Faculty Bibliography

In this review, we outline the biology and management of patients with carcinosarcomas and related malignancies, which are often included under the broader concept of sarcomatoid carcinomas. Carcinosarcomas are unusual tumors that are commonly gynecologic in origin, where they are referred to as malignant mixed Müllerian tumors, but may appear in any anatomic site. Although a variety of hypotheses have been presented as to the biphasic nature of these tumors, carcinosarcomas seem to represent the best example in human cancers of the concept of epithelial-mesenchymal transition (EMT), in which the two parts of the tumor are genomically related to one another, as opposed to the mesenchymal component that represents a second neoplasm or (benign) reactive process. In general, patients with carcinosarcomas fare worse than patients with carcinomas of the same anatomic site. Treatment paradigms for carcinosarcomas generally follow those of carcinomas of the same organ site, except where clinical trials provide more specific options. Agents that block or reverse EMT are worth examination in patients with carcinosarcoma and arguably may be even more effective in carcinomas, given evidence of dependence on EMT to generate successful metastases. Information about EMT may also inform other phase transitions in cancer, such as those between prostate or lung carcinoma and more aggressive tumors with neuroendocrine differentiation.

BACKGROUND: Two recent observations regarding the Warburg effect are that (i) the metabolism of stem cells is constitutive ('aerobic') glycolysis while normal cellular differentiation involves a transition to oxidative phosphorylation and (ii) the degree of glucose uptake of a malignancy as imaged by 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) is associated with histologic measures of tumor differentiation. Combining these observations, we hypothesized that the high levels of glucose uptake observed in poorly differentiated cancers may reflect persistence of the glycolytic metabolism of stem cells in malignant cells that fail to fully differentiate. PATIENTS AND METHODS: Tumor glucose uptake was measured by FDG-PET in 552 patients with histologically diverse cancers. We used normal mixture modeling to explore FDG-PET standardized uptake value (SUV) distributions and tested for associations between glucose uptake and histological differentiation, risk of lymph node metastasis, and survival. Using RNA-seq data, we performed pathway and transcription factor analyses to compare tumors with high and low levels of glucose uptake. RESULTS: We found that well-differentiated tumors had low FDG uptake, while moderately and poorly differentiated tumors had higher uptake. The distribution of SUV for each histology was bimodal, with a low peak around SUV 2-5 and a high peak at SUV 8-14. The cancers in the two modes were clinically distinct in terms of the risk of nodal metastases and death. Carbohydrate metabolism and the pentose-related pathway were elevated in the poorly differentiated/high SUV clusters. Embryonic stem cell-related signatures were activated in poorly differentiated/high SUV clusters. CONCLUSIONS: Our findings support the hypothesis that the biological basis for the Warburg effect is a persistence of stem cell metabolism (i.e. aerobic glycolysis) in cancers as a failure to transition from glycolysis-utilizing undifferentiated cells to oxidative phosphorylation-utilizing differentiated cells. We found that cancers cluster along the differentiation pathway into two groups, utilizing either glycolysis or oxidative phosphorylation. Our results have implications for multiple areas of clinical oncology.

Environmental nutrient levels impact cancer cell metabolism, resulting in context-dependent gene essentiality. Here, using loss-of-function screening based on RNA interference, we show that environmental oxygen levels are a major driver of differential essentiality between in vitro model systems and in vivo tumours. Above the 3-8% oxygen concentration typical of most tissues, we find that cancer cells depend on high levels of the iron-sulfur cluster biosynthetic enzyme NFS1. Mammary or subcutaneous tumours grow despite suppression of NFS1, whereas metastatic or primary lung tumours do not. Consistent with a role in surviving the high oxygen environment of incipient lung tumours, NFS1 lies in a region of genomic amplification present in lung adenocarcinoma and is most highly expressed in well-differentiated adenocarcinomas. NFS1 activity is particularly important for maintaining the iron-sulfur co-factors present in multiple cell-essential proteins upon exposure to oxygen compared to other forms of oxidative damage. Furthermore, insufficient iron-sulfur cluster maintenance robustly activates the iron-starvation response and, in combination with inhibition of glutathione biosynthesis, triggers ferroptosis, a non-apoptotic form of cell death. Suppression of NFS1 cooperates with inhibition of cysteine transport to trigger ferroptosis in vitro and slow tumour growth. Therefore, lung adenocarcinomas select for expression of a pathway that confers resistance to high oxygen tension and protects cells from undergoing ferroptosis in response to oxidative damage.

Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

This session will focus on some new definitions and concepts in the recently published 2015 WHO classification of lung tumors1, some of which were showcased in the 2011 IASLC/ATS/ERS lung adenocarcinoma classification2, while others are introduced for the first time. We will focus on resected lung adenocarcinoma as well resected squamous cell carcinoma, large cell carcinoma and the neuroendocrine tumor spectrum. Adenocarcinoma: In the 2015 WHO classification, the definition of adenocarcinoma has been expanded from a malignant epithelial tumor with glandular differentiation or mucin production to include tumors that also express pneumocyte immunomarkers (e.g. TTF1, Napsin A). This means that undifferentiated carcinomas formerly classified as large cell carcinoma that express pneumocyte immunomarkers, like undifferentiated carcinomas that show mucin expression, are now included in the solid adenocarcinoma category. Invasive adenocarcinomas account for >70% of all surgically resected cases and consist of a complex admixture of histologic subtypes. In an effort to represent this morphologic complexity, comprehensive histologic subtyping was introduced in the 2011 IASLC/ATS/ERS classification. A number of recent studies have demonstrated the utility of comprehensive histologic subtyping in identifying prognostically significant groups of tumors. Studies published both before and after the 2011 IASLC/ATS/ERS classification have highlighted the importance of the secondary patterns in addition to the predominant pattern in resected lung adenocarcinoma. Comprehensive histologic subtyping, its pitfalls and the emerging significance of secondary patterns in tumor recurrence and prognosis will be discussed further in this session. Grading: There is no well-established, internationally accepted grading system in resected lung adenocarcinoma. A simple grading system based on predominant histologic subtype has been proposed due to the prognostic significance of predominant histologic subtype. Other suggested grading schemes include different combinations of mitotic count, second predominant pattern and nuclear features with predominant histologic subtype. The emerging concept of an objective grading system for pulmonary adenocarcinomas will be briefly explored in this session. Another newly introduced concept in the 2015 WHO classification is that of tumor spread through alveolar spaces (STAS) which may occur with micropapillary clusters, solid nests or single cells. STAS was found to be associated with an increased recurrence rate in patients with stage I adenocarcinomas <2cm who underwent sublobar resections.3 Tumor Size and Staging: A recent study confirmed that in resected non-mucinous adenocarcinoma, the size of the invasive component, excluding the lepidic (equated with in situ) component of the tumor, correlates better with patient outcome than total tumor size.4 This finding has been supported by other studies and is expected to be included in the upcoming 8th edition TNM staging system for the T descriptor for pathologic staging in resected non-mucinous adenocarcinoma.5 Squamous Cell Carcinoma (SQCC): SQCC is the second most prevalent non-small cell lung cancer (NSCLC), behind adenocarcinoma. Contrary to the latter where most changes in nomenclature, diagnosis and molecular pathology have occurred, SQCC has a strong association to smoking and remains a challenge for oncologists with few therapeutic advances. To reduce the risk of over diagnosing SQCC, the definition of SQCC became stricter in the 2015 WHO classification. For the diagnosis of this entity it is necessary to have evidence of keratinization and intercellular bridges. For non-keratinizing SQCC it is necessary to demonstrate evidence of squamous differentiation by immunohistochemical (IHC) stain (diffuse positivity for p40 or p63 and absence of adenocarcinoma markers such as TTF-1 and napsin-A). Non-keratinizing SQCC shares a solid pattern of growth with adenocarcinoma; in addition, solid type adenocarcinomas can have squamoid features such as glassy and abundant cytoplasm that can mimic SQCC6, therefore it is recommended the use of IHC for any NSCLC with solid pattern of growth. Presence or absence of mucin is not a criterion for diagnosis of SQCC. Similar to adenocarcinoma, there is no grading system for SQCC. There is evidence that tumor budding is associated with worse prognosis.7 Basaloid carcinoma is now classified in the same group of SQCC and no longer part of large cell carcinoma. In contrast, Lymphoepithelioma-like carcinoma of the lung that share IHC profile with squamous cell carcinoma is grouped in the category of other undifferentiated tumors that also include NUT carcinoma. Large Cell Carcinoma: This remains a separate category; however, the diagnosis of this entity is greatly reduced. Large cell carcinoma is an undifferentiated carcinoma (positive for cytokeratin markers), which lacks evidence of differentiation by morphology and lineage specific immunohistochemical profile (TTF-1/Napsin-A and p40 negative). This classification is supported by molecular profile.8 High Grade Neuroendocrine Carcinomas: This tumor category remains largely the same from previous classification with the exception that Large Cell Neuroendocrine Carcinoma (LCNC) is now grouped with neuroendocrine tumors. It is no longer part of a Large Cell Carcinoma category. Recent studies have suggested that LCNEC is a heterogeneous group ranging in the spectrum from NSCLC-like to small cell carcinoma-like tumors.9 LCNEC that resemble NSCLC have higher incidence of KRAS mutations, whereas those morphologically closer to small cell carcinoma have higher incidence of RB mutation. The significance of these findings for tumor classification and especially for therapeutic options are still unknown as more studies need to be done. There have been several studies on the genetic and epigenetic profile of small cell carcinoma that could lead to new therapeutic options.10 However, the diagnosis and classification of this tumor remains the same. Carcinoid Tumors: Typical and atypical carcinoid tumors maintained the same morphological criteria for diagnosis and classification. Recent molecular studies have showed however that these tumors have distinct molecular profiles from the high grade relatives (LCNC and small cell carcinoma).10

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.