Extensive immunohistochemistry was performed on this case, in the referring community /district hospital. This showed the following results:

Transcription

1 Case 1 The New Classification Helped Keith Kerr Clinical history This 75 year old female patient presented with an increasing cough and some vague symptoms of pain over the left lower chest wall. She is a smoker with a 40+ pack/year history. Investigations (chest radiograph and CT scan) demonstrated a left lower lobe mass but no evidence of mediastinal lymphadenopathy. PET-CT showed a PET-avid lesion. A core biopsy was preformed. Histological Findings The core biopsy shows sheets and clusters of tumour cells which are cohesive. Nuclei show pleomorphism and focal marked atypia. Chromatin is sometimes coarse, nucleoli can be seen. Cytoplasm is focally eosinophilic and moderately abundant. There are some hints of cellular stratification. Occasional vacuoles are seen. The tumour has an abundant fibrous stroma and marked chronic inflammation. There is no evidence of keratin, intercellular bridges or glandular architecture. Extensive immunohistochemistry was performed on this case, in the referring community /district hospital. This showed the following results: Ber-EP4, CK7 and CEA all strongly positive Calretinin, Progesterone receptor, Oestrogen receptor, CDX2, CK20, CK5/6 all negative WT1 cytoplasmic positivity P63 Strong widespread nuclear positivity TTF1 - Strong widespread nuclear positivity In the light of the positivity for TTF1 and p63, the case was referred in consultation. A p40 IHC test and a mucin stain (alcian blue/pas combined) were requested. The p40 IHC is focally positive. The AB/PAS stain also shows some positivity. Diagnosis: Non-small cell carcinoma, probably adenocarcinoma. Subsequently a KRAS mutation was demonstrated in the tumour.

2 Discussion In lung cancer the presence of solid sheets of undifferentiated carcinoma, lacking any evidence of differentiation, is a common finding. If such features are found in a biopsy sample or in cytology, and there are no features of small cell carcinoma, then the appropriate diagnosis, on morphological grounds, is non small cell carcinoma, not otherwise specified (NSCLC-NOS). The case should NOT be signed out as large cell carcinoma. The prevalence of this scenario varies considerably in reports. It appears to be commoner in cytology samples although this is not a universal finding. Up to 25% of biopsy samples and 40% of cytology samples fall into this morphological category but the figure will vary according to sample type and quality, and the bias and experience of the pathologist. Reporting rates are one thing, accuracy of diagnosis is another. Older studies showed that the use of the NSCLC-NOS uncertain category improved accuracy of small sample classification in those cases which were subtyped to adeno or squamous cell carcinoma, which stands to reason. At least 10% of lung carcinomas are morphologically large cell undifferentiated or sarcomatoid tumours and these cases, by definition, must be called NSCLC-NOS since diagnostic criteria for these tumour types are inapplicable to small samples. Thus, in an unselected population, elimination of the NSCLC-NOS category is impossible by morphology, and would always exceed 10% when using H&E or equivalent morphology alone. Many resected squamous cell or adenocarcinomas contain areas lacking the diagnostic differentiation present elsewhere in the tumour, which may be sampled at biopsy or in cytology. Thus an NSCLC-NOS rate of over 10%, by morphology alone, is to be expected. Studies have shown that at least two thirds of NSCLC-NOS cases, when resected, prove to be adenocarcinoma. The new IASLC/ATS/ERS recommendations suggest the use of predictive immunohistochemistry to reduce the rate of NSCLC-NOS diagnosis. This recommendation was based upon existing literature, with more papers published after February 2011 supporting the concept. It was recommended that two antibodies be used, p63 and TTF1, to predict a diagnosis of squamous cell and adenocarcinoma respectively. Other markers have been advocated; for squamous cell carcinoma cytokeratins 34betaE12, and 5/6 (CK5/6), desmocollin 3 (DSC3), S100A7 and p40; for adenocarcinoma CK7 and Napsin A have been used. Data are quite variable but the author s opinion, based practice, comparison with rescted tumour diagnosis and on published work, is as follows:

3 CK-34betaE12, DSC-3 and S100A7 add nothing to the diagnostic problem. S100A7 and DSC3 are usually only positive when a diagnosis can already be made on the H&E. CK-34betaE12 lacks enough specificity for squamous differentiation. CK5/6 fractionally adds to the discriminatory power of p63 as a marker to predict squamous cell carcinoma. Occasional equivocal cases on p63 staining are strong with CK5/6. CK7 lacks specificity for adenocarcinoma and should not be used as a discriminator. If TTF1, p63 and CK5/6 are negative, CK7 positivity would support adenocarcinoma. A CK7 negative tumour is unlikely to be adenocarcinoma. Napsin A seems to add nothing to the power of TTF1 in discriminating adenocarcinoma. Some studies disagree. Napsin A stains alveolar macrophages caution! P40 may be more specific than p63, for discriminating squamous cell carcinoma in lung, but this outcome depends on how p63 is interpreted. Some poorly differentiated adenocarcinomas are p63 positive but usually this is NOT at a level which has been shown to be predictive of squamous cell carcinoma. Adenocarcinoma with p63 at moderate/strong level in more than half the cells is very unusual, CK5/6 would be negative and TTF1 positivity would trump any p63 result. More data on p40 are required. The Alcian blue/pas combined mucin stain lacks sensitivity but is quite specific for a diagnosis of adenocarcinoma, and is useful when TTF1 is negative TTF1 is negative in 20-25% of lung adenocarcinomas. Minimal use of IHC can reduce the NSCLC-NOS rate to under 10% - job done. The IASLC/ATS/ERS recommendations emphasise the minimal and judicious use of predictive IHC, in order to preserve tissue for molecular testing. Most cases of NSCLC-NOS can be sub-typed using the TTF1 & p63 alone, but success of this approach may depend on the population/case mix. If there are larger numbers of undifferentiated or poorly differentiated tumours and non-tru type adenocarcinomas, perhaps in a population with a strong smoking prevalence, the performance of this minimal approach may be less reliable. The author has used TTF1, p63 and CK5/6 plus AB/PAS for mucin, to reduce the NOS rate to 6%, and achieve an overall predictive accuracy of 83%. Recently double staining using p63/ck7 and TTF1/CK5-6 combos has been successful in reducing tissue usage. There are further points worthy of note: None of these markers is specific for a tumour type and certainly not part of the existing defining criteria. Consequently, the outcomes of this IHC approach are predictive of a diagnosis, and not definitive.

