Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy

Full text

Chapter 1
Precise Diagnosis of Histological Type of Lung
Carcinoma: The First Step in Personalized Therapy
Jelena Stojšić
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/intechopen.75316
Provisional chapter
DOI: 10.5772/intechopen.75316
© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Precise Diagnosis of Histological Type of Lung
Carcinoma: The First Step in Personalized Therapy
JelenaStojšić
Additional information is available at the end of the chapter
Abstract
This chapter is a combination of personal experience of a pulmonary pathologist and
available references in the diagnosis of non-small cell lung cancer (NSCLC) types. The
morphological appearance of poorly dierentiated lung carcinoma is not characteristic,
so immunohistochemical staining is used for further dierentiation. In order to save
tumor tissue from paran blocks, the most rational way is to use only two antibodies,
p40 for squamous cell carcinoma and TTF-1 for adenocarcinoma of the lung, and if neces-
sary or if cancer growth is organoid, also one of two neuroendocrine markers (CD56 or
Synaptophysin) can be used. If there is enough tumor tissue in the paran block to con-
rm the diagnosis, NapsinA, p63, Cytokeratin5/6 or Cytokeratin5 can be used. It should
be kept in mind that no antibody is highly specic for one histological type of carcinoma
or its origin and if the immunohistochemical nding is unspecic, it should be concluded
that this is “not otherwise specied” (NOS) carcinoma. The rest of tissue must be pre-
served for current and future molecular testing and predictive immunohistochemical
staining for the purpose of personalized NSCLC therapy.
Keywords: non-small cell lung cancer, diagnosis, immunohistochemical staining,
paran block, TTF-1, p40, p63, CD56, Synaptophysin, NapsinA, Cytokeratin5/6,
Cytokeratin5
1. Introduction
1.1. Epidemiology as basis for developing a strategy in the treatment of advanced
non-small cell lung cancer
Lung carcinoma is the most commonly diagnosed malignancy worldwide. There is a dierent
lung cancer incidence and mortality statistics throughout the world. In male population, lung
© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

cancer has the highest incidence rate, especially in developing countries, while in developed
countries, it is immediately behind the prostate cancer. In female population, the incidence
rate of lung cancer is rising, and it is higher than cervical carcinoma, but still lower than breast
cancer. Lung carcinoma mortality rate is still alarmingly high, both in developing and devel-
oped countries [1–3].
This high mortality rate has led to research of drugs, which will, in the era of personalized
therapy, prolong the survival of diseased patients for more than 5 years and also improve the
quality of their lives during and after the treatment. Individual approach to the treatment of
lung cancer is based on a precisely diagnosed pathohistological type of non-small cell lung
cancer (NSCLC) [4, 5].
1.2. Types of biopsy specimens and pathohistological classication of lung cancer
Two most common histological types of NSCLC are adenocarcinoma and squamocellular car-
cinoma [6–8]. At the time of diagnosis, about 75% of lung cancer is in an inoperable, advanced
stage and less than 15% of patients survive more than 5 years. As these patients cannot be surgi-
cally treated, diagnosis of NSCLC is based on bronchoscopic and ne-needle aspiration biopsy
(fnab) or video-assisted thoracic surgery (VATS). That is why there is a huge responsibility on the
shoulders of the pathologist to diagnose a histological type of NSCLC on a small biopsy speci-
men and preserve paran-embedded carcinoma tissue for further genetic and immunohisto-
chemical testing in order to determine the eective personalized therapy. It aims at prolonging
survival of patients and improving the quality of their lives during and after the therapy [9, 10].
In the nal interpretation of the pathohistological ndings, pathologists use the classication
of the World Health Organization (WHO) from 2004 [6], that is, an improved version from
2015 [7].
According to WHO classication from 2004, histological subtypes of NSCLC are as follows:
• squamous cell carcinoma (Figure 1)
• adenocarcinoma (Figure 2)
• large-cell carcinoma
• adenosquamous carcinoma
• sarcomatoid carcinoma
• carcinoid tumors
• salivary gland tumors.
Each of these histological NSCLC subtypes has its own variants that are diagnosed based on
their morphological picture and specic immunophenotype [6].
In the WHO classication of lung carcinoma from 2015, there have been some changes
because adenocarcinoma took over the rst place from squamous cell carcinoma, which pre-
viously had primacy. The greatest change in this classication compared to the previous one
Lung Cancer - Strategies for Diagnosis and Treatment4

is a grouping of all carcinoma with neuroendocrine dierentiation: carcinoid tumors, typical
carcinomas (TC) and atypical carcinomas (AC), large-cell neuroendocrine carcinomas (LCC-
NEC) and small-cell neuroendocrine carcinomas (SCLC) into one group of carcinomas due
to specic biological behavior and special therapy, regardless of the dierent morphological
picture. Remaining classication is identical to that of 2004 [7].
1.3. Processing of lung carcinoma tissue samples taken during bronchoscopy, FNA
biopsy or VATS method
Bioptized lung samples obtained during bronchoscopy are xed in 10% buered formalin
and brought to the laboratory. It is considered that sampling of tissue is representative if at
least ve biopsy samples have been delivered in diameter larger than 2 mm. Tissue samples
taken during FNA biopsy should be delivered in 10% buered formalin in the form of punc-
tuate cylinder in order to use the whole tissue material for which is believed to contain lung
cancer for morphological analysis and for immunohistochemical staining, while the rest
of the tissue would be preserved for molecular testing (EGFR, optional KRAS), predictive
Figure 1. Keratinizing squamous cell carcinoma of the lung, H&E 100×.
Figure 2. Adenocarcinoma of the lung with lepidic growth paern, H&E 100×.
Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy
http://dx.doi.org/10.5772/intechopen.75316
5

