Recent advances in the pathology and molecular genetics of lung cancer: A practical review for cytopathologists

Accepted Manuscript Recent advances in the pathology and molecular genetics of lung cancer: A practical review for cytopathologists Erika F. Rodriguez, MD, PhD, Sara E. Monaco, MD PII:

S2213-2945(15)30006-5

DOI:

10.1016/j.jasc.2016.02.005

Reference:

JASC 188

To appear in:

Journal of the American Society of Cytopathology

Received Date: 11 October 2015 Revised Date:

21 February 2016

Accepted Date: 23 February 2016

Please cite this article as: Rodriguez EF, Monaco SE, Recent advances in the pathology and molecular genetics of lung cancer: A practical review for cytopathologists, Journal of the American Society of Cytopathology (2016), doi: 10.1016/j.jasc.2016.02.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Recent advances in the pathology and molecular genetics of lung cancer: A practical review for cytopathologists

3

Erika F. Rodriguez MD, PhD1, Sara E. Monaco MD2

4

Departments of Pathology, Johns Hopkins University1 and University of Pittsburgh Medical Center2.

RI PT

1 2

5 6 7

SC

8 9

M AN U

10 11 12 13

15 Address Correspondence to:

17 18 19 20 21 22 23 24 25

Erika F. Rodriguez MD, PhD Department of Pathology Johns Hopkins University Carnegie 469 - Pathology 600 North Wolfe Street Baltimore, MD 21287 Email: [email protected]

AC C

EP

16

TE D

14

For submission to Journal of the American Society of Cytopathology

1

ACCEPTED MANUSCRIPT

Abstract

2

Lung cancer is one of the most common causes of cancer-related death worldwide. Better

3

understanding of the molecular genetic characteristics of non-small cell lung carcinoma

4

(NSCLC), particularly adenocarcinoma, opened the opportunity for targeted therapies. With the

5

different molecular abnormalities and the different responses to new targeted therapies based on

6

the histological subtype of NSCLC, there came a need to further classify NSCLC into squamous

7

cell carcinoma and adenocarcinoma, and to perform the appropriate molecular testing in these

8

different subtypes to guide management decisions. Given that approximately 70% of lung cancer

9

patients have only small biopsies or cytology specimens available, incorporating the testing of

10

these specimens into the cytopathology laboratory has been crucial. Herein, we review current

11

concepts and recommendations on NSCLC subtyping and molecular testing that are relevant for

12

the cytopathology community.

SC

M AN U

TE D

16

Key Words: adenocarcinoma; cytopathology; lung; non-small cell carcinoma; NSCLC; squamous cell carcinoma; targeted therapy

EP

14 15

AC C

13

RI PT

1

2

ACCEPTED MANUSCRIPT

Introduction

2

Cytopathology has been an important part of the diagnostic testing done for lung cancer for

3

decades. This includes the use of both exfoliative and aspiration specimens for the diagnosis,

4

staging, or follow-up of patients with lung cancer. Despite its widespread use, Of historical

5

interest, Papanicolaou received a curious suggestion from Dr. Henry Cromwell of Cornell

6

Medical College and New York Hospital, to use his "technique "on sputum. It was a sample from

7

a patient with a clinical suspicion of lung cancer. According to Papanicolaou words "the smear

8

from the sputum were not particularly good or disclosed nothing unusual except for one group of

9

cells with enlarged heterogeneous and hyperchromatic nuclei. It was impossible to interpret these

10

as anything else but a group of malignant cells"1. Papanicolaou was not the first to study sputum

11

smears, since it has been attempted as far back as 1876, by Hampeln. Nevertheless, one of the

12

first large series of lung cancers diagnosed by cytology (sputum examinations) was published by

13

Wandall in 1945 as a report of 250 cases2. It was not until the 2004 WHO classification of lung

14

cancer when cytology was specifically addressed3. The 2004 WHO classification recommended

15

that small specimens be categorized into two major histologic groups: non-small cell carcinoma

16

(NSCLC) and small cell lung carcinoma (SCLC)3. This was deemed important due to the

17

different management guidelines for NSCLC and SCLC. Better understanding of the molecular

18

genetic characteristics of NSCLC through early focused studies 4-7 and more recently through

19

comprehensive exome/genome sequencing studies, including the Cancer Genome Atlas (TCGA),

20

enhanced the development of many targeted therapies for different subtypes of lung cancer6,8-10.

21

Targeted inhibitors have been used against epidermal growth factor receptor (EGFR) and

22

anaplastic lymphoma kinase (ALK), in tumors with specific EGFR mutations and ALK

23

rearrangements, respectively, and more recently for tumors with translocated ROS1 and RET

AC C

EP

TE D

M AN U

SC

RI PT

1

3

ACCEPTED MANUSCRIPT

1

11,12

2

driver oncogene and are treated with conventional chemoradiation.

3

In 2011, the International Association for the Study of Lung Cancer/American Thoracic

4

Society/European Respiratory Society (IASLC/ATS/ERS) proposed a new classification that

5

also addresses cytology and small biopsies13. In summary, these guidelines suggest that if there

6

are morphologic features of adenocarcinoma or squamous cell carcinoma clearly present, one

7

should diagnose them as adenocarcinoma or squamous cell carcinoma, opposed to NSCLC. If

8

there is no definitive glandular or squamous differentiation, then immunostains should be

9

performed in order to make a diagnosis of NSCLC, favor adenocarcinoma or NSCLC, favor

M AN U

SC

RI PT

. [Table 1] Nevertheless, most pulmonary adenocarcinomas do not have a known identifiable

squamous cell carcinoma13. The recommendation for cases with the term "favor" incorporated

11

into the final diagnosis is for them to be treated as those with definitive subtyping14. The

12

intention of this nomenclature is to facilitate the finding of cases that appear less differentiated

13

(i.e. needed stains to subclassify) from the better differentiated tumors for possible future study

14

purposes. This approach is currently endorsed by the 2015 WHO classification of lung cancer15,

15

and the task of NSCLC subtyping is included in the new quality pathology measures from the

16

Centers for Medicare and Medicaid Services in the United States.16

17

With these new advances in molecular genetics, treatment guidelines, and diagnostic

18

recommendations, the field of cytology has had to respond to these new challenges by

19

incorporating the subclassification of NSCLC into small biopsies, and creating ways to conserve

20

tissue given that a multitude of ancillary studies may be required on cytological or small biopsy

21

samples, in order to guide important treatment decisions. This has been an important shift for

22

cytopathology laboratories to make, given that the majority of lung cancer patients have

23

metastatic disease at the time of presentation, and may only have a small biopsy or cytology

AC C

EP

TE D

10

4

ACCEPTED MANUSCRIPT

specimen for testing. In the present review, we summarize current concepts on the pathology and

2

molecular genetics of lung cancer, with translational implications to the cytology field.

3

Modalities for tumor sampling

4

Selection of the optimal method to obtain diagnostic tissue for diagnosing pulmonary cancer

5

should include careful consideration of a number of factors, including clinical presentation,

6

patient risk factors, results of imaging studies, location of the lesion, and expertise of

7

practitioners within the institution, among others. Patients with advanced disease typically have

8

minimally invasive procedures targeting an easily accessible metastatic site in order to confirm

9

the diagnosis and to stage the patient at the same time. Conversely, patients with early stage or

10

limited disease may undergo more invasive procedures that will provide diagnostic material, as

11

well as potentially curative resection.

12

Minimally invasive procedures for lung cancer diagnosis or staging can be categorized as

13

bronchoscopic and non-bronchoscopic methods. Bronchoscopic approaches may provide

14

diagnostic material from the primary tumor and stage the patient at the same time using

15

exfoliative and aspiration cytology specimens.

16

ultrasound-guided transbronchial fine needle aspiration (EBUS-TBNA), transesophageal

17

ultrasound-guided transbronchial needle aspiration (EUS-TBNA), and electromagnetic

18

navigational bronchoscopy biopsy (EMN-TBNA) have dramatically helped in the staging of

19

NSCLC patients in a minimally invasive way and have become widely available. With these

20

new diagnostic approaches, many cytology laboratories have had to develop ways to deal with

21

staffing and processing of these specimens, in addition to getting familiar with the interpretation

22

of these specimens. [Table 1] In fact, many of these new techniques are becoming more widely

New approaches, such as endobronchial

AC C

EP

TE D

M AN U

SC

RI PT

1

5

ACCEPTED MANUSCRIPT

utilized, as shown in a recent publication by the College of American Pathologists

2

Interlaboratory Comparison Program in Non-Gynecological Cytopathology where 43.2% of

3

respondent laboratories reported interpreting EBUS=-TBNAs and 14% reported interpreting

4

EMN-TBNAs17. This survey serves as a snapshot to illustrate the increasing variety of

5

respiratory cytology specimens that cytology laboratories are confronted with in practice today.

