Andre L. Moreira

Clarifying the Spectrum of Driver Oncogene Mutations in Biomarker-Verified Squamous Carcinoma of Lung: Lack of EGFR/KRAS and Presence of PIK3CA/AKT1 Mutations

Purpose: There is persistent controversy as to whether EGFR and KRAS mutations occur in pulmonary squamous cell carcinoma (SQCC). We hypothesized that the reported variability may reflect difficulties in the pathologic distinction of true SQCC from adenosquamous carcinoma (AD-SQC) and poorly differentiated adenocarcinoma due to incomplete sampling or morphologic overlap. The recent development of a robust immunohistochemical approach for distinguishing squamous versus glandular differentiation provides an opportunity to reassess EGFR/KRAS and other targetable kinase mutation frequencies in a pathologically homogeneous series of SQCC. Experimental Design: Ninety-five resected SQCCs, verified by immunohistochemistry as ΔNp63+/TTF-1−, were tested for activating mutations in EGFR, KRAS, BRAF, PIK3CA, NRAS, AKT1, ERBB2/HER2, and MAP2K1/MEK1. In addition, all tissue samples from rare patients with the diagnosis of EGFR/KRAS-mutant “SQCC” encountered during 5 years of routine clinical genotyping were reassessed pathologically. Results: The screen of 95 biomarker-verified SQCCs revealed no EGFR/KRAS [0%; 95% confidence interval (CI), 0%–3.8%], four PIK3CA (4%; 95% CI, 1%–10%), and one AKT1 (1%; 95% CI, 0%–5.7%) mutations. Detailed morphologic and immunohistochemical reevaluation of EGFR/KRAS-mutant “SQCC” identified during clinical genotyping (n = 16) resulted in reclassification of 10 (63%) cases as AD-SQC and five (31%) cases as poorly differentiated adenocarcinoma morphologically mimicking SQCC (i.e., adenocarcinoma with “squamoid” morphology). One (6%) case had no follow-up. Conclusions: Our findings suggest that EGFR/KRAS mutations do not occur in pure pulmonary SQCC, and occasional detection of these mutations in samples diagnosed as “SQCC” is due to challenges with the diagnosis of AD-SQC and adenocarcinoma, which can be largely resolved by comprehensive pathologic assessment incorporating immunohistochemical biomarkers. Clin Cancer Res; 18(4); 1167–76. ©2012 AACR.

A grading system of lung adenocarcinomas based on histologic pattern is predictive of disease recurrence in stage I tumors.

Currently no objective grading system for pulmonary adenocarcinomas exists. To determine whether specific histologic patterns or combinations thereof could be linked to an objective grading system, the histologic patterns in metastatic tumor deposits was compared with the patterns seen in the corresponding 73 primary tumor to determine whether a specific pattern had higher propensity to metastasize. The concordance of the predominant histologic pattern in the primary tumor and the metastases was of 100% for micropapillary, 86% for solid, 42% for acinar, and 23% for papillary types of adenocarcinoma. Informed by these results, a 3-tier grading system based on the histologic subtypes was established. Grade I, a pattern with low metastatic potential (BAC); Grade II, patterns with intermediate metastatic potential (acinar and papillary); and Grade III, patterns with high metastatic potential (solid and micropapillary). These grades were combined into a number of different scoring systems, whose ability to predict recurrence or death from disease was tested in 366 stage 1 adenocarcinomas. A score based on the 2 most predominant grades was able to stratify patients into low-to-high risk for recurrence or death of disease (P=0.001). The 5-years disease-free survival for patients in the highest score group was of 0.73, compared with 0.84 and 0.92 in the intermediate and lowest score groups. Concordance probability estimate was 0.65 (95% confidence interval 0.57-0.73). Therefore, this scoring system provides valuable information in discriminating patients with different risk of disease-recurrence in a highly homogeneous population of patients with stage I cancer.