immunohistochemical staining (ALK, ROS1 and PDL-1), as well as for uorescent in situ
hybridization (FISH) if this method is accepted by consensus [11].
Basic information about the patient which is to be submied in the biopsy referral for the
pathohistological laboratory is: name and surname, gender, age, place of residence, occupation
and smoking status. It is necessary to deliver clear and concise clinical picture, for example,
whether there is a suspect tumor shadow in the lung, mediastinal lymphadenopathy, superior
vena cava syndrome (SVCS), and so on. Also, it is necessary to note that there is a previously
diagnosed pulmonary or some other kind of malignancy in patient, which histological type of
tumor is diagnosed on that occasion and how long before the current examination. This data
help to set a new pathohistological diagnosis by applying a smaller number of immunohisto-
chemical staining to prove metastatic malignancy or a new primary carcinoma of the lung. An
endoscopic nding of the mode of tumor growth and its localization must also be given. In
this way, we save tumor tissue for methods which would be used in personalized therapy for
advanced lung cancer. If these data are not available, the pathologist should consider that this
is a biopsy of primary lung cancer. Incomplete data because of sloppiness and lack of interest
of the doctor who performed the biopsy can lead to vagueness and diculty in diagnosing
and thus, to disrespect of the patient and pathologist. Correct communication at the patient-
clinician level and clinician-pathologist level is the basis for seing a precise diagnosis.
According to NSCLC morphology, many pathologists would not agree on a denitive diag-
nosis, but after immunohistochemical stainings, the same diagnosis should be made in a high
percentage of them. Also, crush phenomenon that can appear on obtained tissue samples
during bronchoscopy and inadequate xation of them can put pathologist on misdiagnosis.
1.4. Routine treatment of biopsy samples when there is a suspicion of NSCLC
Biopsy samples are routinely xed in a 10% buered formalin and then dehydrated in xylol
and rising alcohol concentrations, embedded into paran block, cut at thickness of 2 μm, using
hematoxylin-eosin stained, covered with medium (Canada balsam) and analyzed under the
microscope. There should be only one, two at most cross sections, on one object plate in order
to determine whether there is cancer in the rst two cross sections by routine hematoxylin-eosin
(H&E) staining and also to assume a histological type of cancer, based on the morphological char-
acteristics of malignant cells. The following sections are cut separately on two respective plates for
immunohistochemical staining in order to determine the precise histological type of NSCLC. The
pathologist concludes that there are no malignant cells on both cross sections and to report this in
his denitive pathohistological nding. If NSCLC is found, two immunohistochemical stainings
are applied, TTF-1 and p40, which determine the histological subtype of two most histological
subtypes of NSCLC, adenocarcinoma and squamous cell carcinoma. In the end, it is desirable for
the pathologist to indicate in his report whether there is enough and how much tissue material
is left for the next molecular testings. The number of plates is 4: for EGFR molecular testing and
ALK, ROS1 and PDL-1 immunohistochemical evaluation (Figure 2). In the era of personalized
therapy, it is desirable to cut eight tissue sections at the same time from paran block: two for
morphological analysis based on H&E stained preparations, two for basic immunohistochemical
staining (TTF-1 and p40) and the last four for molecular and immunohistochemical predictive
staining only in the case that NSCLC is found on H&E stained cross sections [12, 13].
Lung Cancer - Strategies for Diagnosis and Treatment6

2. Immunohistochemistry
2.1. Immunohistochemical staining method
The labeled streptavidin-biotin staining method uses a highly “rened” avidin-biotin com-
plex (ABC) three-stage technique in which a biotinylated secondary antibody reacts with sev-
eral streptavidin molecules conjugated by peroxidase or alkaline phosphatase [12].
2.2. Immunohistochemical staining procedure
Tissue samples for immunohistochemical staining are deparaned according to the pre-
scribed procedure of the manufacturer and then incubated with a specic serum at room
temperature in a damp chamber for a prescribed duration. It is used the labeled streptavi-
din-biotin (LSAB) technique. The antigen-antibody complex is visualized by 3-amino-9-eth-
ylcarbazole or diaminobenzidine hydrochloride solution. Mayer’s hematoxylin is used as a
counterstain. A “positive control” is used to evaluate the eectiveness of a method or reaction.
As already stated, “internal positive control” is used, since there are normal tissue structures
on the preparation itself, in this case, lung, which express the administered antibodies. In the
part of the chapter in which we analyze individual monoclonal antibodies, we will also indi-
cate which lung structures are expressing them regularly [12, 14, 15].
3. Monoclonal antibodies in the diagnosis of non-small cell lung cancer
There is a question, which two monoclonal antibodies should be rationally applied in order to
establish the exact diagnosis of the histological subtype of NSCLC. Currently, these are thy-
reoid-transcription-factor-1 (TTF-1) and p40. These two antibodies have a role in distinguish-
ing two most common histological subtypes, adenocarcinoma and squamous cell carcinoma.
TTF-1 (clone 8G7G3/1, DAKO Cytomation, Denmark) is a diagnostic marker for adenocarci-
noma of the lung and p40 (BC28, Ventana, USA) for squamous cell carcinoma [16, 17].
However, in the past, other less specic antibodies were used in the dierentiation of histolog-
ical subtypes of NSCLC. They can also be used now as an additional conrmation about his-
tological subtype of NSCLC and dierentiation of primary from secondary lung cancer. The
following antibodies are also useful for dierentiation: NapsinA (clone IP64, Novocastra™
HD, Leica Biosystems, UK), p63 (clone 7JUL, Novocastra™ HD, Leica Biosystems, UK),
Cytokeratin5/6 (cloneD5/16B4 DAKO Cytomation, Denmark) or Cytokeratin5 (clone EP1601Y,
Cell Marque RUO, USA) and Cytokeratin7 (clone OV-TL 12/30 DAKO Cytomation, Denmark)
[18, 19]. If the morphological picture of NSCLC has organoid appearance, there is a suspicion
that this is large cell lung carcinoma with neuroendocrine dierentiation (LCC-NEC). To con-
rm this suspicion, it is necessary to use at least one of two neuroendocrine markers, CD56
(clone NCAM-1 Ab-2, Thermo Scientic LabVision, USA) or Synaptophysin (clone 27G1
Novocastra ™ HD, Leica Biosystems, UK) [18, 20]. If TTF-1, p40 and optionally CD56 were
not expressed, for the purpose of storing malignant tissue for molecular testing, it should
Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy
http://dx.doi.org/10.5772/intechopen.75316
7

