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Introduction

Good afternoon, I'm Anja Roden. I'm one of the thoracic pathologists here at Mayo Clinic in Rochester, and I'm also one of the medical directors in the Immunostains Lab. Today, I will give you an update on the WHO 2021 classification for lung tumors. 

Disclosure

I have nothing to disclose. 

Outline

We will be talking about some new entities and also some changes in the terminology in the 2021 WHO classification of lung tumors. Specifically, we will talk about thoracic SMARCA4-deficient undifferentiated tumors and bronchiolar adenoma/ciliated muconodular papillary tumors. We will also discuss the nomenclature in small biopsies. Although this is not new, it often leads to some confusion. And then in the end, we will discuss spread through airspaces or STAS.

Cancer deaths in 2021 (U.S.)

As you know, lung cancer, unfortunately is still the leading cause of death among cancers in the United States, in males and females.

Lung cancer incidence (Canada)

However, the incidence of subtypes of lung cancer have changed over the years, and although this graph is from Canada, the SEER data are very similar. As you can appreciate, large cell carcinomas have decreased in their incidence rate and that is due to us being more able to subtype lung cancers because we have immunostains now. We also see that small cell carcinomas and squamous cell carcinoma incidence is going down. On the other hand, the incidence of lung adenocarcinomas is going up. These changes are thought, at least in part, due to differences in the manufacturing of cigarettes and also due to some smoking habits.

Lung cancer¹

Unfortunately, 26% of non-small cell lung carcinoma patients are only alive five years after the diagnosis. The five-year survival rate for metastatic disease is only 6% with chemotherapy, but it is 15% to 50% if the patients are eligible for targeted therapies or immunotherapies, and therefore, biomarker testing is really important to identify these patients who are actually eligible for these kinds of therapies. 

Lung tumors – 2021 WHO

In regards to the 2021 WHO classification of lung tumors, there have been no major changes. However, there have been a few new entities and some terminology has also changed. New entities include thoracic SMARCA4-deficient undifferentiated tumors and bronchiolar adenoma/ciliated muconodular papillary tumors. Lymphoepithelioma-like carcinomas are now termed lymphoepithelial carcinoma, and the enteric adenocarcinomas are now termed enteric-type adenocarcinomas to keep consistency amongst all thoracic tumors. We are also using the 8th edition of the AJCC/UICC TNM staging that was published in 2017.

Thoracic SMARCA4-deficient undifferentiated tumor

Let's start with a thoracic SMARCA4-deficient undifferentiated tumor. This is an example of a 66-year-old gentleman, who was a smoker, and who presented with a left suprahilar mass. He also was found to have additional masses in the right lung, liver, and adrenal gland. A biopsy was performed, which mainly shows necrosis, as you can appreciate here. However, there were a few areas of viable tumor cells, which you can appreciate here on the right side. These tumor cells are of epithelioid cytology, many with prominent nucleoli. They show a slightly discohesive growth pattern and some of the tumor cells have actually a rhabdoid cytology in that we have an eccentric nucleus with a prominent nucleolus and eosinophilic cytoplasm.

Differential diagnosis

The differential diagnosis included poorly differentiated non-small cell carcinoma, lymphoma because of the slightly discohesive growth pattern, malignant melanoma, and sarcoma. 

OSCAR keratin was negative, as were multiple other keratins. NUT, desmin, SMA, CD20, CD31, CD45, SALL -4, and S100 were also negative, and the expression of INI-1 was preserved. Because of the presentation of the patient, together with the rhabdoid morphology and the immunophenotype, we were thinking about a SMARCA4-deficient undifferentiated tumor, and a BRG1 was performed. BRG1 is a protein that is encoded by SMARCA4, and the expression of BRG1 is lost in the tumor cells with a preserved internal control in the non-neoplastic cells. So here, for instance, the endothelial cells still express BRG1 and that positive internal control is really important in immunostains that look for loss of expression, because otherwise we don't know if the stain actually worked. With that immunophenotype, this was indeed a thoracic SMARCA4-deficient undifferentiated tumor. 

Thoracic SMARCA4-deficient undifferentiated tumor^2-4

These tumors occur at a median age of 39 to 59 years old with a broad age range. There is a male and smoker predominance, and these tumors are rapidly progressive. They most commonly occur in the mediastinum, followed by the pulmonary hilum and the pleura with or without chest wall involvement. They often are already present at multiple sites, and metastatic disease is usually also present at time of presentation. And these are, in general, large tumors. 

SMARCA4-UT

They are often characterized by a rhabdoid morphology or a poorly differentiated morphology, again, rhabdoid here as you can appreciate the pink cytoplasm and the eccentric nucleus. These tumors are variably discohesive, and some of them actually can have a focal myxoid stroma or a desmoplastic small round cell tumor morphology.

