KRAS testing in metastatic colorectal carcinoma: challenges, controversies, breakthroughs, and beyond

Metastatic colorectal cancer harboring a mutation in codon 12 or 13 of the KRAS gene does not benefit from therapy with antibodies targeting the epidermal growth factor receptor (EGFR). The implementation of community KRAS testing is generating a rapid flow of new data that have implications for the pathologist and testing guidelines, besides the physician. Therefore, it seems timely to draw together the threads of this large body of information in order that pathologists can be knowledgeable partners in the multidisciplinary process of targeted cancer therapy, and to help refine current testing guidelines. This review addresses: (1) the most relevant methodological and technical aspects of KRAS testing in terms of sample site (primary/metastatic), test specimens (resection/biopsy/cytology), and the diverse molecular methods available; (2) issues related to daily practice, namely the timing of the test, its turnaround time and the quality control procedures; and (3) the evidence related to the relationship between KRAS genetic intratumoral heterogeneity, clinical sensitivity of mutational detection tools and anti-EGFR treatment outcome. Hopefully, in the near future, elucidation of the potential of biomarker panels, and of the mechanisms underlying primary and acquired resistance to anti-EGFR therapy will refine even further personalized treatment regimes for patients with metastatic colorectal cancer

Approach to cytological indeterminate thyroid nodules.

The indeterminate thyroid nodules diagnosed by fine-needle aspiration cytology (FNAC)represents a problem for both cytopathologists and clinicians. The former sometimes use this diagnostic category as a sort of basket, putting in cases that they do not know exactly how to classify. The latter are faced with a highly variable risk of malignancy and consequently the management remains a challenge. On the histopathological side, the new WHO classification of tumors of the thyroid introduced the concept of tumors with uncertain and low malignant potential, and the concept of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), whose prognosis and management are still to be completely elucidated. While the risk of malignancy of the indeterminate diagnostic category has decreased due to the re-classification of certain types of papillary thyroid carcinomas of the follicular variant into a low malignant potential form (the NIFTP), cases diagnosed cytologically as indeterminate will probably increase in the future to avoid false positive diagnosis. Thus, the indeterminate thyroid diagnostic category still remains a challenge, both at the diagnostic level and for its management. The new version of the Bethesda system for reporting thyroid cytopathology suggests managing these patients with a repeat FNA, diagnostic lobectomy and/or molecular testing

Spatially Resolved Molecular Approaches for the Characterisation of Non-Invasive Follicular Tumours with Papillary-like Features (NIFTPs)

Noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) are low-risk thyroid lesions most often characterised by RAS-type mutations. The histological diagnosis may be challenging, and even immunohistochemistry and molecular approaches have not yet provided conclusive solutions. This study characterises a set of NIFTPs by Matrix-Assisted Laser Desorption/Ionisation (MALDI)-Mass Spectrometry Imaging (MSI) to highlight the proteomic signatures capable of overcoming histological challenges. Archived formalin-fixed paraffin-embedded samples from 10 NIFTPs (n = 6 RAS-mutated and n = 4 RAS-wild type) were trypsin-digested and analysed by MALDI-MSI, comparing their profiles to normal tissue and synchronous benign nodules. This allowed the definition of a four-peptide signature able to distinguish RAS-mutant from wild-type cases, the latter showing proteomic similarities to hyperplastic nodules. Moreover, among the differentially expressed signals, Peptidylprolyl Isomerase A (PPIA, 1505.8 m/z), which has already demonstrated a role in the development of cancer, was found overexpressed in NIFTP RAS-mutated nodules compared to wild-type lesions. These results underlined that high-throughput proteomic approaches may add a further level of biological comprehension for NIFTPs. In the future, thanks to the powerful single-cell detail achieved by new instruments, the complementary NGS-MALDI imaging sequence might be the correct methodological approach to confirm that the current NIFTP definition encompasses heterogeneous lesions that must be further characterised

Molecular Tests for Risk-Stratifying Cytologically Indeterminate Thyroid Nodules: An Overview of Commercially Available Testing Platforms in the United States

The past decade has witnessed significant advances in the application of molecular diagnostics for the pre-operative risk-stratification of cytologically indeterminate thyroid nodules. The tests that are currently marketed in the United States for this purpose combine aspects of tumor genotyping with gene and/or microRNA expression profiling. This review compares the general methodology and clinical validation studies for the three tests currently offered in the United States: ThyroSeq v3, Afirma GSC and Xpression Atlas, and ThyGeNEXT/ThyraMIR

Histological and fine needle aspiration cytological features of Hashimoto thyroditis-associated 'angiomatoid' papillary thyroid carcinoma.