4 Hence the usage of probably or favour as caveat to the final probable subtype, (probably adenocarcinoma, favour squamous cell carcinoma etc) Whatever claims are made in the literature, it is important to monitor the predictive performance of this IHC approach in your own laboratory, in NSCLC-NOS cases, that are subsequently resected. This is the only scenario that matters and is informative. These IHC techniques work perfectly well in appropriately prepared cytology samples. There is no point in performing this IHC in cases that can be confidently classified by the presence of defining morphological criteria on the H&E (or equivalent stain in cytology). Extensive use of IHC can waste time, tissue and laboratory resource when a limited approach is all that is required, supported by adequate clinical information and interdisciplinary dialogue which will limit defensive practice. References 1. 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. J Thorac Oncol 2011; 6, 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. Arch Pathol Lab Med Sep 12. [Epub ahead of print] 3. Yatabe Y, Mitsudomi T, Takahashi T. TTF-1 expression in pulmonary adenocarcinomas. Am J Surg Pathol 2002; 26: Travis WD, Brambilla E, Muller-Hermelink HK et al, (eds). World Health Organisation Classification of Tumours. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart, IARC press, Lyon (2004) 5. Thomas JS, Lamb D, Ashcroft T, Corrin B, Edwards CW, Gibbs AR et al. How reliable is the diagnosis of lung cancer using small biopsy specimens? Report of a UKCCCR Lung Cancer Working Party. Thorax 48, (1993). 6. Burnett RA, Howatson SR, Lang S et al. Observer variability in histopathological reporting of non-small cell carcinoma on bronchial biopsy specimens. J Clin Pathol 49, (1996)

5 7. Cataluña JJ, Perpiñá M, Greses JV et al. Cell type accuracy of bronchial biopsy specimens in primary lung cancer. Chest 109, (1996). 8. Matsuda M, Horai T, Nakamura S et al. Bronchial brushing and bronchial biopsy: comparison of diagnostic accuracy and cell typing reliability in lung cancer. Thorax 41, (1986). 9. Chuang MT, Marchevsky A, Teirstein AS et al. Diagnosis of lung cancer by fibreoptic bronchoscopy : problems in the histological classification of non-small cell carcinomas. Thorax 39, (1984) 10. 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 nonsmall cell category. J Clin Pathol 53, (2000). 11. Loo PS, Thomas SC, Nicolson MC et al. Subtyping of undifferentiated non-small cell carcinomas in bronchial biopsy specimens. J Thorac Oncol. 5, (2010). 12. 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. Am J Surg Pathol 35, (2011) 13. Terry J, Leung S, Laskin J et al. Optimal immunohistochemical markers for distinguishing lung adenocarcinomas from squamous cell carcinomas in small tumor samples. Am J Surg Pathol 34, (2010). 14. 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 117, (2011). 15. Wallace WA, Rassl DM. Accuracy of cell typing in nonsmall cell lung cancer by EBUS/EUS-FNA cytological samples. Eur Respir J. 38, (2011). 16. Rekhtman N, Brandt SM, Sigel CS et al: Suitability of Thoracic Cytology for New Therapeutic paradigm in non-small cell lung carcinoma. J Thorac Oncol 6: Bishop JA, Teruya-Feldstein J, Westra WH et al. p40 (ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol. 25, (2012) 18. Turner BM, Cagle PT, Sainz IM et al. Napsin A, a new marker for lung adenocarcinoma, is complementary and more sensitive and specific than thyroid transcription factor 1 in the differential diagnosis of primary pulmonary carcinoma: evaluation of 1674 cases by tissue microarray. Arch Pathol Lab Med. 2012;136:163-71

6 Case 2- I struggle with the new classification Andre L. Moreira Clinical History: 48 year old Caucasian woman with stable ground glass opacity in the anterior segment of the left lower lobe. The nodule was discovered in a routine examination for an unrelated procedure 3 years ago. The nodule grew slightly in the past year. A CT-guided aspiration biopsy was performed which resulted in the diagnosis of adenocarcinoma. The patient is asymptomatic. She currently smokes cigarettes. A lobectomy was performed. Gross and microscopic examination: A 1.7 cm subpleural nodule was identified in the pulmonary parenchyma. The tumor was composed of neoplastic cells with a hobnail appearance lining the alveolar septae. In some areas there was suggestion of papillary projections, but these were simple structures without secondary or tertiary branches. In another area of the tumor, there was suggestion of a desmoplastic stromal reaction; however, the cells lining the entrapped structures had the same morphological appearance of the rest of the tumor. The tumor cells were positive for TTF-1 and Napsin- A, thus confirming a pulmonary origin. Molecular evaluation of the tumor showed a mutation in KRAs exon 2 G12D. Discussion: The current case is that of a tumor with a predominant lepidic pattern of growth. The differential diagnosis for these tumors includes a pure lepidic adenocarcinoma or adenocarcinoma in situ (AIS), a minimally invasive adenocarcinoma (MIA), and a predominant lepidic invasive adenocarcinoma. AIS is defined by a pure lepidic growth pattern with a continuous growth of neoplastic cells along the alveolar septa without disruption of the alveolar structures, although mild fibrosis or enlargement of the alveolar wall is permitted. MIA is defined as an adenocarcinoma with predominant lepidic pattern with less than or equal to 0.5 cm area of parenchymal invasion. Both diagnoses are excluded if there is any lymphatic, blood vessel, or pleural invasion or areas of necrosis. A predominant lepidic invasive adenocarcinoma has other histological subtypes infiltrating a myofibroblastic stroma. AIS and MIA have been shown to have 100% 5 year recurrence free-survival, whereas lepidic predominant adenocarcinomas can recur. Practically speaking, it is very important to differentiate AIS/MIA from an invasive lepidic predominant adenocarcinoma. There is no clear definition of gross or radiographic features for these 3 tumors types, but there are reports to suggest that all three tumors in this category may have pure ground glass opacity (GGO) or a