They are characterized by the loss of BRG1 and usually also BRM expression. As you can appreciate, here on the right side, the tumor cells are negative for BRG1, again we do have a nice internal positive control. They show preserved expression of INI-1, and they are commonly also positive for SOX2, CD34, and/or SALL4. Keratin staining, may be very focal, claudin 4, TTF-1, and synaptophysin can also be focally expressed, but they are negative for Desmin, NUT, S100, and p40.

SMARCA4-UT^5,6

SMARCA4 is a member of the SWI/SNF chromatin-remodeling complex, which is a tumor suppressor that regulates transcription and promotes cell differentiation. There is a biallelic inactivation of SMARCA4 in these tumors. However, so far, no germline SMARCA4 mutation has been identified in these patients. Often these tumors also show a TP53 mutation.

SMARCA4-UT⁶

The median survival is quite short, often due to local complications, and these tumors only have a very limited response to chemotherapy and surgery. 

SMARCA4-UT — differential diagnosis

The differential diagnosis is broad and includes NUT carcinoma, germ cell tumors, neuroendocrine carcinomas, lymphomas, melanomas, and sarcomas. However, some of the non-small cell lung carcinomas can also be SMARCA4-deficient and there are SMARCA4-deficient carcinomas elsewhere in the body.

Bronchial adenoma/ciliated muconodular papillary tumor^7-10

So let's move on to a bronchial adenoma/ciliated muconodular papillary tumor. This is actually a benign entity. So far there has been no recurrences or metastasis. These are peripheral and peribronchiolar tumors. They are not associated with proximal bronchi. On CT scan they usually show as solid or ground glass nodules. Some may show cavitation. They occur in middle age to elderly patients with a median age of 72 years old without any sex predilection. Usually they are quite small. 

BA/CMPT

Here's an example of such a tumor. You can appreciate the peribronchiolar location. Here is a small airway. The tumor is relatively well circumscribed, and the architecture is either papillary or, as in this case, flat and glandular. 

On higher power, again we see in this case the glandular and flat architecture, and there is some mucin within the glands. 

On high power, in this case, we do have nicely ciliated cells and there is a bi-layered architecture. Inside we have a basal layer, which expresses p40, and a luminal layer, which may express TTF-1. So TTF-1 and p40 might be helpful in these cases.

Bronchial adenoma/ciliated muconodular papillary tumor^7-10

As I said, they are peribronchiolar, they are either papillary or flat, and they are bi-layered with luminal epithelial cells, which are mucous cells and ciliated cells, or cells that may resemble type II pneumocytes and club cells. And then underneath there is a continuous basal cell layer that will be positive for p40, and because of that architecture p40 and TTF-1 may be helpful. There's usually no nuclear atypia, and mitotic activity is really low. However, some of these adenomas, or tumors, actually do show some molecular alterations, including BRAF, EGFR, KRAS, HRAS, ALK, and AKT1. And so BRAF and ALK immunostains might also be helpful in these entities. 

BA/CMPT differential diagnosis

The differential diagnosis certainly includes an adenocarcinoma, including the adenocarcinoma in situ, but these tumors lack the continuum of basal cell layer, and they also lack deciliation. Papillomas occur centrally and endobronchial and not in the vicinity to small airways, and I show you here, a case of peribronchiolar metaplasia. You see the small airway here and the extension of the ciliated epithelium into the adjacent alveoli. This metaplasia lacks the expression of BRAF and ALK. It does have ill-defined borders. It's often multi-centric, because it occurs in a background of interstitial lung disease or small airways disease. On high power, again you can see nicely ciliation, as in the bronchiolar adenomas and tumors; however, this is more irregular and has ill-defined borders. And again, look for the interstitial lung disease or small airways disease in the background. 

Small biopsies 2021 WHO — nomenclature

So, the nomenclature of small biopsies did not change in the 2021 WHO classification, but as I alluded to earlier, it often raises some confusion and therefore, I just want to spend a few minutes on that nomenclature. If the biopsy shows clear morphological features of adenocarcinoma or squamous cell carcinoma, for instance, because we do see a glandular pattern, or we see keratinization and intercellular bridges. In those cases, we should call out adenocarcinoma or squamous cell carcinoma. If you only see a lepidic pattern, we should term it adenocarcinoma with lepidic pattern, see comment, and in the comment we should say, “Although we don't see invasion in the biopsy, these features can be seen in adenocarcinoma in situ, minimally invasive adenocarcinoma, and invasive adenocarcinoma and that distinction requires a resection specimen."

