Stroma AReactive Invasion Front Areas (SARIFA) improves prognostic risk stratification of perioperative chemotherapy treated oesophagogastric cancer patients from the MAGIC and the ST03 trial.

BACKGROUND: Tumour-associated fat cells without desmoplastic stroma reaction at the invasion front (Stroma AReactive Invasion Front Areas (SARIFA)) is a prognostic biomarker in gastric and colon cancer. The clinical utility of the SARIFA status in oesophagogastric cancer patients treated with perioperative chemotherapy is currently unknown. METHODS: The SARIFA status was determined in tissue sections from patients recruited into the MAGIC (n = 292) or ST03 (n = 693) trials treated with surgery alone (S, MAGIC) or perioperative chemotherapy (MAGIC, ST03). The relationship between SARIFA status, clinicopathological factors, overall survival (OS) and treatment was analysed. RESULTS: The SARIFA status was positive in 42% MAGIC trial S patients, 28% MAGIC and 48% ST03 patients after pre-operative chemotherapy. SARIFA status was related to OS in MAGIC trial S patients and was an independent prognostic biomarker in ST03 trial patients (HR 1.974, 95% CI 1.555-2.507, p < 0.001). ST03 patients with lymph node metastasis (ypN + ) and SARIFA-positive tumours had poorer OS than patients with ypN+ and SARIFA-negative tumours (plogrank < 0.001). CONCLUSIONS: The SARIFA status has clinical utility as prognostic biomarker in oesophagogastric cancer patients irrespective of treatment modality. Whilst underlying biological mechanisms warrant further investigation, the SARIFA status might be used to identify new drug targets, potentially enabling repurposing of existing drugs targeting lipid metabolism.

The Concept of Stroma AReactive Invasion Front Areas (SARIFA) as a new prognostic biomarker for lipid-driven cancers holds true in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a 'difficult-to-treat' entity. To forecast its prognosis, we introduced a new biomarker, SARIFA (stroma areactive invasion front areas), which are areas at the tumour invasion front lacking desmoplastic stroma reaction upon malignant invasion in the surrounding tissue, leading to direct contact between tumour cells and adipocytes. SARIFA showed its significance in gastric and colorectal carcinoma, revealing lipid metabolism alternations that promote tumour progression. METHODS: We reviewed the SARIFA status of 166 PDAC cases on all available H&E-stained tumour slides from archival Whipple-resection specimens. SARIFA positivity was defined as SARIFA detection in at least 66% of the available slides. To investigate alterations in tumour metabolism and microenvironment, we performed immunohistochemical staining for FABP4, CD36 and CD68. To verify and quantify a supposed delipidation of adipocytes, adipose tissue was digitally morphometrised. RESULTS: In total, 53 cases (32%) were classified as SARIFA positive and 113 (68%) as SARIFA negative. Patients with SARIFA-positive PDAC showed a significantly worse overall survival compared with SARIFA-negative cases (median overall survival: 11.0 months vs. 22.0 months, HR: 1.570 (1.082-2.278), 95% CI, p = 0.018), which was independent from other prognostic markers (p = 0.014). At the invasion front of SARIFA-positive PDAC, we observed significantly higher expression of FABP4 (p < 0.0001) and higher concentrations of CD68+ macrophages (p = 0.031) related to a higher risk of tumour progression. CD36 staining showed no significant expression differences. The adipocyte areas at the invasion front were significantly smaller, with mean values of 4021 ± 1058 µm2 and 1812 ± 1008 µm2 for the SARIFA-negative and -positive cases, respectively (p < 0.001). CONCLUSIONS: SARIFA is a promising prognostic biomarker for PDAC. Its assessment is characterised by simplicity and low effort. The mechanisms behind SARIFA suggest a tumour-promoting increased lipid metabolism and altered immune background, both showing new therapeutic avenues.

SARIFA as a new histopathological biomarker is associated with adverse clinicopathological characteristics, tumor-promoting fatty-acid metabolism, and might predict a metastatic pattern in pT3a prostate cancer.