The association between Hashimoto thyroiditis (HT) and papillary thyroid carcinoma (PTC) is well known but few reports have described the morphological features of HT-associated PTC.1,2 Di Pasquale et al.3 described a peculiar histological pattern of HT-associated PTC consisting of anastomosing spaces lined with cells having the characteristic nuclear features of PTC. The authors named this HT-associated variant of PTC as angiomatoid and emphasized that these cases may be missed if the cells lining the spaces are not examined at high magnification. 3 We describe the fine needle aspiration (FNA) cytological and histological findings n a case of HT-associated PTC in its angiomatoid variant, in which the diagnosis of PTC was more straightforward on cytology than histology. A 16-year- ld girl complained of diffuse enlargement of the neck. Clinical examination showed a generalized enlargement and increased hardness of the thyroid. Serological data showed high levels of anti-thyroglobulin, anti-thyreoproxidase antibodiesand increased thyroid-stimulating hormone (TSH)levels that were indicative of a HT with subclinical hypothyroidism. An ultrasonography (US) showed the gland to have irregular borders, diffuse micronodulation and hyperechoic bands in both lobes. Moreover, an irregular, 12-mm-sized hypoechoic nodule was detected at the base of the right lobe; no cervical lymph nodes were detected. An US-guided, FNA of the nodule was performed. The Diff-Quik stained smears were highly cellular; follicular cells were organized in very large overlapping groups with a branching appearance. In some monolayered groups, the cells showed plentiful squamoid cytoplasm (Figure 1a); the nuclei were irregular in size and shape, with granular coarse chromatin and inconspicuous nucleoli. Grooves and cytoplasmic intranuclear inclusions were detected (Figure 1a, inset). The background was haemorrhagic, with some lymphoid cells and tangles intermingled with follicular cells; colloid was absent. The cytological diagnosis was PTC in a background of HT. The patient underwent a total thyroidectomy. The gland on the cut section was solid and showed a greyreddish colour, with accentuated lobulation. In the right lobe a haemorrhagic, ill-defined 12-mm nodule was detected. The histological sections showed the classic features of HT, with lymphoid follicles and Hurthle cell metaplasia. The nodule appeared as a haemorrhagic, poorly defined area, and consisted of irregular, spaces, fibrotic bands and inflammatory cells lacking papillary or follicular structures (Figure 1b). The spaces ranged from a roundish to stag-horn pattern and were lined with flat or cuboidal epithelial cells (Figure 1c). Follicular cells showed plentiful cytoplasm, irregular nuclei, sometimes empty, with grooves and cytoplasmic intranuclear inclusions (Figure 1c, inset); these cells were positive for thyroglobulin and thyroid transcription factor-1 (TTF-1) on immunohistochemistry. Because of the coexistence of the classic nuclear features of PTC and the histological features described above, a diagnosis of HT-associated PTC in its angiomatoid variant was made. There is clinical and pathological evidence of a relationship between HT and PTC ranging between some cytological features and RET-PTC rearrangement. 1 Some studies report that over 90% of thyroids with HT express RET ⁄ PTC1 and RET ⁄ PTC3 oncogenes.4 Nevertheless, few reports have described the morphological features of HT-associated PTC

Cytology-based gene mutation tests to predict response to anti-epidermal growth factor receptor therapy: a review.

Recent therapeutic progresses in nonsmall cell lung cancer (NSCLC) and in colorectal cancer (CRC) are based on agents that specifically target the epidermal growth factor receptor (EGFR). To identify the patients most likely to benefit from such therapies, EGFR or KRAS gene mutation tests are mandatory, respectively, in NSCLC and in CRC. In patients with locally advanced or metastatic disease, exploiting cytological samples for these tests avoids not curative surgery. Here, we review the studies that have applied gene mutation assays on cytological samples of NSCLC and CRC to select patients for anti-EGFR therapy. We argue that the standard of quality of gene mutation tests on cytological samples is closely dependent on the extent of the cytopathologist's involvement

Epidermal Growth Factor Receptor Test Performed on Liquid-Based Cytology Lung Samples: Experience of an Academic Referral Center.

Objectives: In this study we reviewed our practice of lung cancer epidermal growth factor receptor (EGFR) mutational testing in an academic centralized laboratory setting, where direct smears and liquid-based cytology (LBC) slides represent the most frequent cytological specimens received. The aim was to assess the differences, if any, between these sample types in terms of DNA yield, adequacy rates and overall EGFR testing performance. Study design: A total of 362 cases were retrieved - received from January 2012 to January 2014 for EGFR testing - including 204 LBC specimens and 158 smears. Exon 19 deletions and the L858R point mutation in exon 21, detected by fragment assay and TaqMan assay, respectively, were confirmed by direct sequencing or by high-resolution melting. Results: Although the direct smears showed a higher DNA yield (60.94 vs. 23.07 ng/µl) and were more frequently cell-rich (54%) than the LBC slides (31.4%), the differences in adequacy (direct smears: 97.4%; LBCs: 94.1%) and in mutant rate (direct smears: 10.3%; LBCs: 14.0%) between the two sample types did not reach statistical significance. Conclusions: Not only direct smears but also LBC slides represent an effective preparation and storage medium for cytological material to be used for EGFR molecular testing

KRAS testing on colo-rectal carcinoma cytological imprints.

Anti-EGFR monoclonal antibodies, cetuximab, and panitumumab, are administrated under the condition that advanced colo-rectal cancer (CRC) carries a wild-type KRAS gene. Thus, clinicians request pathologists to genotype KRAS before treatment. In the near future routine mutation testing at the same time of the surgery may be implemented. The reliability of a rapid KRAS testing on ex vivo cytological samples obtained by direct scraping of the colon tumour tissue is here evaluated. A consecutive series of 20 surgically resected, primary CRC specimens was analysed. Fresh tissue from CRC was scraped with a scalpel blade, smeared on uncoated glass slides, air-dried and Diff-Quik stained to ensure malignant cell presence. The same tissue area was also histologically processed. Exon 2 KRAS gene mutations were evaluated on both cytological and histological specimens by dideoxy sequencing and by the DxS KRAS Mutation Test Kit (DxS, Manchester, England). Data obtained on on imprint cytology and matched histological samples showed full concordance; however, the mutation frequency was slightly higher (35%) by the DxS KRAS Mutation Test Kit than by the dideoxy sequencing (30%). Thus, colon cancer imprint cytology sample is a reliable biospecimen for both dideoxy-sequencing and DxS KRAS Mutation Test Kit analysis and it may be useful to abbreviate the KRAS assay turnaround time