6

Each of these methods has its advantages and disadvantages. Briefly, EBUS-TBNA has a high

7

sensitivity and negative predictive value. This method is most useful for sampling central lung

8

lesions, suspicious/enlarged mediastinal lymph nodes, and almost any FDG-avid mass in the

9

mediastinum. EBUS-TBNA improves upon earlier techniques like the blinded transbronchial

10

biopsy (e.g. Wang needle) by providing real-time image guidance to increase diagnostic yield

11

and to decrease complications. One important limitation of this method is limited sample size

12

and variable yield, which can be operator dependent, and may be somewhat improved by rapid

13

on site evaluation18-20. CT-guided percutaneous/transthoracic fine needle aspiration biopsy (CTG

14

FNA) is also a highly sensitive method that can sample most intraparenchymal lesions as well as

15

mediastinal lymph nodes or anterior mediastinal masses. However, this procedure transverses the

16

pleura and lung parenchyma, leading to higher rates of pneumothorax. Newer modalities like

17

EMN-TBNA use similar technology as global positioning systems(GPS) in automobiles, to

18

localize and sample peripheral lung lesions in an effort to decrease the incidence of

19

pneumothorax that can occur with a transthoracic approach. At the moment, it is a complex

20

diagnostic procedure that is mainly limited to specialized centers19. With the numerous novel

21

ways to approach thoracic lesions, there is the ability to acquire material for ancillary studies in

22

minimally invasive ways, which improves patient care.

23

Preparation of the sample

AC C

EP

TE D

M AN U

SC

RI PT

1

6

ACCEPTED MANUSCRIPT

After the specimen is obtained several types of cytological preparations can be performed, and

2

processing varies based on where you practice. Usually, two smears are prepared after each pass.

3

Part of the material is smeared and either fixed in 95% alcohol or air-dried. Air-dried slides can

4

be stained with Romanowsky-based stains (e.g. Diff-Quik), and may be evaluated on-site for

5

adequacy and preliminary diagnosis. The material fixed in 95% alcohol is typically stained with

6

the Papanicolaou stain at the laboratory. Alternatively concentration cytological methods such as

7

cytospin or thin layer preparations can be made. Thin layer preparations are fixed in alcohol-

8

based media such as ThinPrep or SurePath.

9

Cell blocks (CB) can be prepared from the needle rinses or from additional dedicated FNA

10

passes that are not utilized for smears. Although the method of preparation has evolved, the

11

basic principles for all techniques are the same. It involves fixation, centrifugation and transfer of

12

the cell pellet into a cassette for paraffin embedding and processing similar to that for surgical

13

specimens. Several methods have been described and a careful review of the main

14

methodologies was done by Jain et al20. On a recent survey made by Crapanzano et al21 the most

15

common methods used for cell block processing were Plasma-thrombin, Histogel and Cellient

16

automated cytoblock system21. In their survey 44% of the responders were unsatisfied or

17

sometimes satisfied with their CB. Low-cellularity was the main cause of unsatisfactory CBs.

18

Many factors may influence the suboptimal results including operator, amount of sample,

19

location, type of lesion, and the variability of CB processing20,21. It is important to remember

20

that the variations in fixation can affect immunohistochemistry studies as well as molecular tests,

21

and thus, the tests need to be validated on cytological specimens if they are processed differently

22

than the surgical specimens sent for testing20. Certain fixatives such as Bouin solution, B5 and

23

Zenker fixative, other harsh metal or acidic fixatives and decalcifying agents, adversely affect

AC C

EP

TE D

M AN U

SC

RI PT

1

7

ACCEPTED MANUSCRIPT

the quality of DNA or interfere with the PCR studies, and thus, are not recommended for

2

molecular tests22. Since scant cellularity is also an important issue on cytology/CB specimens, it

3

is important to have a multidisciplinary approach whereby more material is collected upfront on

4

the pre-analytical side (e.g. additional FNA passes for CB or use of larger tissue biopsy), and

5

more tissue is preserved on the backend (e.g. cutting blanks upfront, limiting IHC panels) to

6

maximize diagnostic yield. In addition, having a streamlined approach for processing these

7

pulmonary specimens for molecular testing is important given that the CAP/IASLC/AMP

8

guidelines recommend that results be available within 2 weeks time)22. While everyone would

9

like a perfect standardized procedure for the molecular testing of cytology specimens, the

10

strategy used varies depending on your institution and resources, and the type of specimen

11

received. Some institutions prefer to use cell blocks for molecular testing to avoid having to

12

validate on differently fixed cytological specimens, while others prefer to use aspirate smears for

13

testing or to use aspirate slides only if the cell block fails testing as an alternative back-up

14

specimen. Thus, the decision of how and what to test is a difficult one that has to be considered

15

by each institution offering these important tests, especially since there is an increasing

16

utilization of small biopsies and cytology specimens for these tests.

17

IHC has been successfully performed on unstained direct smear slides or slides that are either

18

air-dried or alcohol fixed, provided that they have been appropriately validated23-25. Knoepp at el

19

report that in their practice they use positively charged slides for preparing smears that can then

20

be used for IHC26. The slides are fixed in formalin for 30 to 60 minutes and followed by antigen

21

retrieval and immunostaining26.

22

In addition to performing molecular studies on formalin fixed paraffin embedded cell blocks,

23

other cytological preparations have been proven to be suitable for molecular testing23,27. Diff-

AC C

EP

TE D

M AN U

SC

RI PT

1

8

ACCEPTED MANUSCRIPT

Quik stained aspirate slides have been shown to be advantageous for molecular testing because

2

they are amenable to adequacy assessment on-site without the need for a cover slip, do not have

3

issues related to fixation, and do not need to be detained prior to analysis28,29. Killian et al30

4

reported that DNA obtained from Diff-Quik stained slides has better preservation and integrity

5

than the material collected from Papanicolaou stained smears. Liquid based preparations (e.g.

6

ThinPrep slides) have also been shown to be adequate for molecular tests31-34. This preparation is

7

an important asset when there is no on-site evaluation. Also, the cell suspension aliquot not used

8

on the slide preparation can be centrifuged and used for mutation tests or IHC31-34. In some

9

studies, the direct smears seem to have higher cellularity and therefore higher yield of DNA

M AN U

SC

RI PT

1

extraction35. ALK rearrangement FISH assay can also be performed on direct smears and liquid

11

based cytology29,36,37.

12

In general, each laboratory should determine the minimum number of tumor cells needed for

13

mutation detection during validation. As a rule of thumb, 100-500 neoplastic cells are usually

14

adequate for sequencing based methodologies 28,38 and it is recommended by The CAP/AMP/IASLC

15

guidelines that a laboratory should be able to detect mutations in specimens with at least 50%

16

tumor cells; however, some studies have shown a lower requirement for cells or proportion of

17

tumor using more sensitive testing methods. 28,31. Fluorescence in situ hybridization assays

18

require a minimum of 50-100 neoplastic nuclei38. Liquid based preparations (e.g. ThinPrep

19

slides) have also been shown to be adequate for molecular tests31-34.

20

Diagnostic evaluation of non-small cell lung carcinoma in cytology and small biopsies

21

The classification of pulmonary adenocarcinoma proposed by IASLC/ATS/ERS in 2011 and

22

recently endorsed by the new 2015 WHO classification introduced new entities such as the

AC C

EP

TE D

10

9

ACCEPTED MANUSCRIPT

concept of adenocarcinoma in situ and minimally invasive adenocarcinoma, and abandons the

2

old terminology of bronchoalveolar carcinoma15,39. It also recognizes five major architectural

3

patterns (i.e. lepidic, acinar, solid, papillary and micropapillary), as well as four cytologic

4

variants (i.e. mucinous, colloid, fetal, and enteric) 40. Cytologic variants, for example mucinous

5

adenocarcinoma, may have any architectural pattern. The importance of this classification is

6

supported by studies showing that the different patterns are associated with varying prognosis41-

7

43

8

micropapillary have unfavorable prognosis41-43. However in up to 90% of cases, pulmonary

9

adenocarcinoma will have a combination of patterns, and the prognosis will be driven by the

RI PT

1

M AN U

SC

. Lepidic, papillary and acinar are considered good/intermediate prognosis, while solid and

primary pattern or by the presence of any solid or micropapillary component 41-43.

11

The current recommendation for small biopsies and cytology specimens is to classify NSCLC

12

into specific categories, such as adenocarcinoma and squamous cell carcinoma. [Table 2] Further

13

subtyping within adenocarcinomas is not standard practice at this time in cytology samples, until

14

further studies on reproducibility and medical necessity are established15,44-46. In a recent study

15

using cytology samples, Sigel et al45,47 suggested several cytomorphologic features, such as

16

nuclear size, chromatin pattern, and nuclear contours, that could be used in a scoring system that

17

correlated well with histologic grade and prognosis45,47. In our experience48, concordant

18

subclassification of adenocarcinoma using the IASLC/ATS/ERS classification between the

19

dominant/single pattern on resection specimens and matched cytology specimens was present in

20

40% of the cases48. Table 3 summarizes some of the morphologic features reported in different

21

patterns of adenocarcinoma49. Although the application of this classification in cytologic

22

specimens is challenging and may even be unreliable in some instances48. Some features such as

23

predominance of 3-dimensional clusters, necrotic background, pleomorphic nuclei, marked

AC C

EP

TE D

10

10

ACCEPTED MANUSCRIPT

irregular nuclear contours and marked nuclear enlargement may be suggestive of the

2

prognostically adverse solid pattern48,49, however these cytologic features are not entirely

3

specific, were studied in a relatively small number of cases, and therefore require further

4

validation50. The micropapillary pattern appears to be even more challenging in cytologic

5

specimens, given that the presence of micropapillary tufts has been shown not to be specific for

6

adenocarcinoma with a micropapillary pattern, and identification is difficult given that the

7

micropapillary pattern is frequently seen in combination with other patterns of adenocarcinoma

8

that may dominate50 . As mentioned above, many of these findings are based on a small number

9

of cases and further studies are recommended.