be concluded that this is non-small-cell lung carcinoma, or “not otherwise specied” (NOS)
carcinoma [7, 16]. It should be noted that no antibody is not highly specic for one organ or
one histological type of cancer, that is, each antibody is specic for more than one histological
types of cancer or more organs [21].
For precise pathohistological diagnosis of histological subtype of NSCLC, it is necessary to
use two antibodies, TTF-1 and p40. If it is estimated that there is enough tumor tissue in a par-
an mold, it is possible to use additional antibodies to conrm the diagnosis and to preserve
it for molecular testing [22, 23].
The advantages and disadvantages of each of these antibodies are discussed in the next section.
3.1. Thyroid transcription factor-1 (TTF-1)
Thyroid Transcription factor-1 (TTF-1) is a nucleic specic protein transcriptional fac-
tor expressed by thyroid gland and thyroid tumors as well as adenocarcinoma of the lung
(Figures 3 and 4). This marker is expressed in the majority of lung adenocarcinoma (75%),
but also in about 10% of squamous cell carcinoma. TTF-1 is signicant in the dierential diag-
nosis between primary and metastatic adenocarcinoma. At a time, when less antibodies were
known, if TTF-1 with Cytokeratin7 was positive and Cytokeratin20 negative, diagnosis of
adenocarcinoma of the lung was established [5, 24–28]. Diagnostic algorithms in the diagnosis
of adenocarcinoma and dierentiation of adenocarcinoma from squamous cell carcinoma are
shown in papers of IASLC/ATS/ERS and Terry et al. [29, 30]. In various studies, TTF-1 was
specic from 88 to 97% [6, 30, 31]. This marker is also expressed in lung cancer with neu-
roendocrine dierentiation, in typical and atypical carcinoids, as well as in more than 90%
of small cell lung carcinomas with neuroendocrine dierentiation and in 50% of large cell
lung carcinomas with neuroendocrine dierentiation [30]. Literature reveals that TTF-1 can
be expressed in breast and ovarian carcinoma [31, 32].
In one of our studies, we found that TTF-1 was expressed in 85.2% and in the other study, in
86% of lung adenocarcinomas [12, 33]. We also found that TTF-1 was also expressed in benign
lung tumors [34, 35].
Figure 3. Adenocarcinoma of the lung with acinar growth, fnab, H&E 100×.
Lung Cancer - Strategies for Diagnosis and Treatment8

3.2. p40
Role of p40 is to distinguish adenocarcinomas from squamous cell carcinomas in small sam-
ples (Figure 5) as well as on cytological smears. Squamous cell carcinoma is conrming with
p40 antibody which is also known as DNp63. p40 is expressed in the nucleus of malignant
cells of squamous cell carcinoma (Figure 6). This antibody is more specic for squamous cell
lung carcinoma from p63. p40 is expressed in a smaller number of lung adenocarcinoma cells
than p63. That is why it is recommended to use p40 instead of p63 for diagnostics of squa-
mous cell carcinoma [36, 37]. If in malignant cells of carcinoma are not expressed TTF-1 and
p40, diagnosis of non-small cell lung carcinoma -not otherwise specied (NSCLC-NOS) on a
small biopsy sample will be established [16].
However, p40 is not a highly specic marker for only squamous cell lung carcinoma. It is
also highly specic marker for urothelial carcinoma. This means that metastatic urothelial
carcinoma in the lung is dicult to distinguish from primary squamous cell lung carcinoma
both for morphological similarity these two carcinomas and similar immunophenotype [38]
Figure 4. Adenocarcinoma of the lung, nucleic expression, TTF-1 200×.
Figure 5. Moderately dierentiated squamous cell carcinoma of the lung on bronchoscopic biopsy, H&E 20×.
Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy
http://dx.doi.org/10.5772/intechopen.75316
9