Virulence of Selected Mycobacterium tuberculosis Clinical Isolates in the Rabbit Model of Meningitis Is Dependent on Phenolic Glycolipid Produced by the Bacilli

Infection with Mycobacterium tuberculosis in humans results in active disease in approximately 10% of immune-competent individuals, with the most-severe clinical manifestations observed when the bacilli infect the central nervous system (CNS). Here, we use a rabbit model of tuberculous meningitis to evaluate the severity of disease caused by the M. tuberculosis clinical isolates CDC1551, a highly immunogenic strain, and HN878 or W4, 2 members of the W/Beijing family of strains. Compared with infection with CDC1551, CNS infection with HN878 or W4 resulted in higher bacillary loads in the cerebrospinal fluid and brain, increased dissemination of bacilli to other organs, persistent levels of tumor necrosis factor-alpha , higher leukocytosis, and more-severe clinical manifestations. This pathogenic process is associated with the production by HN878 of a polyketide synthase-derived phenolic glycolipid (PGL), as demonstrated by reduced virulence in rabbits infected with an HN878 mutant disrupted in the pks1-15 gene, which is required for PGL synthesis.

Histologic features are important prognostic indicators in early stages lung adenocarcinomas.

This study attempts to evaluate the clinicopathologic features of mixed subtype adenocarcinomas and the prognostic implications of histopathology classifications. Surgical specimens from 141 patients with clinical stage I or II lung adenocarcinoma during the period 1992–2004 were included. These cases were classified into four groups defined by the extent of the bronchioloalveolar carcinoma component: group I: pure bronchioloalveolar carcinoma; group II: mixed subtype with predominant bronchioloalveolar carcinoma component and ≤5 mm invasive component; group III: mixed subtype with bronchioloalveolar carcinoma component and >5 mm invasive component; group IV: invasive carcinoma with no bronchioloalveolar carcinoma component. Descriptive statistics were used to examine the groups with respect to age, tumor size, lymph node metastasis, and Ki-67 and p53 expression levels. Death rate for the groups was obtained by patients charts and from the National Death Index database. The population was similar in age, tumor size and lymph node metastasis. Immunohistochemical results showed that the mean Ki-67 labeling and the amount of p53 overexpression had the same trend of increasing mean values or positive results from groups I to IV. The reported proportion of deaths ranged from 0% for groups I and II, 20% in patients with predominant invasive component with bronchioloalveolar carcinoma (group III), and 18% in patients with invasive carcinomas and no bronchioloalveolar carcinoma component (group IV). The difference between the proportion of patients with reported deaths in the time period of this study in the combined greater than 5 mm+pure invasive groups (groups III, IV), and the <5 mm+noninvasive groups (groups I, II) is statistically significant. These results suggest that histological features may be useful in defining categories of lung adenocarcinomas with differing survival and prognostic features. These results are helpful in defining a subcategory of ‘minimally invasive adenocarcinoma’, which has features similar to bronchioloalveolar carcinoma.

A grading system combining architectural features and mitotic count predicts recurrence in stage I lung adenocarcinoma

The International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) has recently proposed a new lung adenocarcinoma classification. We investigated whether nuclear features can stratify prognostic subsets. Slides of 485 stage I lung adenocarcinoma patients were reviewed. We evaluated nuclear diameter, nuclear atypia, nuclear/cytoplasmic ratio, chromatin pattern, prominence of nucleoli, intranuclear inclusions, mitotic count/10 high-power fields (HPFs) or 2.4 mm2, and atypical mitoses. Tumors were classified into histologic subtypes according to the IASLC/ATS/ERS classification and grouped by architectural grade into low (adenocarcinoma in situ, minimally invasive adenocarcinoma, or lepidic predominant), intermediate (papillary or acinar), and high (micropapillary or solid). Log-rank tests and Cox regression models evaluated the ability of clinicopathologic factors to predict recurrence-free probability. In univariate analyses, nuclear diameter (P=0.007), nuclear atypia (P=0.006), mitotic count (P<0.001), and atypical mitoses (P<0.001) were significant predictors of recurrence. The recurrence-free probability of patients with high mitotic count (≥5/10 HPF: n=175) was the lowest (5-year recurrence-free probability=73%), followed by intermediate (2–4/10 HPF: n=106, 80%), and low (0–1/10 HPF: n=204, 91%, P<0.001). Combined architectural/mitotic grading system stratified patient outcomes (P<0.001): low grade (low architectural grade with any mitotic count and intermediate architectural grade with low mitotic count: n=201, 5-year recurrence-free probability=92%), intermediate grade (intermediate architectural grade with intermediate–high mitotic counts: n=206, 78%), and high grade (high architectural grade with any mitotic count: n=78, 68%). The advantage of adding mitotic count to architectural grade is in stratifying patients with intermediate architectural grade into two prognostically distinct categories (P=0.001). After adjusting for clinicopathologic factors including sex, stage, pleural/lymphovascular invasion, and necrosis, mitotic count was not an independent predictor of recurrence (P=0.178). However, patients with the high architectural/mitotic grade remained at significantly increased risk of recurrence (high vs low: P=0.005) after adjusting for clinical factors. We proposed this combined architectural/mitotic grade for lung adenocarcinoma as a practical method that can be applied in routine practice.