M AN U

SC

RI PT

1

In the NSCLC group, tumors that are well differentiated can be subtyped usually without major

11

difficulties. Tumors that have scant cellularity or are poorly differentiated are more challenging,

12

resulting in about 10-35% of small biopsies which are frequently considered NSCLC, not

13

otherwise specified. Immunohistochemical studies are very helpful on further subtyping theses

14

tumors lacking morphological differentiation51,52 and improve the interobserver agreement rate53,

15

especially in poorly differentiated cases of NSCLC. In these cases, the terminology of NSCLC,

16

favor adenocarcinoma or favor squamous cell carcinoma, or NSCLC not otherwise specified (if

17

IHC is indeterminate) are typically used. Further studies will be required to assess the precise

18

prognostic implications of these practical cytologic categories, in order to see if there is a

19

difference

20

adenocarcinoma. Several immunohistochemical panels have been recommended to help in this

21

effort, which underscores the importance of collecting material for a cell block in all lung cancer

22

cases

23

squamous cell markers (e.g. CK5/6, P63 or P40) and adenocarcinoma markers (eg, TTF-1 or

AC C

EP

TE D

10

54,55,56

between

cases

called

adenocarcinoma

and

cases

called

NSCLC,

favor

. Most authors suggest the use of a limited immunohistochemical that incorporates

11

ACCEPTED MANUSCRIPT

Napsin A)51,52,54,57,58. The current WHO recommends the use of limited immunostains (e.g., TTF-

2

1 and P40) when needed for subclassification in order to save material for molecular testings15.

3

However, expanded immunohistochemical studies should be considered in case that stain

4

negative for keratins or TTF1, or in cases with unusual morphology for NSCLC, or in cases with

5

multiple pulmonary nodules with a clinical suspicion of possible metastatic disease.

6

The interpretation of immunocytochemical stains can be challenging in cases with limited

7

cellularity or weak/non-specific staining. Of the adenocarcinoma and squamous cell carcinoma

8

markers, TTF1 and p40 tend to be the more specific, respectively59-62.

9

remember that although TTF-1 is positive in approximately 80% of lung adenocarcinomas, it can

10

also be present in thyroid carcinoma and other metastatic tumors, including endometrial, colon

11

and breast carcinomas59-61. Rare cases of TTF-1 negative, Napsin A positive primary pulmonary

12

adenocarcinoma have been reported;

13

carcinoma, clear cell carcinoma of the gynecologic tract, a subset of thyroid carcinoma,

14

endometrial carcinoma, and hepatocellular carcinoma63,65. A subset of primary pulmonary

15

adenocarcinomas, such as adenocarcinomas with mucinous features, can be TTF-1 and Napsin A

16

negative, while positive for CK7 and CK20. Primary pulmonary adenocarcinoma with enteric

17

differentiation can have morphologic features similar to colonic adenocarcinoma, and may have

18

a similar immunohistochemical profile (CK20, CDX-2 positive and CK-7 negative). However,

19

enteric adenocarcinoma of the lung is a diagnosis of exclusion, and should only be made after

20

careful clinical exclusion of a gastrointestinal tract primary. In addition to distant metastases,

21

careful consideration of a malignant mesothelioma is important in patients presenting with a

22

pleural-based mass, and in this situation, an immunohistochemical panel of adenocarcinoma

23

versus mesothelioma markers is useful.

It is important to

M AN U

SC

RI PT

1

; however, Napsin A is also positive in renal cell

AC C

EP

TE D

63-65

12

ACCEPTED MANUSCRIPT

Napsin A has limitations given that it can show strong staining of alveolar macrophages52, and

2

may be positive in up to 26% of squamous cell carcinoma66 and tumors of non-pulmonary origin.

3

Recently Mukhopadhyay and Katzenstein44 reported that polyclonal, not monoclonal Napsin A

4

antibodies can be positive in a supranuclear location in a variety of metastatic mucinous

5

carcinomas of extrapulmonary origin Rekhtman e all also described similar findings.67Other

6

potential pitfalls include entrapped benign pulmonary epithelium and p63 positive bronchial

7

reserve cells or squamous metaplasia, which may also show cytological atypia due to reactive

8

changes. P40 in particular appears to be more specific for the diagnosis of squamous cell

9

carcinoma than p63 or other markers of squamous cell carcinoma62. However, it must be noted

10

that p40 may rarely stain other carcinomas (e.g. urothelial, myoepithelial and mucoepidermoid

11

carcinoma). Other markers that should be considered in a panel include neuroendocrine markers

12

(eg, synaptophysin) given that large cell neuroendocrine carcinomas can morphologically mimic

13

an adenocarcinoma. NSCLC may also be negative for adenocarcinoma and squamous markers,

14

but if positive for mucin (even as little as two positive cells) the findings should be interpreted as

15

NSCLC, favor adenocarcinoma. It is recognized that a small subset of NSCLCs, even with the

16

use of immunohistochemistry, will not be classifiable. The diagnosis of NSCLC-NOS is

17

appropriate in these cases.39 However, molecular tests (EGFR and ALK) are still recommended

18

in these ambiguous cases46. In summary, a limited immunohistochemical stains (TTF-1 and P40

19

or P63) and in some cases mucicarmine are usually sufficient for the differentiation between

20

adenocarcinoma versus squamous cell carcinomas. When morphology and immunohistochemical

21

stains cannot reliably distinguish the subtypes of NSCLC, and the tumor is clinically presumed

22

to be of lung origin, then the terminology NSCLC-NOS is recommended in order to preserve

23

material for molecular tests, opposed to exhausting material for a long panel of immunostains.

AC C

EP

TE D

M AN U

SC

RI PT

1

13

ACCEPTED MANUSCRIPT

Another important consideration, especially when dealing with adenocarcinomas is the

2

possibility of metastasis, especially in cases where there is a clinical history of malignancy

3

elsewhere or clinical suspicion. Metastatic disease is more common than primary pulmonary

4

carcinoma. Although the typical radiologic finding is multiple peripheral nodules or interstitial

5

thickening, up to 10% of cases can have metastatic disease presenting as a solitary mass68

6

Additionally, the use of dual staining with nuclear and cytoplasmic/membranous stains has been

7

applied in cytopathologic evaluation of NSCLC, in order to maximize the number of antibodies

8

utilized for characterization, while preserving tissue for potential molecular studies

9

instance, TTF-1/Napsin and P63/CK5 have shown high specificity and sensitivity, and are

10

especially useful in specimens with scant cellularity69-71. However, double stains can be difficult

11

to work with slow turn-around-time and may not be available in all laboratories.

12

Ancillary studies, including immunostains, should be used in combination with the

13

cytomorphology for diagnosis and not in isolation. Some diagnostic challenges that may affect

14

interpretation is that adenocarcinoma can be positive for CK5/6 and P63, but usually showing

15

weak to moderate expression, and will usually show staining for TTF1 in the same cells. These

16

cases may best be interpreted as “NSCLC, favor adenocarcinoma”. If the p63/p40 staining and

17

TTF1 staining appear in different tumor cells, then the possibility of an adenosquamous cell

18

carcinoma should be considered. Another potential pitfall is that P63 can be strongly positive in

19

up to 50% of diffuse large B-cell lymphomas involving the lung and the morphology could be

20

confused with a poorly differentiated NSCLC, thus p40 has emerged as a more specific marker72.

21

Table 4 summarizes potential IHC pitfalls.

69-71

. For

AC C

EP

TE D

M AN U

SC

RI PT

1

14

ACCEPTED MANUSCRIPT

Other considerations include the diagnosis of adenosquamous carcinoma of the lung, reserved to

2

surgical specimen where at least 10% of each component should be present. However, on

3

cytology samples, if convincing cytologic features of both components are present, or

4

alternatively positive adenocarcinoma marker/mucin stains and a positive squamous marker in

5

different cell populations, the diagnosis of NSCLC-NOS with concurrent glandular and

6

squamous differentiation may be applied. It is important to not dismiss these cases as simply

7

squamous cell carcinoma, which could exclude the patient from molecular testing and potentially

8

important targeted therapies. Salivary gland type tumors and myoepithelial tumors, such as

9

mucoepidermoid carcinoma and epithelial-myoepithelial carcinomas, should also be considered

M AN U

SC

RI PT

1

in some cases before diagnosing NSCLC-NOS.