(Figures 7 and 8). Therefore, for denitive dierentiation lung squamous cell carcinomafrom
urotothelial carcinoma is also required clinical data on current local status in previously oper-
ated patients with urothelial carcinomas (degree of cancer invasion at the time of surgery,
presence of angioinvasion, state of resection margins, the presence of metastases in local and
remote lymph nodes).
p40 is also expressed in squamous cell carcinoma of other localizations (head and neck, lar-
ings, trachea, cervix, skin). That is why it is not possible to dierentiate primary squamous
cell lung carcinoma from metastatic lung carcinoma of the same histological type by using
p40. Dierentiation of primary from secondary squamous cell carcinoma in the lung is pos-
sible only in clinicopathological correlation [39].
3.3. NapsinA
NapsinA is expressed in the cytoplasm of preserved lung parenchyma in the form of functional
aspartic proteinase, homologous to the polypeptide Tao2 and included in maturation of the
Figure 7. Urothelial carcinoma metastases in the lung, 400×.
Figure 6. p40 expression in the squamous cell lung nuclei, 400×.
Lung Cancer - Strategies for Diagnosis and Treatment10

biologically active surfactant protein B. It also consists of 38-kDa protein, protein chain which is
expressed in type II pneumocytes, alveolar macrophages and renal tubules, secretion channels of
exocrine glands and pancreas. NapsinA is staining as coarse-grained intracytoplasmic marker [40].
There are papers that favor the use of NapsinA in regard to TTF-1 in dierentiation of lung
adenocarcinoma from other histological subtypes of NSCLC. Papers were done on a large
number of lung adenocarcinomas wherein NapsinA showed higher specicity compared to
the TTF-1. It is even recommended double immunohistochemical staining, so-called cocktail,
which would beside NapsinA for cytoplasmic staining, contain and p40 for nuclear staining
for squamous cell lung carcinoma [17, 36, 40, 41]. A simple diagnostic algorithm from 2010
recommends that NapsinA should be applied in order to diagnose adenocarcinoma, if TTF-1
is not exposed in NSCLC and if squamous cell carcinoma is not proved [30]. Our study on
50 adenocarcinomas showed a greater specicity of TTF-1 as compared to NapsinA [34]. If
NapsinA is not expressed and the presence of mucin has not been proven, it should be con-
cluded that it is about NSCLC-NOS. The denitive conclusion is that NapsinA is supplemen-
tal to TTF-1 which serves as the main marker for proving of lung adenocarcinoma (Figure 9).
3.4. p63
This antibody is known in the two isoforms, DNp63 and TAp63. It is expressed in the nucleus
of the malignant cells. According to previous diagnostic algorithms, before occurrence of p40,
p63 was the main marker in the dierentiation of NSCLC on small biopsy samples beside
TTF-1 [29]. Beside TTF-1, NapsinA and Cytokeratin5/6 are considered to be useful mark-
ers for the diagnosis of NSCLC on small biopsy samples [28]. The degree of p63 expression
increases with the decrease in the degree of keratinization, that is, dierentiation of squamous
cell carcinoma [25] (Figure 10 200×; Figure 11 400×).
However, p63 as well as p40 is not a highly specic marker only for squamous cell carcinoma.
It is also a marker for urothelial carcinoma. That is why it is hard to dierentiate metastatic
carcinoma of urothelial type from primary squamous cell lung carcinoma [39] (Figure 12
200×; Figure 13 400×).
Figure 8. p40 expression in urothelial carcinoma metastases in the lung, 400×.
Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy
http://dx.doi.org/10.5772/intechopen.75316
11

Figure 10. NSCLC deposit in the lymphatic tissue, where squamous cell carcinoma was immunohistochemically proven,
H&E 200×.
Figure 11. Strong nuclear expression of p63 in the cells of poorly dierentiated squamous cell lung carcinoma, 400×.
Figure 9. Bronchoscopic biopsy, inltrate of the acinar adenocarcinoma of the lung in the mucosallayer of the bronchi,
proven by using NapsinA 200×.
Lung Cancer - Strategies for Diagnosis and Treatment12

3.5. Cytokeratin5/6 or Cytokeratin5
Cytokeratin 5/6 or Cytokeratin5 is a cytoplasmic marker. Its expression is present at squamous
cell carcinoma (Figure 14). Because of that, this marker can be used as a conrmation for this
histological type, together with p40 and p63, especially if there are no technical conditions for
using one of these antibodies. Except in the regular epithelium of bronchial airways, Cytokeratin
5/6 or Cytokeratin5 is also expressed in regular and reactive mesothelioma cells, but also in the
epithelium cells of the malignant pleural mesothelioma. Ovarian carcinomas, especially those
of serous type, are cytoplasmically expressed antibody [21, 28, 42].
3.6. Neuroendocrine markers
According to WHO recommendations about the classication of lung carcinoma from 2004, in
order to establish the diagnosis of large cell lung carcinoma with neuroendocrine dierentia-
tion, it is necessary for the cells to be large and round in an organoidal arrangement, with a
Figure 12. Urothelial carcinoma with lung metastases, H&E 400×.
Figure 13. p63 expression in urothelial carcinoma with lung metastases, 400×.
Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy
http://dx.doi.org/10.5772/intechopen.75316
13