7 mixed GGO and solid component on CT-scan. The solid component thus represents the scar tissue or invasive histological subtype. Therefore, there are three points that need to be considered for an accurate classification using the proposed IASLC/ERS/ ATS classification. They are: determination of parenchymal invasion, extent of invasion, and presence of other patterns of adenocarcinoma besides the lepidic pattern. Problems with the classification: Determination of parenchymal invasion is by far the most difficult of these parameters. A recent study among thoracic pathologist showed that the reproducibility for the diagnosis of invasion is moderate (kappa 0.55), most of the difficulty comes from determining real invasion from its mimickers like alveolar collapse. There are no good and reproducible parameters to diagnose invasion. Some authors have proposed the use of elastic stains to differentiated collapse (maintenance of the elastic structure) from invasion (destruction of elastic structure). However, these stains may not easy to interpret due to technical problems or variations in the normal elastic patterns. Therefore, the pathologist is left with his/her own interpretation. Similar problem also occurs for the reproducibility of histological patterns. There is a large overlap between the recognition of lepidic with papillary, acinar and micropapillary patterns. The measurement of the extent of invasion becomes complicated if one cannot decide what is invasive or not. MIA for instance is composed of a predominantly lepidic pattern and an invasive component that does not exceed 0.5 cm in greatest diameter. The invasive component can be seen centrally or in the periphery of the tumor mass. The invasive component is more frequently represented by acinar and papillary growth patterns. Scars (areas of fibrosis devoid of infiltrating tumor cells) may be present especially in well-differentiated adenocarcinomas. Some authors have included the measurement of a scar, associated with an invasive component as part of the measurement that defines MIA, however, this criterion is not mentioned in the consensus document. It is not clear if a scar represents collapsed of pulmonary parenchyma due to an invasive carcinoma or it is part of another unrelated process. Until we have clear definitions and guidelines on how to approach and diagnose these tumors, a standardized nomenclature may not be reached. Practical approach to the classification, personal opinion: What is the importance of the diagnosis of MIA compared to AIS or a mixed subtype adenocarcinoma? The diagnosis of AIS and MIA carry the same prognostic significance, when present as a solitary pulmonary nodule. That is a tumor with excellent prognosis with a 100% disease-free survival in 5 years. Therefore, the recognition of MIA gives the pathologist some flexibility in reporting tumors that are not pure lepidic but can still convey to the

8 treating physician the prognostic implications of this histological subtype. Given the areas of uncertainty for the diagnostic criteria of AIS and MIA, I based my diagnosis on the extent of possible invasion. If confronted with an area of uncertainty, either because of different histological pattern (papillary or acinar) that tends to mimic alepidic pattern or an area of scar where invasion is not clear, I tend to measure the area. If smaller than 0.5 cm I tend to make the diagnosis of MIA, if the area of possible invasion is larger than 0.5 cm, I make the diagnosis of invasive adenocarcinoma, predominant lepidic. I reserve the diagnosis of AIS for tumors that fall into the strict criteria set forth by the IASLC/ERS/ATS classification. It is possible that I may be overdiagnosis these two entities (MIA and predominant lepidic adenocarcinomas), but I prefer to leave room for doubt and err in the invasive side. These tumors are by definition only encountered as stage 1a, therefore the patients will not be overtreated, but I believe this approach gives the treating physicians some room for discussion on follow-up and further management. Suggested reading: 1. Noguchi M, Morikawa A, Kawasaki M et al. Small adenocarcinoma of the lung. Histologic characteristics and prognosis. Cancer. 1995;75(12): Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC. Pathology and Genetics. Tumours of the Lung, Pleura, Thymus and Heart. Lyon France: IARC Press; Borczuk AC, Qian F, Kazeros A et al. Invasive size is an independent predictor of survival in pulmonary adenocarcinoma. Am J Surg Pathol. 2009;33(3): Suzuki K, Yokose T, Yoshida J et al. Prognostic significance of the size of central fibrosis in peripheral adenocarcinoma of the lung. Ann Thorac Surg. 2000;69(3): Yim J, Zhu LC, Chiriboga L, Watson HN, Goldberg JD, Moreira AL. Histologic features are important prognostic indicators in early stages lung adenocarcinomas. Mod Pathol. 2007;20(2): Yokose T, Suzuki K, Nagai K, Nishiwaki Y, Sasaki S, Ochiai A. Favorable and unfavorable morphological prognostic factors in peripheral adenocarcinoma of the lung 3 cm or less in diameter. Lung Cancer. 2000;29(3): Yoshizawa A, Motoi N, Riely GJ, Sima CS, Gerald WL, Kris MG, Park BJ, Rusch VW, Travis WD. Impact of proposed IASLC/ATS/ERS classification of lung adenocarcinoma: prognostic subgroups