Subtyping of Non-small Cell Lung Carcinoma: A Comparison of Small Biopsy and Cytology Specimens

Background: There is growing evidence that lung adenocarcinoma and squamous cell carcinoma (SQCC) have distinct oncogenic mutations and divergent therapeutic responses, which is driving the heightened emphasis on accurate pathologic subtyping of non-small cell lung carcinoma (NSCLC). The relative feasibility and accuracy of NSCLC subtyping by small biopsy versus cytology is not well established, particularly in current practice where immunohistochemistry (IHC) is becoming routinely used to aid in this distinction. Methods: Concurrent biopsy and cytology specimens obtained during a single procedure and diagnosed as NSCLC during a 2-year period (n = 101) were reviewed. Concordance of diagnoses in the two methods was assessed. Accuracy was determined based on subsequent resection or autopsy diagnosis (n = 21) or IHC for thyroid transcription factor 1 and p63 on a subset of cases (n = 43). Results: The distribution of definitive versus favored versus unclassified categories (reflecting the degree of diagnostic certainty) was similar for biopsy (71% versus 23% versus 6%, respectively) and cytology (69% versus 19% versus 12%, respectively), p = 0.29. When results from paired specimens were combined, the rate of definitive diagnoses by at least one method was increased to 84% and the unclassified rate was decreased to 4%. NSCLC subtype concordance between biopsy and cytology was 93%. Kappa coefficient (95% confidence interval) for agreement between methods was 0.88 (0.60–0.89) for adenocarcinoma and 0.76 (0.63–0.89) for SQCC. In pairs with discordant diagnoses (n = 7) the correct tumor type was identified with a similar frequency by biopsy (n = 4) and cytology (n = 3). Factors contributing to mistyping were poor differentiation, necrosis, low cellularity, and lack of supporting IHC. All concordant diagnoses for which verification was available (n = 57) were correct. IHC was used more frequently to subtype NSCLC in biopsy than cytology (32% versus 6%; p = 0.0001). Conclusions: Small biopsy and cytology achieve comparable rates of definitive and accurate NSCLC subtyping, and the optimal results are attained when the two modalities are considered jointly. The lower requirement for IHC in cytology highlights the strength of cytomorphology in NSCLC subtyping. Whenever clinically feasible, obtaining parallel biopsy and cytology specimens is encouraged.

Determinants of Response to Interleukin-10 Receptor Blockade Immunotherapy in Experimental Visceral Leishmaniasis

In established Leishmania donovani visceral infection in normal mice, anti-interleukin (IL)-10 receptor (IL-10R) monoclonal antibody (MAb) treatment induced intracellular parasite killing within liver macrophages. IL-10R blockade maintained IL-12 protein 40, markedly increased interferon (IFN)-gamma serum levels, and enhanced tissue inducible nitric oxide synthase (iNOS) expression and granuloma assembly. Optimal MAb-induced killing, including synergism with antimony chemotherapy, required endogenous IL-12 and/or IFN-gamma and at least one IFN-gamma-regulated macrophage mechanism-iNOS or phagocyte oxidase. However, in IFN-gamma knockout mice, anti-IL-10R also induced both granuloma formation and leishmanistatic activity. As judged by IL-10R blockade, endogenous IL-10 primarily regulates killing in L. donovani infection by suppressing production of and responses to the Th1 cell-type cytokines, IL-12, and IFN-gamma. However, because anti-IL-10R also released IFN-gamma-independent effects, IL-10 appears to act more broadly and suppresses multiple antileishmanial mechanisms.