BACKGROUND: Recently, we introduced Stroma-AReactive-Invasion-Front-Areas (SARIFA) as a novel hematoxylin-eosin (H&E)-based histopathologic prognostic biomarker for various gastrointestinal cancers, closely related to lipid metabolism. To date, no studies on SARIFA, which is defined as direct tumor-adipocyte-interaction, beyond the alimentary tract exist. Hence, the objective of our current investigation was to study the significance of SARIFA in pT3a prostate cancer (PCa) and explore its association with lipid metabolism in PCa as lipid metabolism plays a key role in PCa development and progression. METHODS: To this end, we evaluated SARIFA-status in 301 radical prostatectomy specimens and examined the relationship between SARIFA-status, clinicopathological characteristics, overall survival, and immunohistochemical expression of FABP4 and CD36 (proteins closely involved in fatty-acid metabolism). Additionally, we investigated the correlation between SARIFA and biochemical recurrence-free survival (BRFS) and PSMA-positive recurrences in PET/CT imaging in a patient subgroup. Moreover, a quantitative SARIFA cut-off was established to further understand the underlying tumor biology. RESULTS: SARIFA positivity occurred in 59.1% (n = 178) of pT3a PCas. Our analysis demonstrated that SARIFA positivity is strongly associated with established high-risk features, such as R1 status, extraprostatic extension, and higher initial PSA values. Additionally, we observed an upregulation of immunohistochemical CD36 expression specifically at SARIFAs (p = 0.00014). Kaplan-Meier analyses revealed a trend toward poorer outcomes, particularly in terms of BRFS (p = 0.1). More extensive tumor-adipocyte interaction, assessed as quantity-dependent SARIFA-status on H&E slides, is also significantly associated with high-risk features, such as lymph node metastasis, and seems to be associated with worse survival outcomes (p = 0.16). Moreover, SARIFA positivity appeared to be linked to more distant lymph node and bone metastasis, although statistical significance was slightly not achieved (both p > 0.05). CONCLUSIONS: This is the first study to introduce SARIFA as easy-and-fast-to-assess H&E-based biomarker in locally advanced PCa. SARIFA as the histopathologic correlate of a distinct tumor biology, closely related to lipid metabolism, could pave the way to a more detailed patient stratification and to the development of novel drugs targeting lipid metabolism in pT3a PCa. On the basis of this biomarker discovery study, further research efforts on the prognostic and predictive role of SARIFA in PCa can be designed.

The SARIFA biomarker in the context of basic research of lipid-driven cancers.

SARIFA was very recently introduced as a histomorphological biomarker with strong prognostic power for colorectal, gastric, prostate, and pancreatic cancer. It is characterized by the direct contact between tumor cells and adipocytes due to a lack of stromal reaction. This can be easily evaluated on routinely available H&E-slides with high interobserver agreement. SARIFA also reflects a specific tumor biology driven by metabolic reprogramming. Tumor cells in SARIFA-positive tumors benefit from direct interaction with adipocytes as an external source of lipids. Numerous studies have shown that lipid metabolism is crucial in carcinogenesis and cancer progression. We found that the interaction between tumor cells and adipocytes was not triggered by obesity, as previously assumed. Instead, we believe that this is due to an immunological mechanism. Knowledge about lipid metabolism in cancer from basic experiments can be transferred to develop strategies targeting this reprogramed metabolism.

The relationship between Stroma AReactive Invasion Front Areas (SARIFA), Warburg-subtype and survival: results from a large prospective series of colorectal cancer patients.

BACKGROUND: Stroma AReactive Invasion Front Areas (SARIFA) is a recently identified haematoxylin & eosin (H&E)based histopathologic biomarker in gastrointestinal cancers, including colorectal cancer (CRC), defined as direct contact between tumour cells and adipocytes at the tumour invasion front. The current study aimed at validating the prognostic relevance of SARIFA in a large population-based CRC series as well as at investigating the relationship between SARIFA-status and previously established Warburg-subtypes, both surrogates of the metabolic state of the tumour cells. METHODS: SARIFA-status (positive versus negative) was determined on H&E slides of 1,727 CRC specimens. Warburg-subtype (high versus moderate versus low) data was available from our previous study. The associations between SARIFA-status, Warburg-subtype, clinicopathological characteristics and CRC-specific as well as overall survival were investigated. RESULTS: 28.7% (n=496) CRC were SARIFA-positive. SARIFA-positivity was associated with more advanced disease stage, higher pT category, and more frequent lymph node involvement (all p<0.001). SARIFA-positivity was more common in Warburg-high CRC. 44.2% (n=219) of SARIFA-positive CRCs were Warburg-high compared to 22.8% (n=113) being Warburg-low and 33.1% (n=164) being Warburg-moderate (p<0.001). In multivariable-adjusted analysis, patients with SARIFA-positive CRCs had significantly poorer CRC-specific (HRCRC-specific 1.65; 95% CI 1.41-1.93) and overall survival (HRoverall survival 1.46; 95% CI 1.28-1.67) independent of clinically known risk factors and independent of Warburg-subtype. Combining the SARIFA-status and the Warburg-subtype to a combination score (SARIFA-negative/Warburg-high versus SARIFA-positive/Warburg-low versus SARIFA-positive/Warburg-high, and so on) did not improve the survival prediction compared to the use of SARIFA-status alone (SARIFA-negative + Warburg-high: HRCRC-specific 1.08; 95% CI 0.84-1.38; SARIFA-positive + Warburg-low: HRCRC-specific 1.79; 95% CI 1.32-2.41; SARIFA-positive + Warburg-high: HRCRC-specific 1.58; 95% CI 1.23-2.04). CONCLUSIONS: Our current study is the by far largest external validation of SARIFA-positivity as a novel independent negative prognostic H&E-based biomarker in CRC. In addition, our study shows that SARIFA-positivity is associated with the Warburg-high subtype. Further research is warranted to provide a more mechanistic understanding of the underlying tumour biology. Based on our data, we conclude SARIFA-status should be implemented in pathologic routine practice to stratify CRC patients.