9 and implications for further revision of staging based on analysis of 514 stage I cases. Mod Pathol : 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. J Thorac Oncol, 2011; 6: Thunnissen E, Beasley M, Borczuk A, Brambilla E, et al. Reproducibility of histopathological subtypes and invasion in pulmonary adenocarcinoma. An International Interobserver Study. Mod Pathol, :

10 Case 3 - The New Classification Helped Lucian R. Chirieac Clinical History: A 67-year-old woman with a history of moderate to severe COPD underwent a CT scan initially that demonstrated a 2 mm nodule within the left lingula. A followup CT scan six months later showed an increase in size of the left upper lobe nodule to 9 mm. A PET/CT showed a moderate uptake in the left lingular nodule with SUV max of 3.4. No other sites of hypermetabolic activity were noted. A subsequent wedge resection by video-assisted thoracic surgery (VATS) and mediastinoscopy were performed. Morphology The wedge resection revealed two tumor nodules with different morphologies: (1) the Figure 1a Figure 1b first tumor nodule was 1.2 cm in greatest dimension and was consistent with a lung adenocarcinoma, with 60 percent acinar (Figure 1a), and 40 percent lepidic patterns (Figure 1b), and (2) a second nodule classified as a minimally invasive adenocarcinoma (MIA) measuring 1.1 cm with a 0.3 cm focus of invasive acinar Figure 2 adenocarcinoma (Figure 2). Both tumor 0.3 cm nodules have different morphologies, (1) the invasive adenocarcinoma is acinar predominant, and (2) MIA is lepidic predominant.

11 Discussion MIA is a newly defined entity introduced by the IASCL/ATS/ERS Lung Adenocarcinoma Classification to describe a category of tumors in patients with good prognosis. (1-6) MIA is defined as a lung adenocarcinoma smaller than 3 cm in greatest dimension that has an invasive component smaller than 5 mm.(2) Although lepidicpredominant tumors larger than 3 cm may have a prognosis similar to MIA, there are no published studies to date. Therefore, these tumors are categorized as "lepidicpredominant adenocarcinomas" in the new IASCL/ATS/ERS Lung Adenocarcinoma Classification. The invasive component could be any or a combination of the histologic subtypes acinar, papillary, micropapillary or solid or alternatively, isolated tumor cells invading into the fibroblastic stroma Tumor cells in MIA could be mucinous, non mucinous or a combination of both.(7) The presence of lymphvascular and/or pleural invasion excludes a diagnosis of MIA. Diagnosing MIA represents a good alternative to a category of lepidicpredominant adenocarcinomas that display microscopic foci of questionable stromal invasion. Since appreciating stromal invasion versus alveolar collapse is an area of great controversy among pathologists, the introduction of MIA as a new concept is of great help in this scenario. A diagnosis of MIA is based on measuring both the invasive and in situ components and the tumor should be thoroughly sampled and entirely submitted for microscopic examination. Often, correlation between the dimensions measured by optical microscopy and the dimensions of the ground glass-capacity by CT examination might be useful. Impact of the New IASLC/ATS/ERS Classification Patients with MIA have a good prognosis similar to adenocarcinoma in situ (AIS).(4, 8)

12 Since the histologic characteristics of these two adenocarcinomas are different (predominantly acinar vs predominantly lepidic), the two nodules are synchronous primary tumors, and therefore are separately staged as T1aN0, and T1aN0, respectively. Areas of Future Investigation Investigating the outcome of patients with MIA histology greater than 3 cm. More outcome studies are needed to establish if 0.5 mm cutoff size of the invasive component in MIA is an independent predictor of overall survival in multivariate analyses. It is unclear if MIA should be staged as Tis or as T1, and future studies should investigate this issue. References 1. 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. J Thorac Oncol; 6: Travis WD, Brambilla E, Riely GJ. New Pathologic Classification of Lung Cancer: Relevance for Clinical Practice and Clinical Trials. J Clin Oncol. 3. Yoshizawa A, Sumiyoshi S, Sonobe M, et al. Validation of the IASLC/ATS/ERS lung adenocarcinoma classification for prognosis and association with EGFR and KRAS gene mutations: analysis of 440 Japanese patients. J Thorac Oncol; 8: Borczuk AC, Qian F, Kazeros A, et al. Invasive size is an independent predictor of survival in pulmonary adenocarcinoma. Am J Surg Pathol 2009; 33: Yim J, Zhu LC, Chiriboga L, Watson HN, Goldberg JD, Moreira AL. Histologic features are important prognostic indicators in early stages lung adenocarcinomas. Mod Pathol 2007; 20:

13 6. Maeshima AM, Tochigi N, Yoshida A, Asamura H, Tsuta K, Tsuda H. Histological scoring for small lung adenocarcinomas 2 cm or less in diameter: a reliable prognostic indicator. J Thorac Oncol; 5: 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. Mod Pathol; 24: Yokose T, Suzuki K, Nagai K, Nishiwaki Y, Sasaki S, Ochiai A. Favorable and unfavorable morphological prognostic factors in peripheral adenocarcinoma of the lung 3 cm or less in diameter. Lung Cancer 2000; 29:

