Tumor mutational burden (TMB) is an emerging biomarker used to identify patients who are more likely to benefit from immuno-oncology therapy. Aside from various unsettled technical aspects, ...biological variables such as tumor cell content and intratumor heterogeneity may play an important role in determining TMB.
TMB estimates were determined applying the TruSight Oncology 500 targeted sequencing panel. Spatial and temporal heterogeneity was analyzed by multiregion sequencing (two to six samples) of 24 pulmonary adenocarcinomas and by sequencing a set of matched primary tumors, locoregional lymph node metastases, and distant metastases in five patients.
On average, a coding region of 1.28 Mbp was covered with a mean read depth of 609x. Manual validation of the mutation-calls confirmed a good performance, but revealed noticeable misclassification during germline filtering. Different regions within a tumor showed considerable spatial TMB variance in 30% (7 of 24) of the cases (maximum difference, 14.13 mut/Mbp). Lymph node–derived TMB was significantly lower (p = 0.016). In 13 cases, distinct mutational profiles were exclusive to different regions of a tumor, leading to higher values for simulated aggregated TMB. Combined, intratumor heterogeneity and the aggregated TMB could result in divergent TMB designation in 17% of the analyzed patients. TMB variation between primary tumor and distant metastases existed but was not profound.
Our data show that, in addition to technical aspects such as germline filtering, the tumor content and spatially divergent mutational profiles within a tumor are relevant factors influencing TMB estimation, revealing limitations of single-sample–based TMB estimations in a clinical context.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Iron-loaded tumor-associated macrophages (iTAMs) show a pro-inflammatory phenotype, hallmarked by anti-tumorigenic activity and an ability to attenuate tumor growth. Here we explored the relevance of ...these findings in lung cancer patients by investigating the impact of the iTAM content in the tumor microenvironment (TME) on patient survival. We analyzed 102 human non-small cell lung cancer (NSCLC) paraffin-embedded archival tissue samples for iron levels and macrophage numbers. Interestingly, patients with lung adenocarcinoma accumulating iron in the TME show higher numbers of M1-like pro-inflammatory TAMs and a survival advantage compared to iron-negative patients. By contrast, in patients with lung squamous cell carcinoma iron in the TME does not affect survival, suggesting a unique influence of iron on different histological subtypes of non-small cell lung cancer (NSCLC). We conclude that in lung adenocarcinoma iron may serve as a prognostic marker for patient survival and as a potential therapeutic target for anti-cancer therapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Aims
Molecular characterization of non‐small‐cell lung cancer (NSCLC) has revealed multiple druggable mutations for targeted therapies. Recently, chromosomal rearrangements involving c‐ros oncogene ...1, receptor tyrosine kinase (ROS1) were identified, and patients seem to benefit from crizotinib treatment. The aim of this study was to identify the clinicopathological characteristics of NSCLC with ROS1 expression and translocation.
Methods and results
We screened 1478 NSCLCs with a ROS1‐specific antibody, and tested positive cases with FISH. All positive cases were analysed for associated clinicopathological characteristics, including survival and molecular tumour composition. Sixty‐eight cases (4.6%) showed ROS1 immunoreactivity, and ROS1 translocations were confirmed in nine cases (0.6%). ROS1 expression was predominantly found in female adenocarcinoma patients, in patients with low T stages, and in association with TTF1 and napsin expression, and certain histomorphological adenocarcinoma patterns (lepidic, acinar, and solid). ROS1 translocations occurred in conjunction with other driver mutations (EGFR, KRAS, and BRAF). ROS1 expression was found to be a stage‐independent predictor of favourable survival.
Conclusions
ROS1 translocations are rare events in resected NSCLCs from Caucasian patients. Immunohistochemical screening for ROS1 expression and clinicopathological parameters, including female sex, early tumour stages, adenocarcinomas with TTF1 and/or napsin expression, and a distinct histomorphological growth pattern, strongly facilitate case enrichment. Molecularly driven multistep concepts might not be optimal for case selection.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Hypoxia signaling plays a major role in non-malignant and malignant hyperproliferative diseases. Pulmonary hypertension (PH), a hypoxia-driven vascular disease, is characterized by a glycolytic ...switch similar to the Warburg effect in cancer. Ras association domain family 1A (RASSF1A) is a scaffold protein that acts as a tumour suppressor. Here we show that hypoxia promotes stabilization of RASSF1A through NOX-1- and protein kinase C- dependent phosphorylation. In parallel, hypoxia inducible factor-1 α (HIF-1α) activates RASSF1A transcription via HIF-binding sites in the RASSF1A promoter region. Vice versa, RASSF1A binds to HIF-1α, blocks its prolyl-hydroxylation and proteasomal degradation, and thus enhances the activation of the glycolytic switch. We find that this mechanism operates in experimental hypoxia-induced PH, which is blocked in RASSF1A knockout mice, in human primary PH vascular cells, and in a subset of human lung cancer cells. We conclude that RASSF1A-HIF-1α forms a feedforward loop driving hypoxia signaling in PH and cancer.