Distinct profile of driver mutations and clinical features in immunomarker-defined subsets of pulmonary large-cell carcinoma

Pulmonary large-cell carcinoma-a diagnostically and clinically controversial entity-is defined as a non-small-cell carcinoma lacking morphologic differentiation of either adenocarcinoma or squamous cell carcinoma, but suspected to represent an end stage of poor differentiation of these tumor types. Given the recent advances in immunohistochemistry to distinguish adenocarcinoma and squamous cell carcinoma, and the recent insights that several therapeutically relevant genetic alterations are distributed differentially in these tumors, we hypothesized that immunophenotyping may stratify large-cell carcinomas into subsets with distinct profiles of targetable driver mutations. We therefore analyzed 102 large-cell carcinomas by immunohistochemistry for TTF-1 and ΔNp63/p40 as classifiers for adenocarcinoma and squamous cell carcinoma, respectively, and correlated the resulting subtypes with nine therapeutically relevant genetic alterations characteristic of adenocarcinoma (EGFR, KRAS, BRAF, MAP2K1/MEK1, NRAS, ERBB2/HER2 mutations and ALK rearrangements) or more common in squamous cell carcinoma (PIK3CA and AKT1 mutations). The immunomarkers classified large-cell carcinomas as variants of adenocarcinoma (n=62; 60%), squamous cell carcinoma (n=20; 20%) or marker-null (n=20; 20%). Genetic alterations were found in 38 cases (37%), including EGFR (n=1), KRAS (n=30), BRAF (n=2), MAP2K1 (n=1), ALK (n=3) and PIK3CA (n=1). All molecular alterations characteristic of adenocarcinoma occurred in tumors with immunoprofiles of adenocarcinoma or marker-null, but not in tumors with squamous immunoprofiles (combined mutation rate 50% vs 30% vs 0%, respectively; P<0.001), whereas the sole PIK3CA mutation occurred in a tumor with squamous profile (5%). Furthermore, marker-null large-cell carcinomas were associated with significantly inferior disease-free (P<0.001) and overall (P=0.001) survival. In conclusion, the majority (80%) of large-cell carcinomas can be classified by immunomarkers as variants of adenocarcinoma or squamous cell carcinoma, which stratifies these tumors into subsets with a distinct distribution of driver mutations and distinct prognoses. These findings have practical implications for diagnosis, predictive molecular testing and therapy selection.Pulmonary large-cell carcinoma—a diagnostically and clinically controversial entity—is defined as a non-small-cell carcinoma lacking morphologic differentiation of either adenocarcinoma or squamous cell carcinoma, but suspected to represent an end stage of poor differentiation of these tumor types. Given the recent advances in immunohistochemistry to distinguish adenocarcinoma and squamous cell carcinoma, and the recent insights that several therapeutically relevant genetic alterations are distributed differentially in these tumors, we hypothesized that immunophenotyping may stratify large-cell carcinomas into subsets with distinct profiles of targetable driver mutations. We therefore analyzed 102 large-cell carcinomas by immunohistochemistry for TTF-1 and ΔNp63/p40 as classifiers for adenocarcinoma and squamous cell carcinoma, respectively, and correlated the resulting subtypes with nine therapeutically relevant genetic alterations characteristic of adenocarcinoma (EGFR, KRAS, BRAF, MAP2K1/MEK1, NRAS, ERBB2/HER2 mutations and ALK rearrangements) or more common in squamous cell carcinoma (PIK3CA and AKT1 mutations). The immunomarkers classified large-cell carcinomas as variants of adenocarcinoma (n=62; 60%), squamous cell carcinoma (n=20; 20%) or marker-null (n=20; 20%). Genetic alterations were found in 38 cases (37%), including EGFR (n=1), KRAS (n=30), BRAF (n=2), MAP2K1 (n=1), ALK (n=3) and PIK3CA (n=1). All molecular alterations characteristic of adenocarcinoma occurred in tumors with immunoprofiles of adenocarcinoma or marker-null, but not in tumors with squamous immunoprofiles (combined mutation rate 50% vs 30% vs 0%, respectively; P<0.001), whereas the sole PIK3CA mutation occurred in a tumor with squamous profile (5%). Furthermore, marker-null large-cell carcinomas were associated with significantly inferior disease-free (P<0.001) and overall (P=0.001) survival. In conclusion, the majority (80%) of large-cell carcinomas can be classified by immunomarkers as variants of adenocarcinoma or squamous cell carcinoma, which stratifies these tumors into subsets with a distinct distribution of driver mutations and distinct prognoses. These findings have practical implications for diagnosis, predictive molecular testing and therapy selection.