Figure 14. Expression of Cytokeratina5/6 in squamous cell carcinoma, fnab, 200×.
Figure 15. Membrane expression of CD56 into LCLC-NEC cells, 200×.
Figure 16. Cytoplasmic expression of Synaptophysin into NSCLC with partly adenoid growth paern 200×.
Lung Cancer - Strategies for Diagnosis and Treatment14

luminous and large nucleus and noticeable nucleolus and to express at least one neuroendo-
crine marker: CD56 (Figure 15), Synaptophysin (Figure 16) or ChromograninA [5, 6]. It means
that if on a small biopsy sample, non-small cell lung cancer is showing organoid, trabecular or
acinar growth paern, and two neuroendocrine markers, preferably CD56 and Synaptohysin
[43], should be used to conrm this type of cancer (LCLC-NEC) (Figure 17).
4. Discussion
Finally, someone may ask why it is necessary to know of which histological type of non-small
cell lung cancer is about and to save enough malignant tissue in paran block for molecular
testings at the same time.
The answer is that it is necessary to know how to apply certain tests for the application of
personalized oncology therapy. If the evaluation of the tests showed that a positive response
on therapy is expected, the same will be applied.
Globally, adenocarcinoma of the lungs is the most common histologic type of lung cancer.
EGFR molecular testing is used to assess the response to tyrosine kinase inhibitors (TKI)
therapy in patients with adenocarcinoma. Positive response to the TKI therapy is more com-
monly expected in young nonsmoking woman. It is also recommended that this testing is
done in young nonsmoking woman with squamous cell lung cancer because it showed posi-
tive results. The result of this molecular testing depends on the sensitivity of the method and
number of cells remaining after morphological and immunohistochemical diagnostics, more
than 5% of malignant cells in relation to the total number of cells on the cross section. EGFR
mutations are diagnosed in 10–15% of Caucasians patients with NSCLC [22, 44].
ALK testing (clone D5F3, Ventana, USA) is done in patients with adenocarcinoma of the
lungs in which EGFR mutations have not been detected. There is no consensus on whether
this test should be performed only in lung adenocarcinoma or it can be performed in all
Figure 17. The proposed algorithm for the dierential diagnosis of non-small cell lung carcinoma by using immunohisto-
chemical stainings.
Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy
http://dx.doi.org/10.5772/intechopen.75316
15

pathohistological subtypes of NSCLC. Namely, there are several diagnostic algorithms
which include all histological types of NSCLC or only adenocarcinoma of the lungs. There
are data indicating that this genetic rearrangement was detected only in about 4% of patients
with adenocarcinoma of the lungs or in about 2% of patients with NSCLC. In order for this
test to be valid, it is necessary that on the tissue cross section at least 50 malignant cells have
to be present after primary diagnostics and EGFR testing. Then immunochemical testing
would be valid and where there is this type of consensus, to conrm the presence of rear-
rangements, FISH test should also be done [13, 45, 46].
It is necessary to know the fact that about 70% of LCC-NEC is ALK positive, but that its
expression is not in correlation with personalized ALK inhibitors. If NSCLC have an organ-
oid morphological picture, it is necessary to apply CD56 and Synaptophysin on small biopsy
samples in order to exclude LCC-NEC [13, 45, 46].
ROS1 testing (clone D4D6, Ventana, USA) is also done in adenocarcinoma of the lungs where
EGFR and ALK tests did not show positive results. This fusion is present only in 1–2%, pre-
dominantly younger patients, nonsmokers, with adenocarcinoma of the lungs. The presence
of fusion is immunohistochemically determined, and it is necessarily conrmed by the FISH
method, so it is necessary to preserve tissue for these methods after diagnosis of adenocarci-
noma [13, 46].
If any of these tests fail to give results, for the application of immunotherapy, it is applied
PDL-1 testing (clone 22C3, DAKO Cytomation, Denmark), mainly in squamous cell lung can-
cer, but also in adenocarcinomas in which previous test did not show positive results. For this
immunohistochemical testing, it is recommended that tissue cross section contains at least 100
malignant cells [47, 48].
The future of personalized lung cancer treatment is next-generation sequencing (NGS), poly-
merase chain reaction (PCR) method, in which, from the remaining of paran block in which
NSCLS was diagnosed, at the same time is detecting all druggable mutations [23].
Liquid biopsy is a method that detects DNA tumor cells in whole or in its parts. This method
is useful in early detection of lung cancer, but it is false negative if circulating malignant cells
do not possess a mutation [49].
5. Conclusions
Precise diagnosis of NSCLC histological type is based only on morphological characteristics
of the tumor, cell appearance and their growth paern on H&E stained preparation is rarely
possible. Based on morphological ndings, keratinizing squamous cell carcinoma can be
diagnosed with great certainty, without immunohistochemistry. The main disadvantage of all
used antibodies is not highly specicity only for one histological type of cancer or originates
from more than one organ. In order to preserve tumor tissue for molecular testing, one must
use only one, the most specic antibody for one histological type of carcinoma. For adenocar-
cinoma of the lung, highly specic is TTF-1, and for squamous cell carcinoma, it is p40. Other
Lung Cancer - Strategies for Diagnosis and Treatment16