11

Molecular signaling cascades relevant to lung cancer

12

Multiple genetic alterations are now under investigation in lung carcinomas, given the

13

association with response or lack of response with targeted therapies and chemotherapy. A

14

summary of some of the more common mutations is seen below and summarized in Table

15

5. [Table 5]

16

Epidermal Growth Factor Receptor pathway

17

Epidermal growth factor receptor (EGFR) is a cellular transmembrane tyrosine kinase receptor

18

that triggers many signaling cascades that regulate growth, cell survival, proliferation,

19

angiogenesis, cell migration, and differentiation in lung and other cancers. Other components of

20

this signaling cascade that are mutated at various rates in lung cancer include RAS and BRAF.

21

Mutations in the tyrosine kinase domain of the EGFR gene lead to an activation of EGFR in a

22

ligand-independent manner, which increases the activity of survival signaling pathways. EGFR

AC C

EP

TE D

10

15

ACCEPTED MANUSCRIPT

gene mutations are present in 10-20% of patients with pulmonary adenocarcinoma of European

2

ancestry. These mutations are more common in women, non-smokers and patients of Asian

3

descent73-75. Ninety percent of the EGFR mutations involve exon 19 deletions or exon 21 point

4

mutations. They are gain-of-function mutations that lead to an independent activation of EGFR

5

pathway signaling. Targeting the altered gene by tyrosine kinase inhibitors (TKI) causes a

6

sudden cessation of the aberrant signaling cascade and may even induce apoptosis. EGFR

7

tyrosine kinase inhibitors block this activation through regulation of EGFR tyrosine

8

phosphorylation. Of historical interest, patients with lung cancer have been treated with TKIs

9

before knowledge of EGFR mutations. In fact, the improved response in patients using Gefitinib

10

and Erlotininb led to investigation of the EGFR pathway in these tumors and the identification of

11

several mutations76-78.

12

Characterization of EGFR gene mutation status is important in current treatment algorithms for

13

pulmonary adenocarcinoma. Patients with advanced EGFR-mutant tumors will be offered

14

treatment with EGFR TKI. Some advantages of this treatment strategy are tolerability, better

15

quality of life, and longer progression free survival4. Patients with tumors lacking this mutation

16

do not benefit from EGFR-TKIs, and have been shown to have a shorter progression free

17

survival when compared with patients who received conventional chemoradiation therapy,5

18

which highlights the importance of molecular testing in the management of these patients. It is

19

also important to remember that therapeutic response to anti-EGFR therapy is limited to high

20

stage or recurrent disease and is measured in progression free survival. Unfortunately the

21

response is not long-lasting as most patients usually relapse less than 1 year after initiating

22

treatment with TKI inhibitors79. Regarding targeted therapies against mutated EGFR, there are

23

two main strategies: monoclonal antibodies directed against the extracellular ligand-binding

AC C

EP

TE D

M AN U

SC

RI PT

1

16

ACCEPTED MANUSCRIPT

domain of EGFR (ex, cetuximab) and small-molecule TKIs. The most common mechanism of

2

acquired resistance in EGFR-mutated NSCLC being treated with TKIs is the development of

3

exon 20 mutations, including insertions or the T790M point mutation, which are rarely ever

4

found in the absence of prior EGFR TKI treatment. The current CAP/IASCLC/AMP guidelines

5

recommend offering testing for this mutation.

6

Currently, there are several methods for testing for EGFR mutation, the most widespread being

7

direct sequencing from formalin-fixed paraffin-embedded tumor tissue blocks. At the moment,

8

cancer specific panels testing for multiple simultaneous alterations in small tissue samples are

9

feasible through next generation sequencing80. Molecular testing for EGFR mutations has not

M AN U

SC

RI PT

1

been standardized, and therefore current CAP/IASLC/AMP guidelines recommend laboratories

11

choose any assay that performs with equal or better sensitivity or specificity than classic Sanger

12

sequencing22.

13

Antibodies specific for common EGFR mutations have also been developed and applicable to

14

immunohistochemistry. These include monoclonal antibodies targeting the exon 19 (E746-A750)

15

and exon 21 (L858R) deleted proteins. The antibody against exon 21 (L858R) have sensitivity of

16

92% and a positive predictive value of 99%81,82. The antibody against exon 19 has a lower

17

sensitivity for detection81,82. These techniques may hold great promise and create lower cost

18

testing options available on even smaller tumor samples. Hasanovic et al. in their study using

19

cytology samples and small biopsies showed that immunohistochemistry using a stringent

20

interpretation of the staining pattern can be helpful. Although there were no false positive results,

21

a negative result did not completely exclude a mutation83, and therefore would require

22

confirmatory mutational testing. The use of IHC may be feasible as a screening test, in addition

23

to cases where there is not enough material for molecular tests.

AC C

EP

TE D

10

17

ACCEPTED MANUSCRIPT

KRAS mutations

2

K-RAS is a guanosine triphosphate binding protein that transduces growth signals from multiple

3

tyrosine kinases including EGFR and MET. The KRAS gene is the most commonly mutated

4

oncogene in lung adenocarcinoma, present in approximately 20-35% of patients with NSCLC,

5

predominantly smokers84,85. KRAS gene mutations are activating and lead to constitutive

6

downstream signaling and cell proliferation. Of clinical relevance, lung adenocarcinoma with

7

activating KRAS mutations are usually associated with worse prognosis and are resistant to both

8

conventional chemotherapeutic agents and EGFR TKI86,87. KRAS mutations are relatively easy to

9

test for since more than 95% of the mutations are in codons G12 or G13, and are mutually

M AN U

SC

RI PT

1

exclusive with EGFR and ALK alterations84,85. However, the current CAP/IASLC/AMP

11

guidelines do not recommend KRAS testing as a sole determinant of EGFR TKI therapy22,

12

because a significant number of the KRAS wild type tumors also lack EGFR mutations.

13

Therefore, KRAS mutational assays could be performed to exclude KRAS mutant tumors for

14

EGFR testing as part of an algorithm or with next generation sequencing platforms that facilitate

15

testing of multiple genes simultaneously. Therapeutic strategies targeting KRAS-mutated tumors

16

include inhibitors of downstream signaling components (e.g. MEK inhibitors) and are currently

17

under clinical trials.

18

Anaplastic lymphoma kinase (ALK)

19

ALK is a transmembrane tyrosine-kinase receptor expressed in several normal tissues, but not in

20

normal lung. ALK translocations were first described in a subset of anaplastic large cell

21

lymphomas. Subsequently, rearrangements, mutations and amplifications of this gene have been

22

described in several solid tumors. In 2007, Soda described the fusion gene EML4-ALK in mouse

AC C

EP

TE D

10

18

ACCEPTED MANUSCRIPT

models and in patients with NSCLC88. ALK rearrangements are present in approximately 3-5%

2

of patients with pulmonary adenocarcinoma, and the patients are usually younger.7,89 Tumors

3

harboring ALK rearrangements have marked sensitivity to crizotinib, a tyrosine kinase inhibitor

4

that binds to abnormal ALK protein. However, resistance can also develop and second

5

generation ALK inhibitors are currently under investigation.

6

Although many strategies to assess ALK rearrangements with comparable performance have

7

been developed, the CAP/IASLC/AMP guidelines recommend the use of ALK Break Apart FISH

8

Probe Kit, which was the first FDA assay approved as a diagnostic tool for targeted therapy with

9

crizotinib22. Some research has also been done to look at using diagnostic immunohistochemistry

M AN U

SC

RI PT

1

to screen for ALK rearrangements given that is it less complex, more widely available, and

11

cheaper than FISH studies.93, 94, 90. The ALK antibodies that have been validated include the

12

ALK mouse monoclonal antibody, clone 5A4(Novocastra, Newcastle, UK) and a rabbit

13

monoclonal anti-human antibody, CD246 (clones D5F3 and D9E4) from Cell Signaling

14

Technology (Danvers, Massachusetts, USA), which both showed good sensitivity, moderate

15

specificity and variable reproducibility90,91 Thus, some have proposed that follow-up

16

confirmatory FISH studies be performed in indeterminate cases. Recently the Ventana ALK

17

(D5F3) Assay received FDA approval as a diagnostic tool to identify lung cancer patients that

18

would benefit for crizotinib. Since, interpretation of t FISH studies can be challenging and

19

require expert personnel for performance and interpretation of test results, the ability to screen

20

adenocarcinoma cases with an ALK immunostain would dramatically decrease costs associated

21

with performing FISH tests, particularly given that only a small percent of adenocarcinomas are

22

ALK positive. Current guidelines recommend the use of ALK FISH testing, however

23

imunohistochemisty assays, if carefully validated, can be used as a screening methodology 22, 92.

AC C

EP

TE D

10

19

ACCEPTED MANUSCRIPT

ROS1

2

ROS1 is a tyrosine-kinase receptor of the insulin receptor family. ROS1 fusions are present in

3

approximately 2% NSCLC. The patients are usually young and never-smokers. Several different

4

fusion partners have been described11. Although the patient's demographics are similar, ROS1

5

and ALK rearrangements are mutually exclusive11 . Currently there is no formal screening

6

guideline for ROS1 fusions. However, patients with this mutation seem to respond to crizitotinib,

7

the same drug used ALK-positive tumors in initial clinical trials, in addition to FDA-approved

8

RET TKIs that are available (cabozantinib and vandetanib)93. There are several FISH probes

9

commercially available for ROS1, as well as a ROS1 monoclonal antibody that has recently

M AN U

SC

RI PT

1

been developed and validated for use in immunohistochemistry94,95. Of interest, ROS1 alterations

11

are not limited to adenocarcinoma, but are infrequently observed in large cell and squamous cell

12

carcinomas.

