Histopathological Analysis of Lung Squamous Cell Carcinoma

All available hematoxylin and eosin (H&E) stained slides were reviewed by 2 pathologists (K.K. and W.D.T.) using an Olympus BX51 microscope (Olympus, Tokyo, Japan) with a standard 22-mm diameter eyepiece. Both pathologists had no knowledge of those patients’ clinical outcomes.
Tumors were graded by a degree of squamous differentiation into well, moderately, and poorly differentiated, in accordance with the 2004 WHO classification of lung carcinomas.⁷ In the well differentiated tumors, there were tumor nests composed of differentiated keratinocyte-like tumor cells with prominent keratinization (layered and cytoplasmic keratin) and intercellular bridges. In the poorly differentiated tumors, squamous morphology was only noticeable in a small area of the tumor. The moderately differentiated tumors showed an intermediate degree of squamous differentiation that was between well and poorly differentiated tumors.
Histologic subtyping was performed in a similar fashion to nasopharyngeal carcinomas in the 2005 WHO Classification, Pathology and Genetics of Head and Neck Tumours; they were classified as non-keratinizing, keratinizing, and basaloid squamous cell carcinomas.²⁵ The percentage of keratinizing pattern, including layered (Fig. 1A) and cytoplasmic keratinization (Fig. 1B), was recorded and then tumors were classified as having a keratinizing subtype when there was ≥5% keratinizing pattern of the entire tumor while non-keratinizing subtypes were defined as having <5% keratinizing pattern (Fig. 1C). The basaloid pattern was defined as tumor nests showing prominent peripheral palisading of tumor cells with scanty cytoplasm (high nuclear/cytoplasmic ratio) and a greater amount of hyperchromatic nuclei (Fig. 1D).⁷ The percentage of basaloid pattern was recorded and then the tumors were classified as having a basaloid subtype if there was >50% basaloid pattern as previously recommended.^{26 , 27 (link)} The percentage of papillary growth was recorded in 5% increments. Clear cell features were defined as tumor cells with clear cytoplasm and were recorded in 5% increments; it was considered present when ≥5% of the tumor cells had a clear cell pattern. No cases were classified as the small cell variant of squamous cell carcinoma, although occasional basaloid carcinomas had tumor cells that resembled small cell carcinoma.
After scanning through the entire set of tumor slides at intermediate-power fields at ×100 magnification, tumor budding and the size of the smallest tumor nest were assessed at the most invasive area with the maximal number of the smallest tumor nests. Tumor budding was defined as small tumor nests composed of less than 5 tumor cells (Fig.2A and 2B) and they were counted in 10 high-power fields (HPFs) at ×200 magnification.^{9 (link)} According to the number of tumor budding counted in 10 HPFs, tumor budding was assessed 2 ways: 1) the maximum number of tumor budding per HPF among the 10 HPFs (maximum budding /1 HPF) and 2) the total number of tumor budding of 10 HPFs (total budding /10 HPFs). Based on the approach of previously published studies analyzing the prognostic significance of the tumor nest size assessed by the number of tumor cells,^{8 (link), 10 (link), 14 (link), 15 (link)} the size of the smallest invasive tumor nest was classified into large nest (composed of >15 tumor cells), intermediate nest (5–15 tumor cells), small nest (2–4 tumor cells), and single cell invasion (Fig. 2C). The size of the smallest tumor nest was assessed 2 ways: 1) the tumor nests in entire tumor area and 2) the tumor nests infiltrating the tumor edge on the outside of the tumor.
The percentages of tumor necrosis and fibrosis were recorded. Tumor necrosis was considered present when there was ≥10% necrosis in the entire tumor.^{28 (link)} When there was ≥50% fibrosis in the entire tumor, it was considered severe.^{29 (link)} In addition, pleural invasion, which was classified as absent (PL0) or present (PL1, PL2 and PL3)²² , and lymphovascular invasion were investigated.
The nuclear features were evaluated according to the methodologies used in our previous publications.^{21 (link), 30 (link)} They were assessed using a HPF at ×400 magnification (0.237mm² field of view) at the region of the tumor with the greatest abnormal nuclear features. This was done after scanning through the entire set of tumor slides at intermediate-power fields at ×100 magnification. For nuclear diameter, we selected at least 3 HPFs with the largest nuclei and then calculated the average nuclear diameter of at least 100 tumor cells using nearby small lymphocytes (≈4.0 µm) as reference.^{19 (link)} Nuclear atypia was recorded in the area of the tumor with the highest degree of atypia; at least 5% of the entire tumor area needed to be affected. The degree of atypia was assessed using the following gradation: mild atypia - uniform nuclei in size and shape; moderate atypia - nuclei in intermediate size with slight irregularity in shape; and severe atypia - enlarged nuclei of varied sizes and irregular contours with some nuclei at least twice as large as others. The nuclear/cytoplasmic (N/C) ratio was broken down into the following three categories: low N/C ratio (<1/3 nucleus to cytoplasm area), intermediate N/C ratio (1/3–2/3), and high N/C ratio (>2/3). Chromatin pattern was differentiated using two distinctions, finely granular and coarsely granular. The prominence of nucleoli was also broken down into 2 distinct categories: indistinct - inconspicuous at intermediate-power fields at ×100 magnifications, and distinct - conspicuous at intermediate-power fields. Intranuclear inclusions were determined as present or absent in an examination of 50 HPFs. Mitoses were evaluated in the 50 HPF areas that contained the highest mitotic activity and then were calculated as an average of mitotic figures per 10 HPFs (2.37mm² area).^{21 (link), 30 (link)} Atypical mitoses were considered present if any were observed after examination of 50 HPF.^{21 (link), 30 (link)}