Tumor-associated macrophages (TAMs) frequently help to sustain tumor growth and mediate immune suppression in the tumor microenvironment (TME). Here, we identified a subset of iron-loaded, ...pro-inflammatory TAMs localized in hemorrhagic areas of the TME. The occurrence of iron-loaded TAMs (iTAMs) correlated with reduced tumor size in patients with non-small cell lung cancer.
experiments established that TAMs exposed to hemolytic red blood cells (RBCs) were converted into pro-inflammatory macrophages capable of directly killing tumor cells. This anti-tumor effect could also be elicited
iron oxide nanoparticles. When tested
, tumors injected with such iron oxide nanoparticles led to significantly smaller tumor sizes compared to controls. These results identify hemolytic RBCs and iron as novel players in the TME that repolarize TAMs to exert direct anti-tumor effector function. Thus, the delivery of iron to TAMs emerges as a simple adjuvant therapeutic strategy to promote anti-cancer immune responses.
Abstract The chromatin remodeling Switch Sucrose Non-Fermentable (SWI/SNF) complex has been increasingly implicated in the pathogenesis and dedifferentiation of neoplasms from several organs with ...prognostic and potential therapeutic implications. We herein investigated the expression of the SWI/SNF complex catalytic subunits SMARCA4 (BRG1) and SMARCA2 (BRM) in 316 consecutive NSCLC specimens on tissue microarrays (171 adenocarcinomas/ADCA, 130 squamous cell carcinomas/SCC, 9 adenosquamous carcinomas, and 6 large cell carcinomas) excluding undifferentiated/giant cell or rhabdoid carcinomas. Complete loss of SMARCA4 was observed in 8/146 (5.5%) and 6/115 (5.2%) of evaluable pulmonary ADCA and SCC, respectively, while 9/140 (6.4%) ADCA and 2/117 (1.7%) SCC showed SMARCA2 loss, respectively. Two of 6 large cell carcinomas were SMARCA2-deficient. Concurrent loss of both markers was observed in 4 cases (2 ADCA and 2 SCC). Of 15 ADCA with loss of either or both markers, 12 (80%) were TTF1 negative. In conclusion, SMARCA4- and SMARCA2 deficiency is observed in 5.1% and 4.8% of NSCLC, respectively. SMARCB1 expression was intact in all cases. The presence of differentiated histology (glandular or squamous) is a novel aspect among SWI/SNF deficient carcinomas which in other organs generally are associated with undifferentiated/rhabdoid morphology. The predominance of TTF1-negativity among SWI/SNF-deficient pulmonary ADCA (80%) underlines the need to include these two markers in the evaluation of TTF1-negative ADCA of putative pulmonary origin. Given the recently documented potential of SMARCA4 loss as a predictor of chemosensitivity to platinum-based chemotherapy in NSCLC, recognition of the clinicopathological features of SMARCA4-deficient NSCLC in routine surgical pathology practice is recommended.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by patchy scarring of the distal lung with limited therapeutic options and poor prognosis. Here, ...we show that conditional deletion of the ubiquitin ligase Nedd4-2 (Nedd4l) in lung epithelial cells in adult mice produces chronic lung disease sharing key features with IPF including progressive fibrosis and bronchiolization with increased expression of Muc5b in peripheral airways, honeycombing and characteristic alterations in the lung proteome. NEDD4-2 is implicated in the regulation of the epithelial Na
channel critical for proper airway surface hydration and mucus clearance and the regulation of TGFβ signaling, which promotes fibrotic remodeling. Our data support a role of mucociliary dysfunction and aberrant epithelial pro-fibrotic response in the multifactorial disease pathogenesis. Further, treatment with the anti-fibrotic drug pirfenidone reduced pulmonary fibrosis in this model. This model may therefore aid studies of the pathogenesis and therapy of IPF.
PD-(L)1 inhibitors have improved prognosis of non-small-cell lung cancer (NSCLC), but can also cause immune-related adverse events (irAEs) that complicate management.
We analyzed NSCLC patients ...receiving PD-(L)1 inhibitors from 2012 to 2020 in a German academic center.