If we do need stains to make the distinction between adenocarcinoma and squamous cell carcinoma, we should term it non-small cell carcinoma, favor adenocarcinoma, or favor squamous cell carcinoma. In cases that appear to be pleomorphic carcinoma, because we do see spindle cells or giant cell features, we should call it non-small cell carcinoma with spindle and/or giant cell features. In cases where we find an adenocarcinoma component and a squamous cell carcinoma component, or in cases which we cannot further subtype, we should call it non-small cell carcinoma, not otherwise specified. If the features are those of a small cell carcinoma, we can call it a small cell carcinoma. However, if the features are resembling a large cell neuroendocrine carcinoma, we should term it non-small cell carcinoma, with neuroendocrine morphology and positive neuroendocrine markers, possible large cell neuroendocrine carcinoma.

Here's an example of a 77-year-old woman who was found to have a 0.8 cm nodule in the right upper lobe lung that was an incidental finding, and you can appreciate it over here. Because the patient had a medically complex history, she was not a surgical candidate and therefore a needle core biopsy was performed. That biopsy shows clusters of neoplastic cells in a desmoplastic stromal reaction. 

On high power, these cells are of epithelial cytology with ample cytoplasm and round to oval nuclei, many with prominent nucleoli. However, there is no clan formation or keratinization, and we didn't also see any intracellular pitches. Therefore, although this looks like a non-small cell carcinoma, the distinction between adenocarcinoma and squamous cell carcinoma cannot be made on the H&E and therefore TTF-1 and p40 were performed. And as you can appreciate, the tumor cells were positive for TTF-1 and negative for p40, and therefore the tumor was signed out as invasive non-small cell carcinoma favor adenocarcinoma.

Case Study

Here's an example of a 77-year-old woman who clinically was thought to have a recurrent lung carcinoma. On low power, we appreciate nests of tumor cells, so a solid pattern, but there  are clearly also gland formations over here. 

On high power, here are the nests of tumor cells in a desmoplastic stroma reaction. There is no keratinization or gland formation, but in other areas, we do appreciate gland formation and maybe even a micropapillary pattern. So, these solid areas could be a solid component of an adenocarcinoma or a squamous cell carcinoma component. 

Therefore, immunostains were performed, which show that the glandular component is positive for TTF-1, and the solid component is positive for p40. Now this should be signed out as a non-small cell carcinoma not otherwise specified and squamous cell and adenocarcinoma patterns are both present, and then see comment. And in the comment, we can say something like, "Although this could represent an adenosquamous carcinoma, this diagnosis cannot be established on a biopsy.” And that is because you do need at least 10% of each component in a resection specimen to establish that diagnosis.

Small biopsies, tissue preservation

As I talked about before, we really need to try to preserve as much tissue as we can so that we give the patients a chance to have biomarkers tested and to possibly be eligible for targeted therapy or immunotherapy. So, what can we do to preserve as much tissue as possible in these small biopsies? First of all, we should try to divide the cores into at least two tissue blocks. IHC should be only done if morphology requires IHC. We could consider dual stains, such as TTF-1 and p40. And since those two are both nuclear stains, one should be coupled with a red chromogen and one with a brown chromogen, and then we can distinguish TTF-1 staining from p40 staining. We could use p40 in combination with napsin. Napsin is a cytoplasmic stain and p40 a nuclear stain, or we could combine CK5 with TTF-1. TTF-1 is a nuclear stain and CK5, a membranous and cytoplasmic stain, so that would be another good combination. Neuroendocrine markers should only be performed if the morphology is really suggestive of a neuroendocrine neoplasm. If we order stains, we also should get some unstained slides for potential biomarker testing. However, we need to be aware that the unstained slides have only a short shelf-life of optimally, six weeks, maximally, three months, but that is already quite long for immunohistochemistry. We still can use these unstained slides for molecular markers.

We are back to the example of the patient with the adenocarcinoma and the squamous cell carcinoma component. Looking at the dual stain of TTF-1 and p40. TTF-1 is positive in the glandular component. It's indicated by this brown stain. And p40 highlights the squamous cell carcinoma component, as you can see here with the red stain. If you use a combination of TTF-1 and CK5, again, here on the right side, is the glandular component that is highlighted by TTF-1, and on the left side, the squamous component, which is highlighted by CK5. 

