is underdiagnosed and undertreated, with serious consequences not only for the patients and their families, but also in terms of healthcare costs.

Primary biliary cholangitis with normal alkaline phosphatase: A neglected clinical entity challenging current guidelines.

BACKGROUND & AIM: The diagnosis of primary biliary cholangitis (PBC), an uncommon immune-mediated cholestatic liver disease, is based on positive circulating anti-mitochondrial (AMA) and/or PBC-specific anti-nuclear autoantibodies (ANA), coupled with elevated serum alkaline phopsphatase (ALP) levels. Timely initiation of treatment with ursodeoxycholic acid prevents progression to cirrhosis and liver failure. We aimed at investigating liver histology in patients with normal ALP level and positive AMA and/or PBC-specific ANA. METHODS: We searched the Swiss PBC Cohort Study database, which includes subjects with positive PBC autoimmune serology and normal ALP levels, for patients who underwent a liver biopsy. Histological slides were centrally reviewed by an expert liver pathologist, and sera were centrally re-tested for AMA and ANA. RESULTS: 30 patients were included; 90% females, median age 53 (range 27-72) years. Twenty-four (80%) had liver histology typical for (n = 2), consistent with (n = 16) or suggestive of (n = 6) PBC, including three of four AMA-negative ANA-positive patients. Among 22 ursodeoxycholic acid treated patients, 14 had elevated GGT levels before treatment; a significant decrease of the median GGT level between pre- (1.46 x ULN) and post- (0.43 x ULN) treatment (p = 0.0018) was observed. CONCLUSIONS: In our series, a high proportion of AMA positive patients with normal ALP levels have PBC. For the first time we show histological diagnosis of PBC in AMA-negative/PBC-specific ANA-positive subjects and the potential role of GGT as a biomarker in PBC patients with normal baseline ALP levels. Current guidelines for the diagnosis of PBC do not cover the whole extent of PBC presentation, with important clinical implications in terms of timely treatment initiation.

Risk of Malignancy (ROM) of Thyroid FNA Diagnosed as Suspicious for Malignancy or Malignant: an Institutional Experience with Systematic Review and Meta-Analysis of Literature.

A universal recommendation does not exist for thyroid FNA suspicious for malignancy (SFM). In this context, the guidelines have estimated a risk of malignancy (ROM) from 50 to 80% and both total thyroidectomy and lobectomy may be indicated. This study aimed to (1) retrospectively evaluate the SFM (i.e., TIR4) in a single institution to estimate their cancer prevalence at histology, and (2) systematic review the literature to obtain more robust information. The study period was 2015-2018. As a major inclusion criterion, both cytology and histology had to be performed in our institution. Histological diagnosis was the gold standard. For the systematic review, the online databases of Google Scholar, PubMed/MEDLINE, and Scopus were searched for papers using the same classification for thyroid FNA. A proportion meta-analysis was performed to obtain the pooled histological cancer rate among TIR4 and TIR5 (random-effects model). In the institutional database, there were 271 nodules with both histology and FNA and the cancer rate of TIR4 was 88.9%. By systematic review, five studies were selected for the meta-analysis. The pooled cancer rate was 85% in TIR4 and 99% in TIR5 (I2 = 0%; no publication bias). In conclusion, these new findings should prompt the guidelines board to fully revise the estimated ROM of SFM category. Clinical thyroidologists and thyroid surgeons should be aware of these data and the patients with SFM should be informed of their ROM.

Histologic Outcome of Indeterminate Thyroid Nodules Classified at Low or High Risk.

The revised Italian consensus for the classification and reporting of thyroid cytology (ICCRTC) was published in 2014. Very recently, a high reliability of ICCRTC in classifying low and high risk indeterminate nodules (Tir 3A and Tir 3B, respectively) was demonstrated. This finding prompted us to review our case series of thyroid indeterminate lesions to verify these data. Only lesions undergone FNAC from December 2014 to October 2017 with subsequent histology at our institutions were eligible for the study. All cytologic samples had originally been classified according to ICCRTC in the subcategories of indeterminate lesion, such as Tir 3A and Tir 3B by three cytopathologists and another one with more than 10 years experience, when necessary. Sixty-three indeterminate FNAC were diagnosed during the study period, of which 51 were subsequently surgically treated. Overall, 9 carcinomas (7 follicular and 2 papillary) and 42 benign lesions were found at histology. The cancer rate observed in the Tir 3A category (3/40, 7.5%) was significantly (p = 0.0015) lower than that found in Tir 3B (6/11, 54.5%). No significant differences were found in age and size of the sampled nodules between the two subcategories. We can confirm in our series that the Italian reporting system for thyroid cytology shows high reliability in discriminating low risk indeterminate lesions from those at high risk of malignancy.

Deregulation of sialidases in human normal and tumor tissues.

