Rosanna Mezzapelle

Epidermal Growth Factor Receptor Gene Analysis With a Highly Sensitive Molecular Assay in Routine Cytologic Specimens of Lung Adenocarcinoma

Epidermal growth factor receptor (EGFR) gene mutational analysis is critical for guiding the treatment of lung adenocarcinoma. In everyday clinical practice, EGFR testing is frequently centralized in referral laboratories that may receive paucicellular cytologic specimens, often fixed in various ways. We conducted a search for EGFR mutations in 108 cytologic samples of lung adenocarcinoma from different hospitals using the TheraScreen EGFR29 kit. These samples included 80 (74.1%) fine-needle aspirations, 13 (12%) pleural/ascitic fluids, 13 (12%) bronchial washings, and 2 bronchial brushings. The samples were fixed in ethanol (n = 79), Duboscq-Brasil (n = 18) or formalin (n = 10); 1 was unfixed. Ninety-two (85.2%) were amplified, 16 (14.8%) were not. Mutations were detected in 22 (23.9%) of 92 amplified samples, 9 containing less than 200 cancer cells, and 4 with less than 50% cancer cells. DNA was amplified in 12 of 18 Duboscq-Brasil-fixed samples. These findings indicate that cytologic specimens are adequate for EGFR testing when a highly sensitive assay is used, even if they are paucicellular or not optimally fixed.

Mutation analysis of the EGFR gene and downstream signalling pathway in histologic samples of malignant pleural mesothelioma.

Background:As epidermal growth factor receptor (EGFR) is involved in the pathogenesis of malignant pleural mesotheliomas (MPMs), the anti-EGFR drugs may be effective in treating MPM patients. Mutations of the EGFR gene or its downstream effectors may cause constitutive activation leading to cell proliferation, and the inhibition of apoptosis and metastases. Consequently, molecular profiling is essential for select patients with MPM who may respond to anti-EGFR therapies.Methods:After manual macrodissection, genomic DNA was extracted from 77 histological samples of MPM: 59 epithelioid, 10 biphasic, and 8 sarcomatoid. Epidermal growth factor receptor gene mutations were sought by means of real-time polymerase chain reaction (PCR) and direct sequencing, KRAS gene mutations by mutant-enriched PCR, and PIK3CA and BRAF gene mutations by direct sequencing.Results:Gene mutations were identified in nine cases (12%): five KRAS, three BRAF, and one PI3KCA mutation; no EGFR gene mutations were detected. There was no difference in disease-specific survival between the patients with or without gene mutations (P=0.552).Conclusions:Mutations in EGFR downstream pathways are not rare in MPM. Although none of those found in this study seemed to be prognostically significant, they may support a more specific selection of patients for future trials.

High-mobility group box 1 protein orchestrates responses to tissue damage via inflammation, innate and adaptive immunity, and tissue repair

A single protein, HMGB1, directs the triggering of inflammation, innate and adaptive immune responses, and tissue healing after damage. HMGB1 is the best characterized damage‐associated molecular pattern (DAMP), proteins that are normally inside the cell but are released after cell death, and allow the immune system to distinguish between antigens that are dangerous or not. Notably, cells undergoing severe stress actively secrete HMGB1 via a dedicated secretion pathway: HMGB1 is relocated from the nucleus to the cytoplasm and then to secretory lysosomes or directly to the extracellular space. Extracellular HMGB1 (either released or secreted) triggers inflammation and adaptive immunological responses by switching among multiple oxidation states, which direct the mutually exclusive choices of different binding partners and receptors. Immune cells are first recruited to the damaged tissue and then activated; thereafter, HMGB1 supports tissue repair and healing, by coordinating the switch of macrophages to a tissue‐healing phenotype, activation and proliferation of stem cells, and neoangiogenesis. Inevitably, HMGB1 also orchestrates the support of stressed but illegitimate tissues: tumors. Concomitantly, HMGB1 enhances the immunogenicity of mutated proteins in the tumor (neoantigens), promoting anti‐tumor responses and immunological memory. Tweaking the activities of HMGB1 in inflammation, immune responses and tissue repair could bring large rewards in the therapy of multiple medical conditions, including cancer.