IrAE showed comparable frequencies in stage IV (198/894 or 22%)
III (14/45 or 31%, p = 0.15), after anti-PD-(L)1 monotherapy
chemoimmunotherapy (139/483
58/213, p = 0.75), and across treatment lines. In stage IV, irAE occurred after 3.1 months in median, affected multiple organs (median 2) in 27/894 patients and were associated with PD-L1 positivity (25
14%, p = 0.003), lower neutrophil-to-lymphocyte ratios (29
17%, p < 0.001 for NLR dichotomized at 5), better ECOG status (26
18% for 0
1, p = 0.004), but not related to age, sex, smoking and palliative radiotherapy. Two hundred thirty two irAEs occurred mostly in endocrine glands (4.9%), lungs (4.4%), the musculoskeletal system (4.2%), colon (4.1%), liver (3.7%), and skin (2.6%), while pneumonitis was most frequent with durvalumab following definitive chemoradiation (16% or 7/45, p < 0.01). IrAE severity was grade 1 in 11%, 2 in 41%, 3 in 36%, and 4 in 11% events, while two were lethal (<1%, myocarditis and pneumonitis). Therapy was suspended in 72%, while steroids were initiated in 66% and complemented by other immunosuppressants in 6%, with longest treatment duration for rheumatic events (mean >3 months), and average cumulative prednisone doses >700 mg for all organs, except for skin. Patients developing irAE had longer progression-free (PFS) and overall survival (OS) in multivariable 12/14-week landmark analyses including ECOG status, treatment line, treatment type, PD-L1 TPS, and NLR (median PFS 17
10 months, HR = 0.68, p = 0.009; median OS 37
15 months, HR = 0.40, p < 0.001), regardless of grade. OS was longest with skin (95% at 2 years) and shortest with pneumonitis, hepatitis, neurologic, and cardiologic irAE (38, 37, 28, and 0% at 2 years, p < 0.001).
Approximately one-fourth of immunotherapy-treated NSCLC patients develop irAEs, most of which necessitate treatment suspension and steroids. Despite more frequent occurrence with PD-L1 positive tumors, lower NLR, and better ECOG PS, irAEs are independently associated with longer survival, especially when affecting the skin. Lethality is below 1%.
Synaptophysin, chromogranin and CD56 are recommended markers to identify pulmonary tumors with neuroendocrine differentiation. Whether the expression of these markers in pulmonary adenocarcinoma and ...pulmonary squamous cell carcinoma is a prognostic factor has been a matter of debate. Therefore, we investigated retrospectively a large cohort to expand the data on the role of synaptophysin, chromogranin and CD56 in non-small cell lung cancer lacking morphological features of neuroendocrine differentiation.
A cohort of 627 pulmonary adenocarcinomas (ADC) and 543 squamous cell carcinomas (SqCC) lacking morphological features of neuroendocrine differentiation was assembled and a tissue microarray was constructed. All cases were stained with synaptophysin, chromogranin and CD56. Positivity was defined as > 1% positive tumor cells. Data was correlated with clinico-pathological features including overall and disease free survival.
110 (18%) ADC and 80 (15%) SqCC were positive for either synaptophysin, chromogranin, CD56 or a combination. The most commonly positive single marker was synaptophysin. The least common positive marker was chromogranin. A combination of ≤2 neuroendocrine markers was positive in 2-3% of ADC and 0-1% of SqCC. There was no significant difference in overall survival in tumors with positivity for neuroendocrine markers neither in ADC (univariate: P = 0.4; hazard ratio HR = 0.867; multivariate: P = 0.5; HR = 0.876) nor in SqCC (univariate: P = 0.1; HR = 0.694; multivariate: P = 0.1, HR = 0.697). Likewise, there was no significant difference in disease free survival.
We report on a cohort of 1170 cases that synaptophysin, chromogranin and CD56 are commonly expressed in ADC and SqCC and that their expression has no impact on survival, supporting the current best practice guidelines.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background Large cell neuroendocrine carcinoma (LCNEC) represents a rare entity in non-small cell lung cancer, with only partially understood biology and poor survival. A diagnosis is difficult to ...obtain on the basis of small biopsy specimens, but surgical procedures may be indicated in only a small fraction of patients. The aim of this study was to assess the clinical and immunohistochemical features of patients with LCNEC to identify predictors of outcome and long-term survival. Methods The clinical and pathologic data of 57 surgical patients with LCNEC between March 2003 and December 2012 were retrospectively reviewed. The tumor specimens were examined for expression of neuronal specific enolase, synaptophysin, CD 56, chromogranin-A, and the somatostatin receptor by immunohistochemistry. Statistical analysis was performed to determine significant predictors for overall survival and recurrence-free survival. Results Fifty-seven patients (41 men, 16 women) underwent thoracic operations with curative intent. Complete resection was achieved in 91% of cases. The results of staining for CD56, synaptophysin, neuronal specific enolase, chromogranin-A, and somatostatin were positive in 86%, 81%, 68%, 61%, and 21%, respectively. Recurrence occurred in 28 patients (49%). Overall survival and recurrence-free survival were 50% and 45%, respectively, after 3 years. Advanced nodal status (N1, p < 0.025; N2, p < 0.02) and simultaneous expression of CD56 and chromogranin-A ( p < 0.04) were significantly associated with poorer outcome. Conclusions LCNEC is a rare neuroendocrine pulmonary malignancy that is associated with poor prognosis and high recurrence rates. Surgical treatment can achieve satisfactory results in selected cases. Neuroendocrine marker profiles may predict prognosis and may influence the decision for adjuvant therapy or follow-up intervals.
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