14 Case 4 - I struggled with the new classification Yasushi Yatabe Clinical history: A 3-cm nodule was detected in the middle of the right lung of a 67-year-old female non-smoker at a medical check-up. A chest CT revealed a consolidated lesion with an air bronchogram. The lesion was endoscopically examined, but the biopsied tissues contained no tumor cells. A lobectomy was conducted, both for diagnosis and surgical resection. Morphology: Grossly, the nodule showed a well-demarcated lesion with a mucinous lucent cut surface. Indeed, the tumor cells, most of which were comprised of goblet cells or highcolumnar cells, secreted a large amount of mucin around the nodule. The tumor cells with goblet-cell morphology grew partially along the alveolar walls (lepidic growth pattern), whereas acinar patterns were predominant in other solid areas. The former area showing lepidic growth was consistent with mucinous invasive adenocarcinoma (IMA), and the latter solid area could be categorized as acinar-predominant adenocarcinoma. The tumor cells were positive for TTF-1 and CK7 and negative for CK20, CDX2 and HNF4a according to the immunohistochemical staining results. Genotypically, the tumor had a variant- 1 EML4-ALK translocation, whereas the EGFR-TK and KRAS genes were wild type. Discussion Invasive mucinous adenocarcinoma (IMA) is a new category in the new IASLC/ATS/ERS adenocarcinoma classification. 1 It had been formerly diagnosed mostly as mucinous bronchioloalveolar carcinoma (BAC) or mixed adenocarcinoma. In the 1999 World Health Organization classification, BAC was defined as a noninvasive adenocarcinoma without evidence of stromal, vascular, or pleural invasion, and BAC was classified into mucinous and nonmucinous subtypes. However, most lesions that appear to be mucinous BACs can be found to have invasive foci if the lesions are extensively sampled. Indeed, clinical trials on these invasive tumors have been prompted by clinical demand under the term of advanced BACs. Biologically, this group of tumors has characteristics that differentiate it from conventional lung adenocarcinoma: a strong correlation with the KRAS mutation, 2, 3 the lack of EGFR mutation, 4, negative expression of TTF-1, 5, 6 and frequent expression of CK20. 6, 7 In addition, the radiographic features are also distinct from AIS. These differences are summarized in the table. 1

15 Characteristics Invasive Mucinous Adenocarcinoma (Formerly Mucinous BAC) Nonmucinous AIS/MIA/LPA (Formerly Non-mucinous BAC) Female 49/84 (58%) 101/140 (72%) Smoker 39/87 (45%) 75/164 (46%) Radiographic appearance Majority consolidation; airbronchogram; frequent multifocal & multi-lobar presentation. Majority ground-glass attenuation Cell Type Immunophenotype Mucin-filled, columnar and/or goblet cells Type II pneumocytes and/or Clara cells CK7 Mostly positive Positive CK20 Positive Mostly negative TTF-1 Mostly negative Positive Genotype KRAS mutation Frequent Uncommon EGFR mutation Almost none Frequent BAC = bronchioloalveolar carcinoma; AIS=adenocarcinoma in situ; MIA=minimally invasive adenocarcinoma; LPA=lepidic predominant adenocarcinoma; numbers represent the percentage of cases that are reported to be positive. However, isolating this subtype causes the new problems, as noted following, and I am personally struggling to make a definitive diagnosis of the presented tumor. Differentiation of IMA from the other adenocarcinomas with mucinous features. Distinctive histologic appearance of IMA is that tumor cells show a goblet or columnar tumorcell morphology with abundant intracytoplasmic mucin; however, this appearance can also be observed in the other subtypes, including colloid carcinoma, mucinous AIS/MIA and, occasionally, acinar predominant adenocarcinoma. Furthermore, these cancer subtypes are often accompanied by mucinous features (filling mucin in alveolar spaces). Thus, it is challenging to differentiate IMAs from these subtypes. We recently reported that HNF4a could be used as a marker for IMA; nearly all IMAs were positive. 8 However, a subset of acinar-predominant adenocarcinomas also expressed HNF4a. In addition, when adenocarcinomas with goblet cell/high-columnar cell features were selected, the IMA markers (HNF4a, CK20 and CDX2) were frequently positive and the KRAS mutation rate was similar to that of an IMA. 9 These findings suggested that IMA and this subset of adenocarcinoma biologically lie within the same spectrum of the adenocarcinoma subtype. Therefore, it seems to be impossible to distinguish between these adenocarcinomas, biologically. Another reason to separate this tumor into a new category is its radiographic appearance. In IMAs, pneumonia-like infiltrates with multi-focal and multi-lobar presentations are characteristic. There is no doubt that a tumor with such radiographic characteristics will be diagnosed as an IMA when the tumor cells show a goblet/columnar cell morphology with mucinous features. However, when the tumor appears radiographically as a nodular lesion (as in