BACKGROUND: Aberrant sialylation is a characteristic feature associated with cancer. The four types of mammalian sialidases identified to date have been shown to behave in different manners during carcinogenesis. While NEU1, NEU2 and NEU4 have been observed to oppose malignant phenotypes, the membrane-bound sialidase NEU3 was revealed to promote cancer progression. OBJECTIVES: With the aim of improving the knowledge about sialidases deregulation in various cancer types, we investigated the amount of NEU1, NEU3 and NEU4 transcripts in paired normal and tumor tissues from 170 patients with 11 cancer types. METHODS: mRNA was extracted from patients' tissue specimens and retrotranscribed into cDNA, which was quantified by Real-Time PCR. RESULTS: We found NEU1 and NEU3 to be up regulated, while NEU4 was down regulated in most cancer types. In particular, colorectal cancer tissues showed the highest increase in NEU3 expression. Both NEU1 and NEU3 showed a strong up-regulation in ovarian cancer. CONCLUSIONS: Our data show that human sialidases are expressed at different levels in healthy tissues and are strongly deregulated in tumors. Moreover, sialidases expression in our European cohort showed significant differences from Asian populations. Some of these peculiar features open potential applications of sialidases in cancer diagnosis and therapy.

SensiScreen®KRAS exon 2-sensitive simplex and multiplex real-time PCR-based assays for detection of KRAS exon 2 mutations.

Activating mutations in codon 12 and codon 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) gene are implicated in the development of several human cancer types and influence their clinical evaluation, treatment and prognosis. Numerous different methods for KRAS genotyping are currently available displaying a wide range of sensitivities, time to answer and requirements for laboratory equipment and user skills. Here we present SensiScreen® KRAS exon 2 simplex and multiplex CE IVD assays, that use a novel real-time PCR-based method for KRAS mutation detection based on PentaBase's proprietary DNA analogue technology and designed to work on standard real-time PCR instruments. By means of the included BaseBlocker™ technology, we show that SensiScreen® specifically amplifies the mutated alleles of interest with no or highly subdued amplification of the wild type allele. Furthermore, serial dilutions of mutant DNA in a wild type background demonstrate that all SensiScreen® assays display a limit of detection that falls within the range of 0.25-1%. Finally, in three different colorectal cancer patient populations, SensiScreen® assays confirmed the KRAS genotype previously determined by commonly used methods for KRAS mutation testing, and notably, in two of the populations, SensiScreen® identified additional mutant positive cases not detected by common methods.

Pure intraductal carcinoma of the parotid gland: Cytologic findings on FNA sample. Report of one case.

One case of intraductal carcinoma of the parotid gland in a 67-year-old male patient is here introduced. The patient, who had a one-year history of a parotid mass, had undergone ultrasound and MRI examination that disclosed a 13x4x3 mm well delimited nodular mass of the accessory lobe of his left parotid gland. Ultrasound-guided Fine Needle Aspiration (FNA) had been performed by the clinician. The obtained smears showed widespread cellular necrosis in which cellular clusters with moderate and focally severe atypias displayed papillary and cribriform architecture and were admixed with sheets of epithelial cells with less striking nuclear atypias, squamous, or apocrine metaplasia. Histopathological examination disclosed a pure intraductal carcinoma of the parotid gland with classical morphology, which was radically excised. The differential cytological diagnosis of pure intraductal carcinoma of salivary glands may be difficult and comprises mucoepidermoid carcinoma as well as "in situ" carcinomas developping in the context of sclerosing polycystic adenosis, mammary analogue secretory carcinoma (MASC) of the salivary glands and cystic variants of salivary adenocarcinoma NOS (formerly called cystadenocarcinomas).

ALK testing in lung adenocarcinoma: technical aspects to improve FISH evaluation in daily practice.

INTRODUCTION: Anaplastic lymphoma kinase (ALK) gene rearrangement characterizes a subgroup of patients with lung adenocarcinoma who may benefit from ALK inhibitors. Fluorescence in situ hybridization (FISH) with a break-apart/split-signal strategy is the gold standard to investigate ALK. The cutoff to define ALK positivity has been settled at 15% or greater. A subset of patients has ALK borderline status, showing 15% ± 5% positive cells. Several aspects, both biological and technical, might influence signals evaluation, making FISH interpretation a challenging task. To improve ALK evaluation, we classified the different FISH patterns on the basis of the type of the split signals, namely short, long, far away, and deleted. METHODS: We investigated ALK gene status by FISH in 244 lung adenocarcinomas and in a series of ALK negative cell lines samples, collected in three Institutions. RESULTS: ALK positive profile was found in 12% of patients; long, deleted, and far-away splits were the primary patterns observed. ALK borderline profile characterized 10% of samples; long and deleted splits were significantly more frequent in those borderline finally classified as ALK positive, whereas short split were mostly detected in those borderline patients finally classified as ALK negative (p = 3.4 × 10). In the ALK negative control series, short split was the predominant pattern. Concordance was observed among different operators and probes for both samples and controls. CONCLUSIONS: Difficulties in ALK FISH signal interpretation might be bypassed using this detailed scoring system, which is highly reproducible, helps clarify borderline samples (according to split type), and provides experimental evidence that 15% is a reasonable cutoff to overcome the assay-dependent background noise.