13

KIF5B-RET

14

Gene fusions involving RET have been recently been described as a novel oncoprotein in a

15

subset of NSCLC, primarily in young non-smokers. It is present in approximately 1-2% of

16

pulmonary adenocarcinomas, and several kinase inhibitors (e.g. vandetanib, sinitinib and

17

sorafenib) are under clinical trials as a therapeutic strategy96,97 Immunohistochemical antibody is

18

also available95.

19

PI3K/AKT/mTOR pathway abnormalities

20

Approximately 5-7% of lung adenocarcinomas have been shown to have mutations in PIK3CA,

21

which can lead to constitutive activation of the PI-3 kinase pathway. These mutations have been

22

associated with a worse prognosis, and may select patients that could benefit from

AC C

EP

TE D

10

20

ACCEPTED MANUSCRIPT

PI3K/AKT/mTOR pathway inhibitors. An important aspect of these mutations is that they can

2

exist with other mutations, and are therefore not mutually exclusive of other mutations.

3

Current Recommendations for Molecular Testing in Cytology Specimens from NSCLC

4

Given the numerous alterations discovered in NSCLC, particularly adenocarcinoma , guidelines

5

for molecular testing were recently published by the College of American

6

Pathologists(CAP)/International Association for the Study of Lung Cancer (IASLC)/Association

7

from Molecular Pathology (AMP)22. These guidelines recommend EGFR and ALK testing for

8

pulmonary adenocarcinoma and mixed lung cancers with an adenocarcinoma component, such as

9

adenosquamous, carcinosarcoma, pleomorphic carcinoma, or in cases when an adenocarcinoma

M AN U

SC

RI PT

1

component cannot be excluded (NSCLC-NOS).

11

Having enough tissue for molecular assays can be a challenge in cytology and small biopsy

12

samples. The current CAP/IASLC/AMP guideline recommends the use of formalin fixed

13

paraffin-embedded (FFPE) blocks22. However, the use of cells microdissected from cytology

14

smears using alcohol fixed Papanicolaou or Thin Prep slides, or even air dried Diff-Quik stained

15

slides, for molecular testing has been successful and may play an increasing role in the future, as

16

long as the testing is validated in an individual lab. Most laboratories are implementing testing

17

on cell block material, given that most of the tests have been validated on FFPE surgical

18

specimens and the ALK FISH testing is FDA approved for FFPE. In order to conserve tissue for

19

these important molecular tests, a two pronged approach is typically being incorporated,

20

including the use of enhanced collection (eg, additional passes for cell block), triage and

21

conservation of material (eg, cutting 15-20 unstained slides up front to avoid multiple refacing of

22

the paraffin block), and limited immunohistochemical stains. At the moment there are no

AC C

EP

TE D

10

21

ACCEPTED MANUSCRIPT

universal guidelines to test for the rarer alterations (e.g. ROS, RET). However, in larger

2

multidisciplinary and cancer centers, pulmonary adenocarcinomas that are negative for EGFR or

3

ALK alterations may be tested for the rarer mutations (e.g. ROS), as a pre-requisite for specific

4

clinical trials. Furthermore, as the list of biomarkers and actionable molecular targets increases,

5

many academic medical centers are moving towards next generation sequencing to look for a

6

myriad of gene mutations that may be of benefit in lung cancer patients26,30 32,98.

7

For samples with limited cellularity all efforts should be made to save tissue for molecular

8

studies (EGFR and ALK testing). In this setting, targeted mutation methods (e.g. PCR) have the

9

advantage of better analytic sensitivity than Sanger sequencing where increased enrichment of

M AN U

SC

RI PT

1

tumor cell DNA in the samples (30-40% of neoplastic cells) are needed. This issue may be

11

overcome by macro and microdissection, but does increase labor and processing time. FISH is

12

the primary method used to detect ALK, ROS1 and RET fusions. At the moment, IHC is

13

available for ALK testing, as a surrogate for the fusion, and can be attempted in samples with

14

limited cellularity, but positive results may require confirmation by FISH. Also EGFR mutation

15

IHC can be attempted on samples that fail or have limited cellularity for molecular tests, and a

16

positive IHC result may preclude additional sampling.

17

Next generation sequencing (NGS), also known as high-throughput sequencing or massive

18

parallel sequencing, is a term used to describe different, newer sequencing technologies that

19

require smaller amount of input DNA to detect several different molecular alterations. Most

20

commonly utilized NGS platforms involve sequencing by synthesis, where DNA pieces to be

21

tested are bound to a specific array platform and labeled using DNA polymerase in a sequential

22

fashion99. One of the advantages of NGS, compared to traditional Sanger sequencing, is its

23

enhanced ability to detect multiple altered genes on a single platform, which reduces the cost and

AC C

EP

TE D

10

22

ACCEPTED MANUSCRIPT

the amount of diagnostic tissue required. The simultaneous testing of multiple relevant mutations

2

using FFPE tissue and NGS has allowed the development of specific panels that include genes of

3

interest to clinical oncology groups for efficient identification of therapeutic targets.

4

Additionally, NGS is ideally suited to identify the development of resistant mutations during the

5

course of treatment, and in this capacity, optimized small biopsies and cytology specimens will

6

be crucial when a patient relapses. Another potential benefit of NGS platforms is the

7

identification of gene rearrangements, although these may be challenging to detect at times and

8

the practical aspects of the technology are still evolving. Also, FDA approvals for certain drugs

9

(e.g. crizotinib) are linked to specific companions tests (e.g. FISH), and the incorporation of

M AN U

SC

RI PT

1

NGS approaches may require additional regulatory guidelines in the future.

11

Future Directions

12

The enormous advances in our understanding of the molecular basis of pulmonary cancer are

13

starting to have a strong impact on the treatment of these patients for which there was little hope

14

in the past. These advances and the increasing availability of targeted therapies have made the

15

initial interpretation of lung cancer, and optimal utilization of tumor tissue, a priority. New

16

molecular testing applications like next generation sequencing will likely become more popular

17

and widespread as the technology improves and becomes cheaper and more available to smaller

18

hospitals. This will continue to have an impact on cytopathology, since it is currently the first,

19

and sometimes the only, sample available for diagnosis and therapeutic management. This

20

review highlights some of the recent impacts of the molecular testing guidelines and lung cancer

21

classification guidelines on the practice of cytopathology today.

22

Bibliography

AC C

EP

TE D

10

23

ACCEPTED MANUSCRIPT

6. 7. 8. 9. 10. 11.

12. 13.

14.

15. 16.

17.

18.

19.

RI PT

5.

SC

4.

M AN U

3.

TE D

2.

Papanicolaou GN. Diagnostic value of exfoliated cells from cancerous tissues. Journal of the American Medical Association. Jun 1 1946;131:372-378. Wandall HH. A Study of Neoplastic Cells in Sputum as a Contribution to the Diagnosis of Primary Lung Cancer. Acta Chirurgica Scandinavia. 1944 1944(91):1. Travis WD, Brambilla C, Muller-Hermilink H, C. H. Pathology and Genetics Tumours of the Lung, Pleura, Thymus and Heart. Lyon, France: IARC; 2004. Hirsch FR, Janne PA, Eberhardt WE, et al. Epidermal growth factor receptor inhibition in lung cancer: status 2012. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. Mar 2013;8(3):373-384. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. The New England journal of medicine. Sep 3 2009;361(10):947-957. Rudin CM, Durinck S, Stawiski EW, et al. Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer. Nature genetics. Oct 2012;44(10):1111-1116. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. The New England journal of medicine. Oct 28 2010;363(18):1693-1703. Cancer Genome Atlas Research N. Comprehensive molecular profiling of lung adenocarcinoma. Nature. Jul 31 2014;511(7511):543-550. Cancer Genome Atlas Research N. Comprehensive genomic characterization of squamous cell lung cancers. Nature. Sep 27 2012;489(7417):519-525. Peifer M, Fernandez-Cuesta L, Sos ML, et al. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nature genetics. Oct 2012;44(10):1104-1110. Bergethon K, Shaw AT, Ou SH, et al. ROS1 rearrangements define a unique molecular class of lung cancers. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Mar 10 2012;30(8):863-870. Drilon A, Wang L, Hasanovic A, et al. Response to Cabozantinib in patients with RET fusionpositive lung adenocarcinomas. Cancer discovery. Jun 2013;3(6):630-635. Travis WD, Brambilla E, Noguchi M, et al. International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society: international multidisciplinary classification of lung adenocarcinoma: executive summary. Proceedings of the American Thoracic Society. Sep 2011;8(5):381-385. Kerr KM, Bubendorf L, Edelman MJ, et al. Second ESMO consensus conference on lung cancer: pathology and molecular biomarkers for non-small-cell lung cancer. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. Sep 2014;25(9):1681-1690. Travis W. D.; Brambilla E BP, Marx A, Nicholson A. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. 4th ed. Lyon2015. Cagle PT, Allen TC. The 2015 Physician Quality Reporting System reflects pathologists' role in lung cancer biomarker testing. Archives of pathology & laboratory medicine. Apr 2015;139(4):445. Sturgis CD, Marshall CB, Barkan GA, et al. Respiratory Cytology-Current Trends Including Endobronchial Ultrasound-Guided Biopsy and Electromagnetic Navigational Bronchoscopy: Analysis of Data From a 2013 Supplemental Survey of Participants in the College of American Pathologists Interlaboratory Comparison Program in Nongynecologic Cytology. Archives of pathology & laboratory medicine. Jan 2016;140(1):22-28. Karunamurthy A, Cai G, Dacic S, Khalbuss WE, Pantanowitz L, Monaco SE. Evaluation of endobronchial ultrasound-guided fine-needle aspirations (EBUS-FNA): correlation with adequacy and histologic follow-up. Cancer cytopathology. Jan 2014;122(1):23-32. Leong S, Ju H, Marshall H, et al. Electromagnetic navigation bronchoscopy: A descriptive analysis. Journal of thoracic disease. Apr 1 2012;4(2):173-185.