16 the presented case), it is difficult to differentiate the IMA from the acinar-predominant subtype. I am attempting to differentiate an IMA from acinar-predominant adenocarcinomas based on cellular composition. When a tumor consists almost entirely of goblet/columnar cells, my diagnosis of the tumor is IMA. In contrast, a tumor with a mixed cellular composition would be diagnosed as an acinar-predominant adenocarcinoma. As will be mentioned in the next discussion, such a case would include ALK-positive cancers. Some IMA-like tumors harbor the ALK mutation. Some histological features have been reported to be associated with ALK fusion, and these features are exemplified by signet-ring cell morphology, cribriform pattern with mucin, and a solid and acinar structure Interestingly, majority of these characteristics are associated with mucin production. Indeed, some IMA-like tumors could have features of ALK fusion, as in the presented case; however, a tumor with ALK fusion is biologically distinct from an IMA. Tumors with ALK fusion are always positive for TTF-1, in contrast to IMAs, which are negative. In terms of histologic features, although the background mucin lake formations, goblet/columnar cell morphology and acinar growth patterns are similar, the tubular/acinar structure in ALK-fusion cancers tends to be closed, so the tumors frequently show closed tubular or cribriform nests in the background mucin lake. 13 In the case of IMAs, the tumor cells are open to the alveolar spaces and typically show lepidic growth. The diagnosis with a small biopsy specimen Because goblet/columnar cells in IMA and IMA-like tumors have cytologically inconspicuous atypia, diagnosis is often difficult for small biopsy/cytology specimens. Two characteristics of these tumors are helpful for diagnosis. First is the finding that continuous goblet/columnar cells grow along thin delicate alveolar septa. It is common to see goblet cells in the bronchus and bronchioles, but these cells are usually lined with other non-goblet epithelial cells. Generally, uniform cellular composition is a hallmark of neoplasia. The goblet/columnar cells are also observed in association with inflammatory reactions, such as bronchiolization. In this case, the background stroma show inflammatory fibrosis, and bronchiolization never develops on the thin alveolar septa. These histological findings are helpful, when mucin is seen filling alveolar spaces (usually identified by foamy macrophages in the center of those spaces). Another finding is HNF4a expression. This marker is negative for all cellular components in the normal lung, with the exception of patchy positive reactions in reactive type-ii pneumocytes. When this marker shows continuous, intense reactions, the positive cells are most likely related to this adenocarcinoma subset, although only a limited number of tumor cells are observed. References 1. 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. J Thorac Oncol 2011; 6(2): Kobayashi T, Tsuda H, Noguchi M, et al. Association of point mutation in c-ki-ras oncogene in lung adenocarcinoma with particular reference to cytologic subtypes. Cancer 1990; 66(2): Marchetti A, Buttitta F, Pellegrini S, et al. Bronchioloalveolar lung carcinomas: K-ras mutations are constant events in the mucinous subtype. J Pathol 1996; 179(3): Marchetti A, Martella C, Felicioni L, et al. EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. J Clin Oncol 2005; 23(4): Lau SK, Desrochers MJ, Luthringer DJ. Expression of thyroid transcription factor-1, cytokeratin 7, and cytokeratin 20 in bronchioloalveolar carcinomas: an immunohistochemical evaluation of 67 cases. Mod Pathol 2002; 15(5): Goldstein NS, Thomas M. Mucinous and nonmucinous bronchioloalveolar adenocarcinomas have distinct staining patterns with thyroid transcription factor and cytokeratin 20 antibodies. Am J Clin Pathol 2001; 116(3):

17 7. Shah RN, Badve S, Papreddy K, et al. Expression of cytokeratin 20 in mucinous bronchioloalveolar carcinoma. Hum Pathol 2002; 33(9): Sugano M, Nagasaka T, Sasaki E, et al. HNF4alpha as a Marker for Invasive Mucinous Adenocarcinoma of the Lung. Am J Surg Pathol 2013; 37(2): Yatabe Y, Koga T, Mitsudomi T, Takahashi T. CK20 expression, CDX2 expression, K-ras mutation, and goblet cell morphology in a subset of lung adenocarcinomas. J Pathol 2004; 203(2): Inamura K, Takeuchi K, Togashi Y, et al. EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers. J Thorac Oncol 2008; 3(1): Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009; 27(26): Yoshida A, Tsuta K, Nakamura H, et al. Comprehensive histologic analysis of ALK-rearranged lung carcinomas. Am J Surg Pathol 2011; 35(8): Murakami Y, Mitsudomi T, Yatabe Y. A Screening Method for the ALK Fusion Gene in NSCLC. Front Oncol 2012; 2:24.

18 THE IASLC/ERS/ATS ADENOCARCINOMA CLASSIFICATION RATIONALE AND STRENGTHS PULMONARY PATHOLOGY SOCIETY USCAP, BALTIMORE, March 2, 2013 William D. Travis, M.D. Dept of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY Worldwide, lung cancer is the most common cause of major cancer mortality in men and the second most common in women. Recent therapeutic advances have led to a revolution in the lung cancer field in discovering therapeutically tractable oncogene dependency, that have major implications for patient evaluation and approach to diagnosis. The 2011 IASLC/ATS/ERS Classification of Lung Adenocarcinoma addresses these issues. 1 This classification was developed based on an evidence-based approach by an international multidisciplinary panel including pathologists, oncologists/respiratory physicians, radiologists, molecular biologists, and thoracic surgeons. 1 Multiple paradigm shifts are outlined that have a major impact on clinical practice for pathologists as well as the entire multidisciplinary team caring for lung cancer patients. Two review articles one on small biopsies/cytology specimens and the other on resection specimens addressed primarily to pathologists are about to be published and are currently available online. 2, 3 Since 70% of lung cancer patients present in advanced stages, their diagnosis is usually established based on small biopsies and/or cytology specimens and the primary therapy is chemotherapy; the remaining patients are usually resectable and surgery is the primary treatment. In order to address all types of cancer patients, there are two major components to the classification based on the type of specimen: 1) small biopsies and cytology or 2) resection. Because previous WHO classifications did not provide specific criteria and terminology for pathologic diagnosis of lung cancer in small biopsies and cytology, 4 this new classification is more clinically relevant as it addresses these small specimens and addresses molecular testing. In recent years, three therapeutic advances for advanced NSCLC have made accurate histologic diagnosis a critical step in developing a personalized approach to management. The first, relates to tyrosine kinase inhibitors as first line therapy in patients with advanced lung adenocarcinoma with EGFR mutations. 5 For this reason, in the new classification EGFR mutation testing is recommended for advanced lung cancer patients with a histologic diagnosis of adenocarcinoma. Second, patients with adenocarcinoma or NSCLC, not otherwise specified (NSCLC-NOS) are more responsive to pemetrexed than those squamous cell carcinoma. 6 Third, squamous cell carcinoma is associated with life threatening hemorrhage in patients treated with bevacizumab therefore it is contraindicated in lung cancer patients with this histology. 7 Fourth, crizotinib is an FDA-approved therapy for advanced adenocarcinomas with ALK rearrangements Both EGFR mutations and ALK rearrangements are almost exclusively seen in lung adenocarcinomas. So a pathologic diagnosis of adenocarcinoma or squamous cell carcinoma will determine patient eligibility for EGFR mutation testing and for specific therapies. In all of these clinical trials the pathologic diagnoses were based on light microscopy with or without mucin stains but not on the basis of immunohistochemical stains. 5-7 While much progress has been made in identifying validated molecular targets for lung adenocarcinoma, only recently have potential targets been identified for squamous cell carcinoma