KRAS mutations are associated with solid growth pattern and tumor-infiltrating leukocytes in lung adenocarcinoma

KRAS mutations define a clinically distinct subgroup of lung adenocarcinoma patients, characterized by smoking history, resistance to EGFR-targeted therapies, and adverse prognosis. Whether KRAS-mutated lung adenocarcinomas also have distinct histopathological features is not well established. We tested 180 resected lung adenocarcinomas for KRAS and EGFR mutations by high-sensitivity mass spectrometry-based genotyping (Sequenom) and PCR-based sizing assays. All tumors were assessed for the proportion of standard histological patterns (lepidic, acinar, papillary, micropapillary, solid, and mucinous), several other histological and clinical parameters, and TTF-1 expression by immunohistochemistry. Among 180 carcinomas, 63 (35%) had KRAS mutations (KRAS+), 35 (19%) had EGFR mutations (EGFR+), and 82 (46%) had neither mutation (KRAS−/EGFR−). Solid growth pattern was significantly over-represented in KRAS+ carcinomas: the mean±s.d. for the amount of solid pattern in KRAS+ carcinomas was 27±34% compared with 3±10% in EGFR+ (P<0.001) and 15±27% in KRAS−/EGFR− (P=0.033) tumors. Furthermore, at least focal (≥20%) solid component was more common in KRAS+ (28/63; 44%) compared with EGFR+ (2/35; 6%; P<0.001) and KRAS−/EGFR− (21/82; 26%; P=0.022) carcinomas. KRAS mutations were also over-represented in mucinous carcinomas and were significantly associated with the presence of tumor-infiltrating leukocytes and heavier smoking history. EGFR mutations were associated with non-mucinous non-solid patterns, particularly lepidic and papillary, lack of necrosis, lack of cytological atypia, hobnail cytology, TTF-1 expression, and never/light smoking history. In conclusion, extended molecular and clinicopathological analysis of lung adenocarcinomas reveals a novel association of KRAS mutations with solid histology and tumor-infiltrating inflammatory cells and expands on several previously recognized morphological and clinical associations of KRAS and EGFR mutations. Solid growth pattern was recently shown to be a strong predictor of aggressive behavior in lung adenocarcinomas, which may underlie the unfavorable prognosis associated with KRAS mutations in these tumors.

The cribriform pattern identifies a subset of acinar predominant tumors with poor prognosis in patients with stage I lung adenocarcinoma: a conceptual proposal to classify cribriform predominant tumors as a distinct histologic subtype

The 2011 International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) lung adenocarcinoma classification emphasizes the prognostic significance of histologic subtypes. However, one limitation of this classification is that the highest percentage of patients (∼40%) is classified as acinar predominant tumors, and these patients display a spectrum of favorable and unfavorable clinical behaviors. We investigated whether the cribriform pattern can further stratify prognosis by histologic subtype. Tumor slides from 1038 patients with stage I lung adenocarcinoma (1995–2009) were reviewed. Tumors were classified according to the IASLC/ATS/ERS classification. The percentage of cribriform pattern was recorded, and the cribriform predominant subtype was considered as a subtype for analysis. The log-rank test was used to analyze the association between histologic variables and recurrence-free probability. The 5-year recurrence-free probability for patients with cribriform predominant tumors (n=46) was 70%. The recurrence-free probability for patients with cribriform predominant tumors was significantly lower than that for patients with acinar (5-year recurrence-free probability, 87%; P=0.002) or papillary predominant tumors (83%; P=0.020) but was comparable to that for patients with micropapillary (P=0.34) or solid predominant tumors (P=0.56). The recurrence-free probability for patients with ≥10% cribriform pattern tumors (n=214) was significantly lower (5-year recurrence-free probability, 73%) than that for patients with <10% cribriform pattern tumors (n=824; 84%; P<0.001). In multivariate analysis, patients with acinar predominant tumors with ≥10% cribriform pattern remained at significantly increased risk of recurrence compared with those with <10% cribriform pattern (P=0.042). Cribriform predominant tumors should be considered a distinct subtype with a high risk of recurrence, and presence (≥10%) of the cribriform pattern is an independent predictor of recurrence, identifying a poor prognostic subset of acinar predominant tumors. Our findings highlight the important prognostic value of comprehensive histologic subtyping and recording the percentage of each histologic pattern, according to the IASLC/ATS/ERS classification with the addition of the cribriform subtype.