EP

1.

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

24

ACCEPTED MANUSCRIPT

24.

25.

26. 27.

28. 29.

30.

31.

32. 33. 34.

35.

36.

RI PT

SC

23.

M AN U

22.

TE D

21.

Jain D, Mathur SR, Iyer VK. Cell blocks in cytopathology: a review of preparative methods, utility in diagnosis and role in ancillary studies. Cytopathology : official journal of the British Society for Clinical Cytology. Dec 2014;25(6):356-371. Crapanzano JP, Heymann JJ, Monaco S, Nassar A, Saqi A. The state of cell block variation and satisfaction in the era of molecular diagnostics and personalized medicine. CytoJournal. 2014;11:7. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Archives of pathology & laboratory medicine. Jun 2013;137(6):828-860. Hookim K, Roh MH, Willman J, et al. Application of immunocytochemistry and BRAF mutational analysis to direct smears of metastatic melanoma. Cancer cytopathology. Feb 25 2012;120(1):52-61. Knoepp SM, Hookim K, Placido J, Fields KL, Roh MH. The application of immunocytochemistry to cytologic direct smears of metastatic merkel cell carcinoma. Diagnostic cytopathology. Aug 2013;41(8):729-733. Roh MH, Schmidt L, Placido J, et al. The application and diagnostic utility of immunocytochemistry on direct smears in the diagnosis of pulmonary adenocarcinoma and squamous cell carcinoma. Diagnostic cytopathology. Nov 2012;40(11):949-955. Knoepp SM, Roh MH. Ancillary techniques on direct-smear aspirate slides: a significant evolution for cytopathology techniques. Cancer cytopathology. Mar 2013;121(3):120-128. Gailey MP, Stence AA, Jensen CS, Ma D. Multiplatform comparison of molecular oncology tests performed on cytology specimens and formalin-fixed, paraffin-embedded tissue. Cancer cytopathology. Jan 2015;123(1):30-39. Roh MH. The Utilization of Cytologic Fine-Needle Aspirates of Lung Cancer for Molecular Diagnostic Testing. Journal of pathology and translational medicine. Jul 2015;49(4):300-309. Betz BL, Dixon CA, Weigelin HC, Knoepp SM, Roh MH. The use of stained cytologic direct smears for ALK gene rearrangement analysis of lung adenocarcinoma. Cancer cytopathology. Sep 2013;121(9):489-499. Killian JK, Walker RL, Suuriniemi M, et al. Archival fine-needle aspiration cytopathology (FNAC) samples: untapped resource for clinical molecular profiling. The Journal of molecular diagnostics : JMD. Nov 2010;12(6):739-745. Malapelle U, Bellevicine C, De Luca C, et al. EGFR mutations detected on cytology samples by a centralized laboratory reliably predict response to gefitinib in non-small cell lung carcinoma patients. Cancer cytopathology. Oct 2013;121(10):552-560. Malapelle U, de Rosa N, Rocco D, et al. EGFR and KRAS mutations detection on lung cancer liquid-based cytology: a pilot study. Journal of clinical pathology. Jan 2012;65(1):87-91. Rivera C, Cancalon C, Schmidt A, et al. [Hospital undertaking of patients with a resection of lung cancer]. Revue des maladies respiratoires. Sep 2013;30(7):529-536. Reynolds JP, Tubbs RR, Minca EC, et al. EGFR mutational genotyping of liquid based cytology samples obtained via fine needle aspiration (FNA) at endobronchial ultrasound of non-small cell lung cancer (NSCLC). Lung cancer. Nov 2014;86(2):158-163. Bellevicine C, Malapelle U, Vigliar E, de Luca C, Troncone G. Epidermal growth factor receptor test performed on liquid-based cytology lung samples: experience of an academic referral center. Acta cytologica. 2014;58(6):589-594. Minca EC, Lanigan CP, Reynolds JP, et al. ALK status testing in non-small-cell lung carcinoma by FISH on ThinPrep slides with cytology material. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. Apr 2014;9(4):464-468.

EP

20.

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

25

ACCEPTED MANUSCRIPT

42.

43.

44.

45.

46.

47. 48.

49.

50.

RI PT

SC

40. 41.

M AN U

39.

TE D

38.

Proietti A, Ali G, Pelliccioni S, et al. Anaplastic lymphoma kinase gene rearrangements in cytological samples of non-small cell lung cancer: comparison with histological assessment. Cancer cytopathology. Jun 2014;122(6):445-453. Dacic S. Molecular genetic testing for lung adenocarcinomas: a practical approach to clinically relevant mutations and translocations. Journal of clinical pathology. Oct 2013;66(10):870-874. Travis WD, Brambilla E, Noguchi M, et al. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. Feb 2011;6(2):244-285. Travis WD. Classification of lung cancer. Seminars in roentgenology. Jul 2011;46(3):178-186. Russell PA, Wainer Z, Wright GM, Daniels M, Conron M, Williams RA. Does lung adenocarcinoma subtype predict patient survival?: A clinicopathologic study based on the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary lung adenocarcinoma classification. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. Sep 2011;6(9):1496-1504. Warth A, Muley T, Meister M, et al. The novel histologic International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification system of lung adenocarcinoma is a stage-independent predictor of survival. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. May 1 2012;30(13):1438-1446. Yoshizawa A, Motoi N, Riely GJ, et al. Impact of proposed IASLC/ATS/ERS classification of lung adenocarcinoma: prognostic subgroups and implications for further revision of staging based on analysis of 514 stage I cases. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. May 2011;24(5):653-664. Travis WD, Brambilla E, Noguchi M, et al. Diagnosis of Lung Cancer in Small Biopsies and Cytology: Implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Classification. Archives of pathology & laboratory medicine. Sep 12 2012. Sigel CS, Moreira AL, Travis WD, et al. Subtyping of non-small cell lung carcinoma: a comparison of small biopsy and cytology specimens. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. Nov 2011;6(11):1849-1856. Travis WD, Brambilla E, Noguchi M, et al. Diagnosis of lung cancer in small biopsies and cytology: implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Archives of pathology & laboratory medicine. May 2013;137(5):668-684. Sigel CS, Rudomina DE, Sima CS, et al. Predicting pulmonary adenocarcinoma outcome based on a cytology grading system. Cancer cytopathology. Feb 25 2012;120(1):35-43. Rodriguez EF, Monaco SE, Dacic S. Cytologic subtyping of lung adenocarcinoma by using the proposed International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) adenocarcinoma classification. Cancer cytopathology. Nov 2013;121(11):629-637. Rodriguez EF, Dacic S, Pantanowitz L, Khalbuss WE, Monaco SE. Cytopathology of pulmonary adenocarcinoma with a single histological pattern using the proposed International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) classification. Cancer cytopathology. May 2015;123(5):306-317. Rudomina DE, Lin O, Moreira AL. Cytologic diagnosis of pulmonary adenocarcinoma with micropapillary pattern: does it correlate with the histologic findings? Diagnostic cytopathology. May 2009;37(5):333-339.

EP

37.

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

26

ACCEPTED MANUSCRIPT

55.

56. 57.

58.

59.

60.

61. 62.

63.

64.

65.

RI PT

SC

54.

M AN U

53.

TE D

52.