Spread through airspace (STAS)^11,12

Our last entity we want to discuss is spread through airspace or STAS. Now STAS has been already mentioned in the 2015 WHO classification. But now we do have to comment on that in resection specimens, and since this is a somewhat new terminology, I just wanted to make sure we are all familiar with it. STAS means there are tumor cells within air spaces beyond the edge of the main tumor. This can be present in 15% to over 50% of non-small cell carcinomas, but it also can be found in small cell carcinomas. Patterns include micropapillary structures, solid tumor cell nests, and discohesive single tumor cells. As I said, they can occur in adenocarcinomas, squamous cell carcinomas, any subtype of neuroendocrine neoplasms or pleomorphic carcinomas. STAS should not be included into the tumor size. 

And here's an example. You can appreciate the adenocarcinoma and the green line shows you the edge of the tumor. And clearly there are some tumor cell clusters in air spaces that are away from the main edge of the tumor. 

If we look at higher power, here are the tumor cell clusters in airspaces and they are clearly beyond the edge of the tumor. So, this is STAS.

Spread through airspace (STAS)¹³

STAS has been associated with reduced recurrence free and overall survival in any stage lung adenocarcinomas. And it has also been associated with more aggressive pathologic features, KRAS mutations in stage I and II adenocarcinoma, amongst other associations. Limited resections, such as wedge resections or segmentectomy, may be at an increased risk for recurrence when compared to lobectomy, if STAS is present. And if STAS is present in a sublobar resection, such as segmentectomy or wedge resection, this might be an indication for subsequent lobectomy. However, more studies really need to be performed to be certain about that.

Take home message

So in summary, we talked about thoracic SMARCA4 deficient undifferentiated tumors and bronchiolar adenoma/ciliated muconodular papillary tumors. We discussed small biopsies and their nomenclature, and also talked a little bit about how we may be able to preserve tissue in these biopsies. And then in the end, we discussed spread through airspace. 

Thank you

And with that I would like to thank you for your attention. If you ever have any questions, please don't hesitate to contact me. And if you want to look at any references, here they are. Thank you very much.

References

1. Ettinger D, Wood D, Aisner D, et al: NCCN Guidelines Insights: Non-Small Cell Lung Cancer, Version 2.2021. J Natl Compr Canc Netw. 2021 Mar 2;19(3):254-266.
2. Sauter, J., Graham, R., Larsen, B. et al. SMARCA4-deficient thoracic sarcoma: a distinctive clinicopathological entity with undifferentiated rhabdoid morphology and aggressive behavior. Mod Pathol 30, 1422–1432 (2017).  
3. Hung YP et al. Adv Anat Pathol. 2018. 25:374-86.
4. Perret R, Chalabreysse L, Watson S, et al: SMARCA4-deficient Thoracic Sarcomas: Clinicopathologic Study of 30 Cases With an Emphasis on Their Nosology and Differential Diagnoses. Am J Surg Pathol. 2019 Apr;43(4):455-465.
5. Yoshida A, Kobayashi E, Kubo T, et al: Clinicopathological and molecular characterization of SMARCA4-deficient thoracic sarcomas with comparison to potentially related entities. Mod Pathol. 2017 Jun;30(6):797-809.
6. Le Loarer F, Watson S, Pierron G et al: SMARCA4 inactivation defines a group of undifferentiated thoracic malignancies transcriptionally related to BAF-deficient sarcomas. Nat Genet. 2015 Oct;47(10):1200-5.
7. Taguchi R, Higuchi K, Sudo M, et al: A case of anaplastic lymphoma kinase (ALK)-positive ciliated muconodular papillary tumor (CMPT) of the lung. Pathol Int, 67: 99-104. 
8. Ude E et al. Diagn Pathol 2017; 12:62. 
9. Kamata T, Sunami K, Yoshida A, et al: Frequent BRAF or EGFR Mutations in Ciliated Muconodular Papillary Tumors of the Lung. J Thorac Oncol. 2016 Feb;11(2):261-5.
10. Chang JC, Montecalvo J, Borsu L, et al: Expansion of the Concept of Ciliated Muconodular Papillary Tumors With Proposal for Revised Terminology Based on Morphologic, Immunophenotypic, and Genomic Analysis of 25 Cases. Am J Surg Pathol. 2018 Aug;42(8):1010-1026.
11. Travis WD, Brambilla E, Nicholson AG, et al: The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. J Thorac Oncol. 2015 Sep;10(9):1243-1260.
12. Jia M, Yu S, Yu J. et al: Comprehensive analysis of spread through air spaces in lung adenocarcinoma and squamous cell carcinoma using the 8^th edition AJCC/UICC staging system. BMC Cancer 2020;20:705.
13. Han YB, Kim H, Mino-Kenudson M, et al: Tumor spread through air spaces (STAS): prognostic significance of grading in non-small cell lung cancer. Mod Pathol. 2021 Mar;34(3):549-561.Reviewed in Mino-Kenudson M. Transl Lung Cancer Res. 2020; 9:847-59.

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