case_images: PMC4531126_01
case_images: Contrast enhanced CT scan of case presentation. (A) osteolytic mass of the eleventh left rib (white arrows) invading surrounding soft tissues; (B) bulky lymph-nodal metastasis (white arrow) of the right posterior cervical space dislocating the right common carotid artery; (C) lower paratracheal, 4R according to IASLC, lymph-nodal metastasis (white arrow); (D) subpleural mass in the posterior segment of inferior right lobe; (E) Bone scintigraphy showing active pathological uptake of the left eleventh rib (black arrow) and of S2 vertebral body (black arrow head).
case_images: can-9-559fig1.jpg
case_images: PMC4531126_01_can-9-559fig1.jpg
case_images: figure1
case_images: A full-body computed tomography (CT) scan confirmed a 60 mm large laterocervical lymphadenopathy, also revealing a right lung lesion, a rib metastasis, and a voluminous neoplastic involvement of the first sacral vertebra (Figure 1), whose extension went beyond the spinal canal, infiltrating the radicular fibres.
case_images: Microscopic presentation of the tumor showing enteric-type adenocarcinoma. (A) Cluster of neoplastic cells arranged in acinar pattern show atipical nuclei and clear cytoplasmatic droplets of mucin; (B) Immunohistochemically cells are negative to TTF-1; (C) Expression of CDX2 and (D) cytokeratin 7; (E) No immunoreactivity is evident for Cytokeratin 20. (A) Hematoxylin and Eosin, original magnification X40 - (B) X40 - (C) X20 - (D) X40 - (E) X40.
case_images: can-9-559fig2.jpg
case_images: PMC4531126_01_can-9-559fig2.jpg
case_images: figure2
case_images: Histologic revision from a pathologist (EP) of the Perugia Hospital (PG, Italy) supported the diagnosis of pulmonary enteric-type adenocarcinoma (Figure 2).
case_images: Biologic profile of the tumor. (A) Sequence analysis of the KRAS exon 2 performed on DNA extracted from microdissected area of paraffin-embedded tumor sections, shows a heterozygous point mutation: a C to A transversion (black arrow), resulting in a substitution of Glutamine, wild type (CAG) for Lysine (AAG) at codon 22 (p.Q22K, c.64C>A ). The mutation was named according to recommendations of the Nomenclature System for Human Gene Mutations. NCBI Reference Sequence: NM_004985.4 of KRAS gene was used as a reference; (B) Sequence analysis of the KRAS exon 2, performed on DNA extracted from peripheral white blood cells, shows the sequence wild type (CAG) resulting in normal glutamine at codon 22; (C) Dual colour FISH analysis (original magnification 60X) was performed using a CEP12 alpha satellite probe (12p11-q11) labelled in SpectrumOrange (Vysis, Downers Grove, IL. USA) and a BAC (Bacterial Artificial Chromosome) genomic probe RP11-707G18 (12p12.1) spanning an approximately 176 kb region encompassing the K-Ras gene, labelled in SpectrumGreen; (D) Representative data from KRAS and IL22 relative quantification (RQ) using TaqMan Copy Number Assays run on ViiA7 instrument. The number of copies of the KRAS and IL22 is determined by relative quantitation (RQ) using the comparative CT (DeltaDeltaCT) method. This method measures the CT difference (DeltaCT) between target and reference gene (RNase P), then compares the DeltaCT values of test samples to a calibrator sample(s) known to have two copies of the target sequence. The copy number of the target is calculated to be two times the relative quantification.
case_images: can-9-559fig3.jpg
case_images: PMC4531126_01_can-9-559fig3.jpg
case_images: figure3
case_images: While no EGFR mutation was detected, a Q22K point mutation was described in the KRAS gene (Figure 3A). Such a mutation was not found in the peripheral white blood cells, thus indicating its somatic origin in the tumor (Figure 3B). To assess whether the KRAS Q22K mutation was accompanied by an increased KRAS gene copy number, KRAS FISH analysis was performed, which documented the presence of polysomy (>= 4 copies in 10-40% of cells) for the KRAS gene (Figure 3C). In addition, quantitative polymerase chain reaction (qPCR) was carried out, which confirmed a copy number gain of 4 (Figure 3D).