additional antibodies can be applied only if there is a greater amount of bioptic tumor mate-
rial (fnab biopsy or VATS biopsy). NapsinA is an additional antibody, which can be applied in
proving adenocarcinoma of the lung and p63 for squamous cell carcinoma. When LCLC-NEC
is suspected, it is recommended to use only one neuroendocrine marker, and the most reliable
is CD56. For saving, in order to apply all the appropriate antibodies and to preserve tissue for
molecular testing, it is necessary to cut only one cross section on one plate.
Author details
Jelena Stojšić
Address all correspondence to: dr.jelenastoj@sezampro.rs
Department of Thoracopulmonary Pathology, Service of Pathology, Clinical Centre of
Serbia, Belgrade, Serbia
References
[1] Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide
burden of cancer in 2008: GLOBOCAN 2008. International Journal of Cancer. 2010;
127(12):2893-2917. DOI: 10.1002/ijc.25516
[2] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA: A
Cancer Journal for Clinicians. 2011;61(2):69-90. DOI: 10.3322/caac.20107
[3] Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman
D, Bray F. Cancer incidence and mortality worldwide: Sources, methods and major pat-
terns in GLOBOCAN 2012. International Journal of Cancer. 2015;136(5):E359-E386. DOI:
10.1002/ijc.29210
[4] Mollberg N, Surati M, Demchuk C, Fathi R, Salama AK, Husain AN, Hensing T, Salgia R.
Mind-mapping for lung cancer: towards a personalized therapeutics approach. Advances
in Therapy. 2011;28(3):173-194. DOI: 10.1007/s12325-010-0103-9
[5] Kerr KM. Personalized medicine for lung cancer: New challenges for pathology. His-
topathology. 2012;60(4):531-546. DOI: 10.1111/j.1365-2559.2011.03854.x
[6] Travis WD, Brambilla E, Müller-Hermelink HK, Harris CC, editors. World Health
Organisation Classication of Tumours. Pathology and Genetics of Tumours of the
Lung, Pleura, Thymus and Heart. Lyon: IARC; 2004. ISBN: 9283224183
[7] Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG, editors. World Health
Organisation Classication of Tumours of the Lung, Pleura, Thymus and Heart. Lyon:
IARC; 2015. ISBN: 9789283224365
[8] Janssen-Heijnen M, Coebergh JW. The chainging epidemiology in Europe. Lung Cancer.
2003;41(3):245-258. DOI: 10.1016/S0169-5002(03)00230-7
Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy
http://dx.doi.org/10.5772/intechopen.75316
17

[9] Cagle PT, Allen TC, Dacic S, Beasley MB, Borczuk AC, Chirieac LR, Laucirica R, Ro JY,
Kerr KM. Revolution in lung cancer: New challenges for the surgical pathologist. Archives
of Pathology & Laboratory Medicine. 2011;135(1):110-116. DOI: 10.1043/2010-0567-RA.1
[10] Hirsch FR, Franklin WA, Gazdar AF, Bunn PA Jr. Early detection of lung cancer: Clinical
perspectives of recent advances in biology and radiology. Clinical Cancer Research. 2001;
7(1):5-22. PMID: 11205917
[11] Cheng L, Alexander RE, Maclennan GT, Cummings OW, Montironi R, Lopez-Beltran
A, Cramer HM, Davidson DD, Zhang S. Molecular pathology of lung cancer: Key to
personalized medicine. Modern Pathology. 2012;25(3):347-369. DOI: 10.1038/modpathol.
2011.215
[12] Dabbs DJ. Diagnostic Immunohistochemistry Theranostic and Genomic Applications.
3rd ed. Philadelphia: Saunders Elsevier; 2010. ISBN: 9781416057666
[13] Sound TM, Hirsch ER, Yatabe Y, editors IASLC Atlas of ALK and ROS1 testing in lung
cancer. North Fort Myers, FL; Editorial Rx Press; 2016
[14] Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-xed, paran-embedded tis-
sues: An enhancement method for immunohistochemical staining based on microwave
oven heating of tissue sections. The Journal of Histochemistry and Cytochemistry.
1991;3449(6):741-748. DOI: 10.1177/39.6.1709656
[15] Stojsic J, Jovanic I, Markovic J, Gajic M. Contribution of immunohistochemistry in the
dierential diagnosis of non-small cell lung carcinomas on small biopsy samples. Journal
of BUON. 2013;18(1):176-187. PMID: 23613404
[16] Walia R, Jain D, Madan K, Sharma MC, Mathur SR, Mohan A, Iyer VK, Kumar L. p40
& thyroid transcription factor-1 immunohistochemistry: A useful panel to character-
ize non-small cell lung carcinoma-not otherwise specied (NSCLC-NOS) category. The
Indian Journal of Medical Research. 2017;146(1):42-48. DOI: 10.4103/ijmr.IJMR_1221_15
[17] Ikeda S, Naruse K, Nagata C, Kuramochi M, Onuki T, Inagaki M, Suzuki K. Immu-
nostaining for thyroid transcription factor 1, Napsin A, p40, and cytokeratin 5 aids in
dierential diagnosis of non-small cell lung carcinoma. Oncology Leers. 2015;9(5):2099-
2104. DOI: 10.3892/ol.2015.3045
[18] Micke P, Masson JS, Djureinovic D, Nodin B, Jirström K, Tran L, Jönsson P, Planck M,
Botling J, Brunnström H. The impact of the fourth edition of the WHO classication of
lung tumours on histological classication of resected pulmonary NSCCs. Journal of
Thoracic Oncology. 2016;11(6):862-872. DOI: 10.1016/j.jtho.2016.01.020
[19] Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, Beer DG,
Powell CA, Riely GJ, Van Schil PE, Garg K, Austin JH, Asamura H, Rusch VW, Hirsch FR,
Scaglioi G, Mitsudomi T, Huber RM, Ishikawa Y, Je J, Sanchez-Cespedes M, Sculier
JP, Takahashi T, Tsuboi M, Vansteenkiste J, Wistuba I, Yang PC, Aberle D, Brambilla
C, Flieder D, Franklin W, Gazdar A, Gould M, Hasleton P, Henderson D, Johnson
B, Johnson D, Kerr K, Kuriyama K, Lee JS, Miller VA, Petersen I, Roggli V, Rosell R,
Lung Cancer - Strategies for Diagnosis and Treatment18