19 Travis WD Lung Adenocarcinoma Classification including FGFR1 amplification and DDR2 mutations which may render these patients sensitive to FGFR 11, inhibition and dasatinib respectively. The Cancer Genome Atlas (TCGA) project sponsored by the National Cancer Institute has identified molecular alterations that may represent molecular targets in the majority of lung squamous cell carcinomas. 13 In the future this may lead to effective targeted therapies for lung squamous cell carcinomas, which would only increase the importance of accurate pathologic classification in small biopsies and cytology. CLASSIFICATION BASED ON SMALL BIOPSIES AND CYTOLOGY Now that lung cancer therapy is personalized for individual patients based on the histologic type of lung cancer and molecular status, the pathologist s role and approach to lung cancer diagnosis in small biopsies and cytology have been affected dramatically. The need to classify non-small cell carcinoma (NSCLC) further to distinguish squamous cell carcinoma from adenocarcinoma has not existed until recently so the frequency of NSCLCnot otherwise specified (NOS) has been increasing up to 20-40%. Now pathologists need to make an effort to make a more specific diagnosis using special stains. Tumors that show morphologic squamous or adenocarcinoma differentiation can be classified as squamous cell carcinoma or adenocarcinoma, respectively. However, for those tumors that lack clear differentiation by morphology and would be classified as NSCLC-NOS in the past, now need to be evaluated by immunohistochemistry. The recommendation is to use a single adenocarcinoma marker such as TTF-1 and a single squamous marker such as the recently described p40 antibody. 14 If a NSCLC-NOS by light microscopy shows a staining pattern that clearly points to adenocarcinoma (TTF-1 positive, p40 negative), the tumor can be classified as NSCLC, favor adenocarcinoma. If such a tumor stains with a squamous pattern (p40 positive, TTF-1 negative), then it can be called NSCLC, favor squamous cell carcinoma. If the tumor does not show clear differentiation by immunohistochemistry, it should remain as NSCLC-NOS. These terms and criteria will help track the former NOS tumors that are being reclassified with the addition of special stains for future clinical trials. Advanced stage tumors classified as adenocarcinoma, NSCLC, favor adenocarcinoma or NSCLC-NOS should be tested for EGFR mutation; mutation positive patients are eligible for tyrosine kinase inhibitor therapy. If there is no EGFR mutation, recent data suggests these patients are candidates for EML4-ALK fusion testing and if positive they can receive the FDA approved drug crizotinib, 10 and if negative these patients are eligible for pemetrexed or bevacizumab based chemotherapeutic regimens. In the new classification, a limited use of special stains is recommended for NSCLC- NOS by light microscopy in order to try to classify these tumors further. Minimizing special stains is helpful to maximize the amount of tissue available for molecular testing. 1 A new responsibility for pathologists, in addition to making a correct diagnosis, is to manage these small biopsies and cytology specimens strategically so there is sufficient tissue preserved for molecular studies. A new emphasis is made on the need for a multidisciplinary approach to lung cancer diagnosis. One of the central proposals in this classification is that each institution needs to have a multidisciplinary strategy that addresses how to obtain these small specimens, how to process them in the pathology laboratory, how to preserve material for molecular testing, sending specimens to the molecular laboratory for expedited testing and the reporting the results in a pathology report. It may be useful to have a multidisciplinary committee to develop this strategy and to monitor issues in an ongoing fashion. 2

20 Travis WD Lung Adenocarcinoma Classification CLASSIFICATION BASED ON RESECTION SPECIMENS Major changes are also recommended for adenocarcinomas diagnosed in resection specimens: 1 1) the term bronchioloalveolar carcinoma (BAC) should not be used anymore, because tumors previously classified under as BAC are represented by five different tumors in this classification; 2), for solitary tumors measuring 3cm, new concepts of adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) have been introduced for lesions that have no invasion or 5mm invasion, respectively; these patients should have 100% or near 100% disease free survival (DFS); 3) for invasive adenocarcinomas, comprehensive histologic subtyping is recommended for evaluation with classification according to the predominant subtype; 4) micropapillary adenocarcinoma is proposed as a new subtype with a poor prognosis; 5) the term lepidic replaces BAC for tumors with a predominant component formerly called nonmucinous BAC, and the term lepidic predominant adenocarcinoma is recommended along with discontinuing the term mixed subtype ; 6) invasive mucinous adenocarcinoma (IMA) is the term used to replace those formerly classified as mucinous BAC. IMA are strongly correlated with KRAS mutation Recently strong survival correlations were demonstrated using this classification in Stage I adenocarcinomas with the following 5-year DFS: AIS and MIA (100%), lepidic (90%), acinar (84%), papillary (83%), and there was a poor prognostic group: micropapillary (67%), solid (70%), colloid (71%) predominant tumors as well as invasive mucinous adenocarcinoma (75%). 15 Comprehensive histologic subtyping is performed by making semiquantitative estimation of each of the patterns in 5% increments. A deliberate choice needs to be made to give one pattern the largest percentage. It is useful to record in diagnostic reports each adenocarcinoma subtype that is present with the percentages. This approach may also provide a basis for architectural grading of lung adenocarcinomas Early reproducibility studies have shown moderate to substantial inter-observer agreement among pathologists for the predominant pattern. A reproducibility study of classical and difficult selected images of the major lung adenocarcinoma subtypes circulated among a panel of 26 expert lung cancer pathologists documented kappa values of / and /- 0.14, respectively. 18 A recent study of reproducibility for predominant pattern showed moderate to good κ-values of 0.44 to 0.72 for pulmonary pathologists. For untrained pathologists κ-values were expectedly lower ranging from 0.38 to 0.47, but these improved after a training session to 0.51 to 0.66 and reevaluation by the same reviewers led to very high κ-values between Reproducibility improves after training sessions. However, work is needed to improve separation of difficult problems such as lepidic versus acinar or papillary and micropapillary versus papillary patterns. 18, Since this classification was initially published, there are a growing number of studies of resected lung adenocarcinomas that have demonstrated its utility in identifying significant prognostic subsets and molecular correlations according to the predominant 15, 16, patterns. In summary this classification outlines many paradigm shifts in lung cancer diagnosis that crystalize the importance of histology and genetics in personalized medicine for lung cancer patients. Evidence-based recommendations are made that will transform the clinical practice of all physicians involved with lung cancer diagnosis. Reference List 1. Travis WD, Brambilla E, Noguchi M et al. The New IASLC/ATS/ERS international multidisciplinary lung adenocarcinoma classification. J Thoracic Oncol 2011;6(2):