Rekhtman N, Ang DC, Sima CS, Travis WD, Moreira AL. Immunohistochemical algorithm for differentiation of lung adenocarcinoma and squamous cell carcinoma based on large series of whole-tissue sections with validation in small specimens. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. Oct 2011;24(10):1348-1359. Mukhopadhyay S, Katzenstein AL. Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: Utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6. The American journal of surgical pathology. Jan 2011;35(1):15-25. Thunnissen E, Noguchi M, Aisner S, et al. Reproducibility of histopathological diagnosis in poorly differentiated NSCLC: an international multiobserver study. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. Sep 2014;9(9):13541362. Rekhtman N, Brandt SM, Sigel CS, et al. Suitability of thoracic cytology for new therapeutic paradigms in non-small cell lung carcinoma: high accuracy of tumor subtyping and feasibility of EGFR and KRAS molecular testing. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. Mar 2011;6(3):451-458. Edwards SL, Roberts C, McKean ME, Cockburn JS, Jeffrey RR, Kerr KM. Preoperative histological classification of primary lung cancer: accuracy of diagnosis and use of the non-small cell category. Journal of clinical pathology. Jul 2000;53(7):537-540. Cataluna JJ, Perpina M, Greses JV, Calvo V, Padilla JD, Paris F. Cell type accuracy of bronchial biopsy specimens in primary lung cancer. Chest. May 1996;109(5):1199-1203. Ocque R, Tochigi N, Ohori NP, Dacic S. Usefulness of immunohistochemical and histochemical studies in the classification of lung adenocarcinoma and squamous cell carcinoma in cytologic specimens. American journal of clinical pathology. Jul 2011;136(1):81-87. Righi L, Graziano P, Fornari A, et al. Immunohistochemical subtyping of nonsmall cell lung cancer not otherwise specified in fine-needle aspiration cytology: a retrospective study of 103 cases with surgical correlation. Cancer. Aug 1 2011;117(15):3416-3423. Siami K, McCluggage WG, Ordonez NG, et al. Thyroid transcription factor-1 expression in endometrial and endocervical adenocarcinomas. The American journal of surgical pathology. Nov 2007;31(11):1759-1763. Penman D, Downie I, Roberts F. Positive immunostaining for thyroid transcription factor-1 in primary and metastatic colonic adenocarcinoma: a note of caution. Journal of clinical pathology. Jun 2006;59(6):663-664. Robens J, Goldstein L, Gown AM, Schnitt SJ. Thyroid transcription factor-1 expression in breast carcinomas. The American journal of surgical pathology. Dec 2010;34(12):1881-1885. Bishop JA, Teruya-Feldstein J, Westra WH, Pelosi G, Travis WD, Rekhtman N. p40 (DeltaNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. Mar 2012;25(3):405-415. Bishop JA, Sharma R, Illei PB. Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Human pathology. Jan 2010;41(1):20-25. Yang M, Nonaka D. A study of immunohistochemical differential expression in pulmonary and mammary carcinomas. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. May 2010;23(5):654-661. Mukhopadhyay S, Katzenstein AL. Comparison of monoclonal napsin A, polyclonal napsin A, and TTF-1 for determining lung origin in metastatic adenocarcinomas. American journal of clinical pathology. Nov 2012;138(5):703-711.

EP

51.

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

27

ACCEPTED MANUSCRIPT

70.

71.

72. 73.

74.

75.

76.

77.

78. 79.

RI PT

SC

69.

M AN U

68.

TE D

67.

Pereira TC, Share SM, Magalhaes AV, Silverman JF. Can we tell the site of origin of metastatic squamous cell carcinoma? An immunohistochemical tissue microarray study of 194 cases. Applied immunohistochemistry & molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry. Jan 2011;19(1):10-14. Rekhtman N, Kazi S. Nonspecific reactivity of polyclonal napsin a antibody in mucinous adenocarcinomas of various sites: a word of caution. Archives of pathology & laboratory medicine. Apr 2015;139(4):434-436. Seo JB, Im JG, Goo JM, Chung MJ, Kim MY. Atypical pulmonary metastases: spectrum of radiologic findings. Radiographics : a review publication of the Radiological Society of North America, Inc. Mar-Apr 2001;21(2):403-417. Sethi S, Geng L, Shidham VB, et al. Dual color multiplex TTF-1 + Napsin A and p63 + CK5 immunostaining for subcategorizing of poorly differentiated pulmonary non-small carcinomas into adenocarcinoma and squamous cell carcinoma in fine needle aspiration specimens. CytoJournal. 2012;9:10. Fatima N, Cohen C, Lawson D, Siddiqui MT. Combined double CK5/P63 stain: useful adjunct test for diagnosing pulmonary squamous cell carcinoma. Diagnostic cytopathology. Nov 2012;40(11):943-948. Fatima N, Cohen C, Lawson D, Siddiqui MT. TTF-1 and Napsin A double stain: a useful marker for diagnosing lung adenocarcinoma on fine-needle aspiration cell blocks. Cancer cytopathology. Apr 25 2011;119(2):127-133. Hallack Neto AE, Siqueira SA, Dulley FL, Ruiz MA, Chamone DA, Pereira J. p63 protein expression in high risk diffuse large B-cell lymphoma. Journal of clinical pathology. Jan 2009;62(1):77-79. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proceedings of the National Academy of Sciences of the United States of America. Sep 7 2004;101(36):13306-13311. Tang X, Shigematsu H, Bekele BN, et al. EGFR tyrosine kinase domain mutations are detected in histologically normal respiratory epithelium in lung cancer patients. Cancer research. Sep 1 2005;65(17):7568-7572. Lopez-Chavez A, Thomas A, Rajan A, et al. Molecular profiling and targeted therapy for advanced thoracic malignancies: a biomarker-derived, multiarm, multihistology phase II basket trial. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Mar 20 2015;33(9):1000-1007. Fukuoka M, Yano S, Giaccone G, et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected]. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Jun 15 2003;21(12):2237-2246. Kris MG, Natale RB, Herbst RS, et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. Jama. Oct 22 2003;290(16):2149-2158. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al. Erlotinib in previously treated non-small-cell lung cancer. The New England journal of medicine. Jul 14 2005;353(2):123-132. Eberhard DA, Giaccone G, Johnson BE, Non-Small-Cell Lung Cancer Working G. Biomarkers of response to epidermal growth factor receptor inhibitors in Non-Small-Cell Lung Cancer Working Group: standardization for use in the clinical trial setting. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Feb 20 2008;26(6):983-994.

EP

66.

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

28

ACCEPTED MANUSCRIPT

85.

86. 87. 88. 89.

90. 91.

92.

93.

94.

95.

RI PT

84.

SC

83.

M AN U

82.

TE D

81.

Lin MT, Mosier SL, Thiess M, et al. Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing. American journal of clinical pathology. Jun 2014;141(6):856-866. Brevet M, Arcila M, Ladanyi M. Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR. The Journal of molecular diagnostics : JMD. Mar 2010;12(2):169-176. Yu J, Kane S, Wu J, et al. Mutation-specific antibodies for the detection of EGFR mutations in non-small-cell lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. May 1 2009;15(9):3023-3028. Hasanovic A, Ang D, Moreira AL, Zakowski MF. Use of mutation specific antibodies to detect EGFR status in small biopsy and cytology specimens of lung adenocarcinoma. Lung cancer. Aug 2012;77(2):299-305. Guin S, Ru Y, Wynes MW, et al. Contributions of KRAS and RAL in non-small-cell lung cancer growth and progression. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. Dec 2013;8(12):1492-1501. Suda K, Tomizawa K, Mitsudomi T. Biological and clinical significance of KRAS mutations in lung cancer: an oncogenic driver that contrasts with EGFR mutation. Cancer metastasis reviews. Mar 2010;29(1):49-60. Macerelli M, Caramella C, Faivre L, et al. Does KRAS mutational status predict chemoresistance in advanced non-small cell lung cancer (NSCLC)? Lung cancer. Mar 2014;83(3):383-388. Pao W, Wang TY, Riely GJ, et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS medicine. Jan 2005;2(1):e17. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. Aug 2 2007;448(7153):561-566. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with nonsmall-cell lung cancer who harbor EML4-ALK. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Sep 10 2009;27(26):4247-4253. Moreira AL, Hasanovic A. Molecular characterization by immunocytochemistry of lung adenocarcinoma on cytology specimens. Acta cytologica. 2012;56(6):603-610. Mino-Kenudson M, Chirieac LR, Law K, et al. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry. Clinical cancer research : an official journal of the American Association for Cancer Research. Mar 1 2010;16(5):1561-1571. Leighl NB, Rekhtman N, Biermann WA, et al. Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement of the College of American Pathologists/International Association for the study of lung cancer/association for molecular pathology guideline. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. Nov 10 2014;32(32):3673-3679. Bos M, Gardizi M, Schildhaus HU, et al. Complete metabolic response in a patient with repeatedly relapsed non-small cell lung cancer harboring ROS1 gene rearrangement after treatment with crizotinib. Lung cancer. Jul 2013;81(1):142-143. Sholl LM, Sun H, Butaney M, et al. ROS1 immunohistochemistry for detection of ROS1rearranged lung adenocarcinomas. The American journal of surgical pathology. Sep 2013;37(9):1441-1449. Lee SE, Lee B, Hong M, et al. Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. Apr 2015;28(4):468-479.

EP

80.

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

29

ACCEPTED MANUSCRIPT

96. 97. 98.

99. 100.