Saijo N, Thunnissen E, Tsao M, Yankelewi D. International association for the study
of lung cancer/American Thoracic Society/European Respiratory Society: International
multidisciplinary classication of lung adenocarcinoma. Journal of Thoracic Oncology.
2011;6(2):244-285. DOI: 10.1513/pats.201107-042ST
[20] Fasano M, Della Corte CM, Papaccio F, Ciardiello F, Morgillo F. Pulmonary large-cell
neuroendocrine carcinoma: From epidemiology to therapy. Journal of Thoracic Onco-
logy. 2015;10(8):1133-1141. DOI: 10.1097/JTO.0000000000000589
[21] Stojsic J, Spasic Z, Velinovic M, Adzic T, Maric D, Todorovic V, Drndarevic N. Diagnostic
procedures in pleural malignant mesothelioma: Our experience. Journal of BUON.
2004;9:423-426 17415849
[22] Penzel R, Sers C, Chen Y, Lehmann-Mühlenho U, Merkelbach-Bruse S, Jung A, Kirchner
T, Büner R, Kreipe HH, Petersen I, Dietel M, Schirmacher P. EGFR mutation detection
in NSCLC — Assessment of diagnostic application and recommendations of the German
Panel for Mutation Testing in NSCLC. Virchows Archiv. 2011;458:95-98. DOI: 10.1007/
s00428-010-1000-y
[23] Padmanabhan V, Steinme HB, Rizzo EJ, Erskine AJ, Fairbank TL, de Abreu FB, Tsongalis
GJ, Tafe LJ. Improving adequacy of small biopsy and ne-needle aspiration specimens
for molecular testing by next-generation sequencing in patients with lung cancer: A qual-
ity improvement study at Dartmouth-Hitchcock Medical Center. Archives of Pathology
& Laboratory Medicine. 2017;141(3):402-409. DOI: 10.5858/arpa.2016-0096-OA
[24] Stojsić J, Adzić T, Marić D, Subotić D, Milovanović I, Milenković B, Radojicić J, Marković
J, Dimitrijević D. Histological types and age distribution of lung cancer operated patients
over a 20-year period: A pathohistological based study. Srpski Arhiv za Celokupno
Lekarstvo. 2011;139(9-10):619-624. DOI: 10.1097/JTO.0b013e3181d40fac
[25] Loo PS, Thomas SC, Nicolson MC, Fyfe MN, Kerr KM. Subtyping of undierentiated
non-small cell carcinomas in bronchial biopsy specimens. Journal of Thoracic Oncology.
2010;5(4):442-447
[26] Conde E, Angulo B, Redondo P, Toldos O, Garsia-Garsia E, Suarez A, Rubio-Viqueira
B, Marron C, et al. The use of p63 immunohistochemistry for the identication of squa-
mous cell carcinoma of the lung. PLoS One. 2010;5(8):e12209. DOI: 10.1371/journal.
pone.0012209
[27] Tan D, Zander DS. Immunohistochemistry for assessment of pulmonary and pleural
neoplasms: A review and update. International Journal of Clinical and Experimental
Pathology. 2008;1:19-31. PMCID: PMC2480532
[28] Mukhopadhyay S, Kaenstein A-LA. Subclassication of non-small cell lung carcino-
mas lacking morphologic dierentiation on biopsy specimens: Utility of an immunohis-
tochemical panel containing TTF-1, Napsin A, p63, and CK5/6. The American Journal of
Surgical Pathology. 2011;35:15-25. DOI: 10.1097/PAS.0b013e3182036d05
Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy
http://dx.doi.org/10.5772/intechopen.75316
19

