Cribriform Adenocarcinoma of the Lung: Clinicopathologic, Immunohistochemical, and Molecular Analysis of 15 Cases of a Distinctive Morphologic Subtype of Lung Adenocarcinoma

Alexander C Mackinnon Jr; Arturo Luevano; Lisley C de Araujo; Nagarjun Rao; Min Le; Saul Suster


Mod Pathol. 2014;27(8):1063-1072. 

In This Article

Materials and Methods


A total of 15 cases were identified during a retrospective review of lung adenocarcinomas from the surgical pathology files of the Medical College of Wisconsin, Milwaukee, Wisconsin (six cases), and the Ohio State University, Columbus, Ohio (nine cases), during a period from 1990 to 2010. The study was conducted with Institutional Review Board approval from both institutions. Clinical information and follow-up data were obtained from the patients' records or by contacting the responsible physicians. Criteria for inclusion were the presence of >70% cribriform pattern of growth of the tumor and an appropriate immunohistochemical profile consistent with a primary lung adenocarcinoma (CK7/TTF1-positive; CK20/CDX2-negative). For pathologic evaluation, from 5 to 15 slides stained with hematoxylin and eosin were available for review in all cases.

Paraffin blocks for immunohistochemical studies were available in all cases. Four-micrometer sections were stained using a Dako Autostainer Plus according to the manufacturer's protocol. Slides were dried at 60 °C for 1 h and deparaffinized. Heat-induced epitope retrieval was performed with Dako Envision FLEX target retrieval solution (high pH Tris/EDTA) at 100 °C for 20 min. Primary antibodies (DAKO, Carpinteria, CA, USA) for: CK7 (OV-TL 12/30), CK20 (Ks20.8), TTF1 (8G7G3/1), thyroglobulin (DAK Tg6), CDX-2 (DAK-CDX2), and ALK (5A4, Novacastra-Leica, Richmond, IL, USA) were incubated at room temperature for 60 min. Signals were detected using a Dako FLEX detection kit. Counterstaining was performed with Envision FLEX hematoxylin for 7 min at room temperature. Nuclear immunoreactivity was considered positive for TTF-1 and CDX-2. Cytoplasmic positivity was considered positive for ALK, thyroglobulin, CK7, and CK20. Appropriate positive and negative controls were run concurrently for all antibodies tested.

DNA Extraction

Six cases (Medical College of Wisconsin cohort) were amenable to molecular analysis. Hematoxylin and eosin-stained slides were used to identify areas in which tumor cells represented greater than 50% of total nucleated cells. DNA extraction was performed using Zymo Pinpoint DNA isolation kit (Zymo Research Corporation, Orange, CA, USA) in 10 μm unstained sections according to the manufacturer's guidelines. DNA was obtained from both tumor and adjacent, histologically normal appearing areas of each specimen.

EGFR and KRAS Sequencing

M13-tailed primers were used to amplify exons 18, 19, 20, and 21 of EGFR and a portion of KRAS including codons 12 and 13 (Table 1) by PCR. The PCR amplicons were confirmed by electrophoresis in 2% agarose gel. PCR products were sequenced in both directions using the BigDye® Direct Cycle Sequencing Kit according to the manufacturer's conditions. After the sequencing step, the BigDye Xterminator® Purification Kit was used to clean up the dye excess. Sequence data were obtained with a 3500 genetic analyzer (Applied Biosystems, Foster City, CA, USA). Sequencing results were analyzed using SeqScape v2.7 (Applied Biosystems).

Fluorescence In-Situ Hybridization (FISH)

FISH was performed on six cases using break apart probes for either ALK, ROS1, or RET (Empire Genomics, Buffalo, NY, USA) according to manufacturer's instructions. Four-micrometer-thick sections of formalin-fixed and paraffin-embedded tumor tissue were deparaffinized, dehydrated, immersed in 0.2 N HCl, and washed. The sections were immersed in 10 mM citrate buffer and boiled in a microwave for 5 min, treated with pretreatment reagent (Abbott Molecular) followed by protease digestion. After applying the probe, the samples were incubated in a humidified atmosphere using HybriditeTM (Abbott Molecular). Slides were washed with 2 × saline sodium citrate and counterstained with 4,6-diamino-2-phenylindole II and antifade (p-phenylamide). Signals for each probe were evaluated under a microscope equipped with a double-pass filter (green/orange; Abbott Molecular) and an oil immersion objective lens. Greater than 50 nuclei were analyzed for each case. ALK, ROS1, and RET FISH-positive cases were defined as those presenting more than 15% split signals (>1 signal diameter separation); in addition, loss of the 5′ ROS1 probe with retention of the 3′ probe was interpreted as ROS1 FISH positive.[25]