Converging deep learning and human-observed tumor-adipocyte interaction as a biomarker in colorectal cancer.

BACKGROUND: Tumor-Adipose-Feature (TAF) as well as SARIFA (Stroma AReactive Invasion Front Areas) are two histologic features/biomarkers linking tumor-associated adipocytes to poor outcomes in colorectal cancer (CRC) patients. Whereas TAF was identified by deep learning (DL) algorithms, SARIFA was established as a human-observed histopathologic biomarker. METHODS: To study the overlap between TAF and SARIFA, we performed a systematic pathological review of TAF based on all published image tiles. Additionally, we analyzed the presence/absence of TAF in SARIFA-negative CRC cases to elucidate the biologic and prognostic role of a direct tumor-adipocyte contact. TCGA-CRC gene expression data is investigated to assess the association of FABP4 (fatty-acid binding protein 4) and CD36 (fatty-acid translocase) with both TAF and CRC prognosis. RESULTS: By investigating the TAF/SARIFA overlap, we show that many TAF patches correspond to the recently described SARIFA-phenomenon. Even though there is a pronounced morphological and biological overlap, there are differences in the concepts. The presence of TAF in SARIFA-negative CRCs is not associated with poor outcomes in this cohort, potentially highlighting the importance of a direct tumor-adipocyte interaction. Upregulation of FABP4 and CD36 gene expression seem both linked to a poor prognosis in CRC. CONCLUSIONS: By proving the substantial overlap between human-observed SARIFA and DL-based TAF as morphologic biomarkers, we demonstrate that linking DL-based image features to independently developed histopathologic biomarkers is a promising tool in the identification of clinically and biologically meaningful biomarkers. Adipocyte-tumor-cell interactions seem to be crucial in CRC, which should be considered as biomarkers for further investigations.

Different methods exist in assessing samples removed from cancer patients during surgery. We linked two independently established tissue-based methods for determining the outcome of colorectal cancer patients together: tumor adipose feature (TAF) and Stroma AReactive Invasion Front Areas (SARIFA). SARIFA as biological feature was observed solely by humans and TAF was identified by the help of a computer algorithm. We examined TAF in many cancer slides and looked at whether they showed similarities to SARIFA. TAF often matched SARIFA, but not always. Interestingly, these methods could be used to predict outcomes for patients and are associated with specific gene expression involved in tumor and fat cell interaction. Our study shows that combining computer algorithms with human expertize in evaluating tissue samples can identify meaningful features in patient samples, which may help to predict the best treatment options.

A20 haploinsufficiency disturbs immune homeostasis and drives the transformation of lymphocytes with permissive antigen receptors.

Genetic TNFAIP3 (A20) inactivation is a classical somatic lymphoma lesion and the genomic trait in haploinsufficiency of A20 (HA20). In a cohort of 34 patients with HA20, we show that heterozygous TNFAIP3 loss skews immune repertoires toward lymphocytes with classical self-reactive antigen receptors typically found in B and T cell lymphomas. This skewing was mediated by a feed-forward tumor necrosis factor (TNF)/A20/nuclear factor κB (NF-κB) loop that shaped pre-lymphoma transcriptome signatures in clonally expanded B (CD81, BACH2, and NEAT1) or T (GATA3, TOX, and PDCD1) cells. The skewing was reversed by anti-TNF treatment but could also progress to overt lymphoma. Analysis of conditional TNFAIP3 knock-out mice reproduced the wiring of the TNF/A20/NF-κB signaling axis with permissive antigen receptors and suggested a distinct regulation in B and T cells. Together, patients with the genetic disorder HA20 provide an exceptional window into A20/TNF/NF-κB-mediated control of immune homeostasis and early steps of lymphomagenesis that remain clinically unrecognized.