[29] Terry J, Leung S, Laskin J, Leslie KO, Gown AM, Ionescu DN. Optimal immunohis-
tochemical markers for distinguishing lung adenocarcinomas from squamous cell car-
cinomas in small tumor samples. The American Journal of Surgical Pathology. 2010;
34:1805-1811. DOI: 10.1097/PAS.0b013e3181f7dae3
[30] Sterlacci W, Fiegl M, Hilbe W, Auberger J, Mikuz G, Tzankov A. Clinical relevance of
neuroendocrine dierentiation in non-small cell lung cancer assessed by immunohis-
tochemistry: A retrospective study on 405 surgically resected cases. Virchows Archiv.
2009;455(2):125-132. DOI: 10.1007/s00428-009-0812-0
[31] Klingen TA, Chen Y, Gundersen MD, Aas H, Westre B, Sauer T. Thyroid transcription
factor-1 positive primary breast cancer: A case report with review of the literature. Diag-
nostic Pathology. 2010;5:37-41. DOI: 10.1186/1746-1596-5-37
[32] Graham AD, Williams AR, Salter DM. TTF-1 expression in primary ovarian epithelial
neoplasia. Histopathology. 2006;48(6):764-765. DOI: 10.1111/j.1365-2559.2006.02365.x
[33] Stojsic J. Immunohistochemical approach to the diagnosis of adenocarcinoma of the
lung. Journal of Cytology and Histology. 2014;5(3):229. DOI: 10.4172/2157-7099.1000229
[34] Stojsić J, Milenković B, Radojicić J, Percinkovski M. Alveolar adenoma—A rare lung
tumour. Srpski Arhiv Za Celokupno Lekarstvo. 2007;135(7-8):461-464. Serbian. PMID:
17929540
[35] Stojsić J, Milenković V, Radojicić J, Percinkovski M. Pulmonary sclerosing haeman-
gioma—Case report. Srpski Arhiv Za Celokupno Lekarstvo. 2007;135(9-10):569-571.
Serbian. PMID: 18088044
[36] Nishino M, Mai P, Hoang MP, Della Pelle P, Morales-Oyarvide V, Huynh TG, Mark
EJ, Mino-Kenudson M. Napsin A/p40 antibody cocktail for subtyping non-small cell
lung carcinoma on cytology and small biopsy specimens. Cancer Cytopathology. 2016;
124:472-478. DOI: 10.1002/cncy.21707
[37] Bishop JA, Teruya-Feldstein J, Westra WH, Pelosi G, Travis WD, Rekhtman N. p40
(ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma.
Modern Pathology. 2012;25(3):405-415. DOI: 10.1038/modpathol.2011.173
[38] Leivo MZ, Elson PJ, Tacha DE, Delahunt B, Hansel DE. A combination of p40, GATA-3
and uroplakin II shows utility in the diagnosis and prognosis of muscle-invasive urothe-
lial carcinoma. Pathology. 2016;48(6):543-549. DOI: 10.1016/j.pathol.2016.05.008
[39] Tacha D, Bremer R, Haas T, Qi W. An immunohistochemical analysis of a newly
developed, mouse monoclonal p40 (BC28) antibody in lung, bladder, skin, breast,
prostate, and head and neck cancers. Archives of Pathology & Laboratory Medicine.
2014;138(10):1358-1364. DOI: 10.5858/arpa.2013-0342-OA
[40] Turner BM, Cagle PT, Sainz IM, Fukuoka J, Shen SS, Jagirdar J. Napsin A, A new marker
for lung adenocarcinoma, is complementary and more sensitive and specic than thy-
roid transcription factor 1 in the dierential diagnosis of primary pulmonary carcinoma.
Evaluation of 1674 cases by tissue microarray. Archives of Pathology & Laboratory
Medicine. 2012;136:163-171. DOI: 10.5858/arpa.2011-0320-OA
Lung Cancer - Strategies for Diagnosis and Treatment20

[41] Jagirdar J. Application of immunohistochemistry to the diagnosis of primary and meta-
static carcinoma to the lung. Archives of Pathology & Laboratory Medicine. 2008;132:384-
396. DOI: 10.1043/1543-2165(2008)132[384:AOITTD]2.0.CO;2
[42] Ricciardelli C, Lokman NA, Pyragius CE, Ween MP, Macpherson AM, Ruszkiewicz A,
Homann P, Oehler MK. Keratin 5 overexpression is associated with serous ovarian
cancer recurrence and chemotherapy resistance. Oncotarget. 2017;8(11):17819-17832.
DOI: 10.18632/oncotarget.14867
[43] Wallace WA. The challenge of classifying poorly dierentiated tumours in the lung.
Histopathology. 2009;54(1):28-42. DOI: 10.1111/j.1365-2559.2008.03181.x. Review
[44] Chan BA, Bre GM, Hughes BGM. Targeted therapy for non-small cell lung cancer:
Current standards and the promise of the future. Translational Lung Cancer Research.
2015;4(1):36-54. DOI: 10.3978/j.issn.2218-6751.2014.05.01
[45] Thunnissen E, Bubendorf L, Dietel M, Elmberger G, Kerr K, Lopez-Rios F, Moch H,
Olszewski W, Pauwels P, Penault-Llorca F, Rossi G. EML4-ALK testing in non-small cell
carcinomas of the lung: A review with recommendations. Virchows Archiv. 2012;461:245-
257. DOI: 10.1007/s00428-012-1281-4
[46] Scarpinoa S, Vinciguerraa GLR, Di Napolia A, Focheia F, Uccinia S, Iaconob D, Mar-
cheib P, Rucoa L. High prevalence of ALK+/ROS1+ cases in pulmonary adenocarci-
nomaof adoloscents and young adults. Lung Cancer. 2016;97:95-98. DOI: 10.1016/j.
lungcan.2016.04.022
[47] Ilie M, Hofman V, Dietel M, Soria J-C, Hofman P. Assessment of the PD-L1 status by
immunohistochemistry: Challenges and perspectives for therapeutic strategies in lung
cancer patients. Virchows Archiv. 2016;468:511-525. DOI: 10.1007/s00428-016-1910-4
[48] Tsao MS, Kerr KM, Dacic S, Yatabe Y, Hirsch FR. IASLC Atlas of PD-L1 Immu-
nohistochmeistry Testing in Lung Cancer. North Fort Myers, FL, Editorial Rx Press; 2017.
ISBN: 978-0-9832958-7-7
[49] Duréndez-Sáez E, Azkárate A, Meri M, Calabuig-Fariñas S, Aguilar-Gallardo C, Blasco
A, Jantus-Lewintre E, Camps C. New insights in non-small-cell lung cancer: Circulating
tumor cells and cell-free DNA. Journal of Thoracic Disease. 2017;9(Suppl 13):S1332-S1345.
DOI: 10.21037/jtd.2017.06.112
Precise Diagnosis of Histological Type of Lung Carcinoma: The First Step in Personalized Therapy
http://dx.doi.org/10.5772/intechopen.75316
21