KRAS mutational analysis in ductal adenocarcinoma of the pancreas and its clinical significance

Mutations of KRAS are detectable in 70-90% of pancreatic duct adenocarcinomas (PDAC), using direct sequencing. We used a highly sensitive molecular method in order to investigate: (a) the frequency and prognostic significance of different KRAS mutations and, (b) whether the presence of KRAS mutations in histologically-negative resection margins of PDAC could explain local tumor recurrence after surgery. Twenty-seven patients with histologic diagnosis of PDAC, radical pancreaticoduodenectomy and histologically-negative margins were evaluated. KRAS mutations were searched for mutant-enriched PCR in tumor and negative resection margins. KRAS mutations were detected in 85.2% of the cases; the most frequent mutation was G12D (48.1%). Shorter OS was found in patients with G12D (25 months; 95% CI, 20.5-29.5), vs patients with other mutations (31.5 months; 95% CI, 25.6-37.1) (N.S.). KRAS mutation in histologically-negative margins was detected in one patient who died of locoregional recurrence; six patients had tumor recurrence but no mutations in surgical margins. The high frequency of KRAS mutations suggests a search for KRAS status to improve the diagnosis in suspected cases; the G12D mutation could be related to poor prognosis, but without statistical significance. No correlation was found between the frequency of cancer recurrence and KRAS mutations in surgical margins.

Mutation analysis of KRAS in primary colorectal cancer and matched metastases by means of highly sensitivity molecular assay

Mutation analysis of KRAS is needed before starting treatment with anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer (CRC). In most of the cases, testing is performed on primary tumors, assuming that KRAS mutation status does not change in metastasis although correlation studies gave conflicting results. We evaluated the KRAS status concordance rate between primary tumors and related metastasis using a highly sensitive molecular assay. Forty-five primary tumors and related metastases from patients with CRC (28/45 male-62.2% and 17/45 female-37.8%; mean age 66.4 years) were analyzed by using TheraScreen: KRAS mutational kit. Metastatic samples were collected from lymph nodes (8/45-17.8%) and visceral sites (37/45-82.2%); 23 were synchronous (49%) and 22 were metachronous (51%), obtained after a mean of 30.8 months after the first diagnosis of CRC. Twenty-eight patients had KRAS mutations in both primary CRC and related metastases (62.2%). No differences in type and frequency of mutations were identified, despite different metastatic sites and time of onset of metastatic disease. Our results indicate that the mutation status of KRAS is the same in primary CRC and metastasis, suggesting that in clinical practice, KRAS testing can be performed on both tumor tissues when using a highly sensitive molecular assay.

Extra-osseous Ewing sarcoma of the thyroid gland mimicking lymphoma recurrence: A case report

Extra-osseous Ewing sarcomas/peripheral primitive neuroectodermal tumors (EOES/pPNETs) are high-grade malignant tumors found in various organs, such as the lung, skin, intestine, kidney and female genital tract; however, to the best of our knowledge, only two cases have previously been identified in the thyroid gland. We describe a case of primary EOES/PNET of the thyroid gland in a 66-year-old man with a previous history of large B cell lymphoma. During a routine follow-up examination, the patient underwent an ultrasound cervical scan showing a solid nodule of the left thyroid lobe. The fine-needle aspiration biopsy of the nodule suggested a neuroendocrine tumor. Histological and immunohistochemical examination of the surgical specimen supported a diagnosis of EOES/PNET, which was further confirmed by the demonstration of EWSR1 gene translocation by means of fluorescent in situ hybridization and by the detection of glycogen particles and neurosecretory granules by means of electron microscopy. Total body computed tomography and magnetic resonance imaging excluded the involvement of other sites, and therefore a diagnosis of primary EOES/PNET of the thyroid gland was made.This paper also discusses the main differential diagnoses, including lymphoma recurrence, other small round cell tumors (primary or metastatic), and a thyroid localization of an EWS/PNET from another organ.