21 Travis WD Lung Adenocarcinoma Classification 2. Travis WD, Brambilla E, Noguchi M et al. Lung Cancer Diagnosis in Small Biopsies and Cytology: Implications of the 2011 IASLC/ATS/ERS Lung Adenocarcinoma Classification. Arch Pathol Lab Med 2013;e pub ahead. 3. Travis WD, Brambilla E, Noguchi M et al. Diagnosis of Lung Adenocarcinoma in Resected Specimens: Implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Lung Classification. Arch Pathol Lab Med 2013;e pub ahead. 4. Travis WD, Brambilla E, Müller-Hermelink HK, Harris CC. Pathology and Genetics: Tumours of the Lung, Pleura, Thymus and Heart. Lyon: IARC; Mok TS, Wu YL, Thongprasert S et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361(10): Scagliotti GV, Parikh P, von PJ et al. Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 2008;26(21): Johnson DH, Fehrenbacher L, Novotny WF et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 2004;22(11): Sasaki T, Janne PA. New Strategies for Treatment of ALK Rearranged Non-Small Cell Lung Cancers. Clin Cancer Res 2011;17(23): Shaw AT, Solomon B. Targeting anaplastic lymphoma kinase in lung cancer. Clin Cancer Res 2011;17(8): Kwak EL, Bang YJ, Camidge DR et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363(18): Dutt A, Ramos AH, Hammerman PS et al. Inhibitor-Sensitive FGFR1 Amplification in Human Non- Small Cell Lung Cancer. PLoS ONE 2011;6(6):e Weiss J, Sos ML, Seidel D et al. Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. Sci Transl Med 2010;2(62):62ra Hammerman PS, Hayes DN, Wilkerson MD et al. Comprehensive genomic characterization of squamous cell lung cancers. Nature 2012;489(7417): 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. Mod Pathol 2012;25(3): 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. Mod Pathol 2011;24(5):

22 Travis WD Lung Adenocarcinoma Classification 16. Sica G, Yoshizawa A, Sima CS et al. A grading system of lung adenocarcinomas based on histologic pattern is predictive of disease recurrence in stage I tumors. Am J Surg Pathol 2010;34(8): Kadota K, Suzuki K, Kachala SS et al. A grading system combining architectural features and mitotic count predicts recurrence in stage I lung adenocarcinoma. Mod Pathol 2012;25(8): Thunnissen FB, Beasley MB, Borczuk A et al. Reproducibility of histopathological subtypes and invasion in pulmonary adenocarcinoma. An international interobserver study. Mod Pathol 2012;25(12): Warth A, Stenzinger A, von Brunneck AC et al. Interobserver variability in the application of the novel IASLC/ATS/ERS classification. Eur Respir J 2012;40(5): Warth A, Stenzinger A, von Brunneck AC et al. Interobserver variability in the application of the novel IASLC/ATS/ERS classification. Eur Respir J 2012;40(5): Warth A, Cortis J, Fink L et al. Training increases concordance in classifying pulmonary adenocarcinomas according to the novel IASLC/ATS/ERS classification. Virchows Arch 2012;461(6): Warth A, Muley T, Meister M et al. The novel histologic IASLC/ATS/ERS classification system of invasive pulmonary adenocarcinoma is a stage-independent predictor of survival. J Clin Oncol 2012;30(13): 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. J Thorac Oncol 2011;6(9): Yoshizawa A, Sumiyoshi S, Sonobe M et al. Validation of the IASLC/ATS/ERS Lung Adenocarcinoma Classification for Prognosis and Association with EGFR and KRAS Gene Mutations: Analysis of 440 Japanese Patients. J Thorac Oncol 2013;8(1): Shim HS, Lee dh, Park EJ, Kim SH. Histopathologic characteristics of lung adenocarcinomas with epidermal growth factor receptor mutations in the international association for the study of lung cancer/american thoracic society/european respiratory society lung adenocarcinoma classification. Arch Pathol Lab Med 2011;135(10): Sterlacci W, Savic S, Schmid T et al. Tissue-Sparing Application of the Newly Proposed IASLC/ATS/ERS Classification of Adenocarcinoma of the Lung Shows Practical Diagnostic and Prognostic Impact. Am J Clin Pathol 2012;137(6):