Takeuchi K, Soda M, Togashi Y, et al. RET, ROS1 and ALK fusions in lung cancer. Nature medicine. Mar 2012;18(3):378-381. Kohno T, Ichikawa H, Totoki Y, et al. KIF5B-RET fusions in lung adenocarcinoma. Nature medicine. Mar 2012;18(3):375-377. Lindeman N. Molecular diagnostics of lung cancers at the Brigham and Women's Hospital and Dana-Farber Cancer Institute: technology in rapid evolution. Archives of pathology & laboratory medicine. Oct 2012;136(10):1198-1200. Gagan J, Van Allen EM. Next-generation sequencing to guide cancer therapy. Genome medicine. 2015;7(1):80. Travis WD, Rekhtman N. Pathological diagnosis and classification of lung cancer in small biopsies and cytology: strategic management of tissue for molecular testing. Seminars in respiratory and critical care medicine. Feb 2011;32(1):22-31.

RI PT

1 2 3 4 5 6 7 8 9 10 11 12

SC

13 14

AC C

EP

TE D

M AN U

15

30

ACCEPTED MANUSCRIPT

Table 1-Summary of minimally invasive sampling techniques.

EUS-FNA

6 7

Intraparenchymal lung lesions (more peripheral lesions) and anterior mediastinal lymph nodes or mass; lung cancer diagnosis or diagnosis of anterior mediastinal mass, when other sampling techniques fail Tumors that invade the posterior and inferior mediastinum Paratracheal (2,4) And posterior subcarinal lymph nodes (3,7,8 and 9); lung or esophageal cancer diagnosis and staging; avoids mediastinoscopy in subset of patients Peripheral lesions Lymph nodes 12 and 14

Higher rate of pneumothorax than EMN-TBNA Bleeding Low negative predictive value

RI PT

Small samples Low negative predictive value Variable operator proficiency Availability Time consuming No access to retrotracheal, periaortic, paraesophageal and pulmonary ligament lymph nodes station (3a, 5, 6, 8, 9)

Small samples Low negative predictive value Variable operator proficiency Availability Time consuming Not able to visualize airway and access tumor mass No sampling of the main lesion Lower rate of pneumothorax than CTGFNA Complex technique Available in few institutions Low diagnostic yield

Legend: EBUS-TBNA, endobronchial ultrasound guided transbronchial needle aspiration; CTG-FNA, CTguided fine needle aspiration; EUS-FNA, transesophageal endoscopic ultrasound guided fine needle aspiration; EMN-TBNA, electromagnetic navigational bronchoscopy guided transbronchial needle aspiration

AC C

2 3 4 5

Central lung lesions and mediastinal lymphadenopathy; lung cancer diagnosis and staging; avoids mediastinoscopy in subset of patients

EP

EMN-TBNA

Limitations

SC

CTG-FNA

Indications & Advantages

M AN U

Sampling modality EBUS-TBNA

TE D

1

8

31

ACCEPTED MANUSCRIPT

Table 2- Summary of morphologic features ADC versus SCC100 Squamous cell carcinoma

Adenocarcinoma

Architecture

Large syncytial or crowded sheets with frayed border

Honeycomb, 3-dimensional cell balls, picket fence, acinar formation

Cytoplasm

Dense with distinct cell border, frequent spindle shaped cells

Delicate and finely vacuolated, targetoid mucin vacuoles

Nuclear

Hyperchromatic, coarse nuclear chromatin

Eccentric nucleus, open pale chromatin, prominent nucleoli

SC

RI PT

Morphologic features

M AN U

1

2

AC C

EP

TE D

3

32

ACCEPTED MANUSCRIPT

Table 3. Summary table of cytological findings seeing the 5 main ADC patterns based on prior 48,49 published data (modified from ) Suggestive Cytological Features Tumor cells distributed in a glandular pattern with central lumen formation Two-dimensional sheets Moderate pleomorphism Clean background

RI PT

ADC Pattern Acinar

M AN U

SC

Pitfall: Broken or incomplete acinar structures on the cell block can mimic a “string of pearls”, as described with lepidic pattern Solid

More complex three-dimensional clusters of cells No definitive cribriform or papillary architecture More pleomorphism

Background necrosis or inflammation

Papillary

TE D

Pitfall: Some clusters may have a vague acinar architecture when falling apart or appearing in smaller groups with less complexity Elongated or finger-like clusters of cells

EP

Nuclear palisading along the edges and endothelial cells streaming through the fibrovascular core Intranuclear inclusions and/or grooves may be seen

AC C

1 2

Lepidic

Pitfall: Some papillary tufts with central clearing and an absence of a definitive fibrovascular core can be difficult to distinguish from an acinar or lepidic pattern of growth Intranuclear inclusions and/or grooves can be seen in the lepidic pattern as well. Strips of cells with mild pleomorphism Hobnailing or a “string of pearl” arrangement on the cell block. Absence of marked pleomorphism and necrosis 33

ACCEPTED MANUSCRIPT

Intranuclear inclusions and/or grooves may be seen

RI PT

Pitfall: Clusters may show back-to-back collapsed strips resembling cribriform or acinar features. Intranuclear inclusions and/or grooves can also be seen in the papillary pattern Micropapillary

Small tight tufts of cells without discrete fibrovascular cores or lumen

SC

Pitfall: Micropapillary tufts may be seen in acinar, lepidic and papillary ADC 1

M AN U

2 3 4 5

AC C

EP

TE D

6

34

ACCEPTED MANUSCRIPT

3 4

Napsin A

~ 80% of lung ADC (cytoplasmic stain)

To confirm lung origin or adenocarcinoma subtype

P63

~ 97% of SCC (nuclear stain)

To subtype poorly differentiated NSCLC and confirm squamous cell carcinoma

P40

~ 90% of SCC (nuclear stain)

Pitfalls: Positivity in other tumors Thyroid, endometrial, biliary, colon, breast, squamous cell carcinoma (6%) Large cell neuroendocrine tumors Small cell carcinoma

RI PT

Diagnostic use in Lung Cytology To confirm lung origin or adenocarcinoma subtype

Carcinoid tumors Renal cell carcinoma, clear cell carcinoma of the gynecologic tract, subset of thyroid carcinoma, endometrial carcinoma and hepatocellular carcinoma Mucinous adenocarcinoma of multiple sites (supranuclear location), squamous cell carcinoma (26%), macrophages Lung ADC ~20% Small cell carcinoma (30-40%) Diffuse large B-cell lymphoma Urothelial carcinoma, Myoepithelial & salivary gland type tumors Trophoblastic tumors Thymic epithelial neoplasm Ovarian, endometrial, breast and colorectal carcinomas Bronchial reserve cells Urothelial carcinoma Thymic neoplasmSmall cell carcinoma (<5%)

SC

Positivity in NSCLC ~80% of lung ADC (nuclear stain)

TE D

M AN U

Immunohistochemical stain TTF-1

To subtype poorly differentiated NSCLC and confirm squamous cell carcinoma

EP

2

Table 4-Potential immunohistochemical pitfalls.

Legend: SCC, squamous cell carcinoma; ADC, adenocarcinoma

AC C

1

35

ACCEPTED MANUSCRIPT

Table 5. Practical Summary of Relevant Molecular Alterations in Pulmonary Adenocarcinoma

10-20%

ALK

6%

MET

6%

MET amplification

BRAF/PIK3

2%

BRAF (V600E)

HER2/MEK

2%

HER2 activation by exon 20 in-frame insertion mutation

ROS1

2%

KIF5B-RET

1%

Exon 19 (E746-A750) Exon 21 (L858R) (Cell Signaling Technology)

Crizotinib*

D5F3 (Cell Signaling Technology) 5A4 (Novocastra) ALK1 M7195 (Dako) NA

TE D

Onartuzumab**, ilotumumab**, Cabozantinib**, tivantinib**, Crizotinib** Vemurafenib***, GSK2118436**** AZD6244****

Crizotinib** RET-TKIs**

RET rearrangement KIF5B-RET

Sunitinib***, sorafenib***, Vandetanib***, cabozantinib*** Rapamycin, everolimus****

PI3K mutations

Immunohistochemistry NA

Erlotinib, gefitinib, afatinib*

ROS1 rearrangement

EP

5-7%

Targeted Therapy Selumetinib

RI PT

EGFR

PI3K/AKT/mTOR pathway Unknown

Main mechanism single amino acid substitutions in codons 12, 13, or 61 Exon 19 del Exon 21 point mutation Exon 20 (resistance mutations) EML4-ALK fusion gene

SC

Frequency 30%

M AN U

Gene/Pathway KRAS

AC C

1

V600 Clone VE1 (Ventana) Not useful in lung cancer since the mechanism of activation is mutation rather than amplification, in contrast to breast cancer D4D6 (Cell Signaling Technology) ab134100 (Abcam)

N/A

40%

2 3 4 5

* FDA approved in NSCLC, **FDA not approved for this molecular subtype yet, but approved for other subtype, *** drugs approved in other cancers.**** Drugs in clinical development (at the time of preparation of manuscript)

36

ACCEPTED MANUSCRIPT

-Major advances in diagnostic and treatment of lung cancer have been made, particularly in the identification of key somatic genetic drivers and targeted therapy - In a large percentage of lung cancers patients, cytology specimens are the only specimen available for diagnosis and to guide treatment

AC C

EP

TE D

M AN U

SC

RI PT

- Importance of the correct nomenclature as well as strategies to save tissue for molecular studies is critical