Frequency of O6‐methylguanine‐DNA methyltransferase promoter methylation in cytological samples from small cell lung cancer

Background. In a phase II study for patients with relapsed small cell lung cancer (SCLC), the administration of Temozolomide, an alkylating agent used in gliomas and anaplastic astrocytoma, showed a effective activity when O6‐methylguanine‐DNA methyltransferase (MGMT) gene promoter was methylated. Methods. We tested the feasibility of MGMT promoter status evaluation in small biopsies and cytological specimens routinely processed for diagnostic purposes. We tested samples from 56 patients with SCLC: 30 tissue biopsies, 17 fine‐needle aspiration biopsy, 8 bronchial washing, and 1 was a sputum. Biopsies and fine‐needle aspiration biopsy were fixed in formalin, bronchial washing and sputum in Dubosq Brazil. DNA was extracted after macrodissection of the areas containing the maximum number of cancer cells. MGMT promoter methylation status was assessed by methylation specific PCR. Results. Methylation analysis was obtained in 54 samples (54/56) and failed in two bronchial wash. MGMT promoter was methylated in 35.2% of the cases without any significant difference between histological and cytological samples (37.9% vs. 32%). Conclusion. MGMT promoter methylation is present in SCLC and cytological samples are perfectly adequate for methylation analysis, even if they were taken during routine diagnostic procedures, using different fixative and with low number and percentage of cancer cells. Diagn. Cytopathol. 2015;43:947–952.

192PPROGNOSTIC BIOMARKERS IN MALIGNANT PLEURAL MESOTHELIOMA

ABSTRACT Aim: The standard of care for malignant pleural mesothelioma (MPM) includes combination of platinum compounds and pemetrexed. Despite this, the median survival of MPM patients is still bad (12 months). Excision Repair Cross Complementing Group 1 (ERCC1) acts removing DNA adducts formed by platinum compounds, whereas Pemetrexed inhibits Thymidylate Synthase (TS), a folic enzyme also involved in ex novo synthesis of DNA. This study aims to analyse retrospectively, in a large series of MPM specimens, the ERCC1 and TS gene and protein expression to investigate their prognostic and predictive role. Methods: Pleural biopsies were collected from140 consecutive MPM patients (98 males and 42 females, mean age 68 years, range 27-90). Histologic subtypes were: epithelioid (#103, 73%), sarcomatoid (#19, 14%) and biphasic (#18, 13%). At the end of the study 113 patients were died (average disease specific survival (DSS) 13 months; range 1-60); mRNA was extracted from formalin fixed paraffin embedded blocks and reverse transcribed in cDNA to perform gene expression assay by qRT-PCR. Immunohistochemistry was performed by anti-ERCC1 and anti-TS mAbs and the results were scored from 0 to 3 depending on the percentage of positive tumour cells and staining intensity. The results were statistically correlated with the clinical features and with the DSS of the patients respectively by Fishers exact test and Logrank test. Results: Of 140 patients 102 received platinum and pemetrexed (in single or in combination), the remaining were untreated due to bad performance status, advanced age or refusing therapy. Statistically significant correlation was found between the lack of protein expression of ERCC1 and DSS in the whole cohort (P = .03). Low levels of TS mRNA were significantly correlated with the DSS in the whole cohort (P = .01) and in the untreated subgroup (including patients not receiving pemetrexed) (P = .04), whereas did not in the subgroup of pemetrexed treated patients Conclusions: ERCC1 protein expression, scored by IHC, and TS gene expression, evaluated by mRNA analysis, seem both important prognostic markers; we suggest their evaluation in routine biopsies from patients with MPM. Disclosure: All authors have declared no conflicts of interest.