Formalin-fixed Paraffin-embedded Surgical Specimens Research Articles

Abstract 5659: Origin of synchronous or metachronous multiple pancreatic cancers

Abstract [Introduction] The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Additionally, synchronous multiple pancreatic cancers have been frequently documented. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. This study is designed to elucidate the origin of multiple pancreatic cancers through an unbiased detection of somatic mutations in primary and metachronous cancers, along with adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 20 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. The investigation into the shared and private mutations across different samples aimed to unveil the clonal evolution history of these lesions. [Results] We analyzed 20 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 16 metachronous, and 2 synchronous tumors. In metachronous cases, all patients exhibited margin-negative pathology for the primary cancer, with a median interval of 38.9 months (ranging from 7 to 85 months) between the initial and second surgery. In addition, a total of 20 pancreatic intraepithelial neoplasia (PanIN) from 5 of these cases were analyzed. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=19), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. [Conclusions] In conclusion, our study suggests that even in early pancreatic cancer, dissemination within the pancreas may occur, contributing to the development of synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Takayuki Anazawa, Kazuyuki Nagai, Toshihiko Masui, Sachiko Minamiguchi, Hironori Haga, Takeshi Tanaka, Atsuhiro Masuda, Yuzo Kodama, Norimitsu Uza, Hiroshi Seno, Satoru Miyano, Hiroko Tanaka, Seishi Ogawa. Origin of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5659.

Fine-needle aspiration biopsy possibilities in studying the molecular genetic landscape of breast tissue

Background. Core biopsy of the breast is currently considered to be the standard method of obtaining material for morphological and molecular genetic methods. Unfortunately, this method is associated with a number of problems, primarily the risk of complications (bleeding, pneumothorax) and discomfort during manipulation.Aim. To analyze transcriptional signatures of breast tissue samples obtained by fine-needle aspiration biopsy. Materials and methods. Using reverse transcriptase polymerase chain reaction, we studied the mRNA expression level of 60 target genes in 60 samples obtained by fine-needle aspiration biopsy and in 60 corresponding formalin-fixed paraffin-embedded (FFPE) surgical specimens of breast. Samples were obtained from the tumor, adjacent tissue, the so-called tumor bed and formally normal tissue at a distance from the primary lesion.Results. A comparative analysis of transcriptional signatures in samples obtained by fine-needle aspiration biopsy and FFPE specimens (120 samples in total) reveled the strongest correlations between transcriptional signatures in biopsy samples and FFPE specimens of tumors. Very strong correlation in tumor samples was established for one gene (CTSL2); strong for 18 genes (MKI67, MYBL2, NAT1, PTEN, TPX2, PTTG1, UBE2T, CCNB1, ESR1, CCND1, MYC, SCGB2A2, MIA, TRAC, FGFR4, ANLN, GSTM1, PRLR); averages for 28 genes (PGR, AURCA, KRT5, FOXA1, SFRP1, EMSY, EXO1, PAK1, KIF14, ERBB2, MMP11, BCL2, BAG1, TMEM45B, BIRC5, CD274/PDL1, ZNF703, TYMS, CCNE1, TPT1, TMEM45A, BRCA1, BRCA2, ESR2, STS, TNFSF11/RANKL, TNFRSF11B/OPG, TNF); weak for 4 genes (GRB7, EGFR, PGRMC1, CYP19A). The presence of correlations between transcriptional signatures in biopsy samples and FFPE specimens can be established in case of sufficient material corresponding to sample intake control (SIC) ≥5 lg for B2M gene.Conclusion. The ability to conduct molecular genetic research on small samples of breast tissue makes it possible to obtain the material using the most minimally invasive method. And this, in turn, expands the possibilities of “genetic monitoring” of cancer, as well as the possibility of more accurate assessment the risks of malignant tumor development in the settings of benign conditions in women with fibrocystic disease and increased mammographic density.

Prediction of Adjuvant Gemcitabine Sensitivity in Resectable Pancreatic Adenocarcinoma Using the GemPred RNA Signature: An Ancillary Study of the PRODIGE-24/CCTG PA6 Clinical Trial.

GemPred, a transcriptomic signature predictive of the efficacy of adjuvant gemcitabine (GEM), was developed from cell lines and organoids and validated retrospectively. The phase III PRODIGE-24/CCTG PA6 trial has demonstrated the superiority of modified folinic acid, fluorouracil, irinotecan, and oxaliplatin (mFOLFIRINOX) over GEM as adjuvant therapy in patients with resected pancreatic ductal adenocarcinoma at the expense of higher toxicity. We evaluated the potential predictive value of GemPred in this population. Routine formalin-fixed paraffin-embedded surgical specimens of 350 patients were retrieved for RNA sequencing and GemPred prediction (167 in the GEM arm and 183 in the mFOLFIRINOX [mFFX] arm). Survival analyses were stratified by resection margins, lymph node status, and cancer antigen 19-9 level. Eighty-nine patients' tumors (25.5%) were GemPred+ and were thus predicted to be gemcitabine-sensitive. In the GEM arm, GemPred+ patients (n = 50, 30%) had a significantly longer disease-free survival (DFS) than GemPred- patients (n = 117, 70%; median 27.3 v 10.2 months, hazard ratio [HR], 0.43 [95% CI, 0.29 to 0.65]; P < .001) and cancer-specific survival (CSS; median 68.4 v 28.6 months, HR, 0.42 [95% CI, 0.27 to 0.66]; P < .001). GemPred had no prognostic value in the mFFX arm. DFS and CSS were similar in GemPred+ patients who received adjuvant GEM and mFFX (median 27.3 v 24.0 months, and 68.4 v 51.4 months, respectively). The statistical interaction between GEM and GemPred+ status was significant for DFS (P = .008) and CSS (P = .004). GemPred+ patients had significantly more adverse events of grade ≥3 in the mFFX arm (76%) compared with those in the GEM arm (40%; P = .001). This ancillary study of a phase III randomized trial demonstrates that among the quarter of patients with a GemPred-positive transcriptomic signature, survival was comparable with that of mFOLFIRINOX, whereas those receiving adjuvant gemcitabine had fewer adverse events.

The changes of HER3 expression in head and neck cancer patients treated with induction chemotherapy.

e18004 Background: Erb-b2 receptor tyrosine kinase 3 (HER3) is broadly expressed in various types of cancer and is associated with poor prognosis. Recent evidence supports HER3 plays a role in acquired resistance to chemotherapy. The change of HER3 expression by chemotherapy in head and neck squamous cell carcinoma (HNSCC) remains unclear. Methods: We evaluated 95 patients with locally advanced HNSCC who had received induction chemotherapy (ICT) between 2009 and 2019. Immunohistochemical staining for HER3 was performed using formalin-fixed paraffin-embedded surgical specimens and biopsy samples. H-score of membrane staining was evaluated. Results: Median age of patients was 64 years, and 85% were male. Primary locations included oropharynx (54.8%), hypopharynx (21.5%), oral cavity (17.2%), and salivary gland (6.5%). Mean H-score of HER3 in tumor tissues was 77.6 ± 7.9. H-score of HER3 in HPV-positive tumors was higher than HPV-negative tumors (p = 0.019), which was more pronounced in oropharynx and hypopharynx tumors. While, H-score of HER3 in adjacent normal tissues of these tumors did not significantly differ. Compared with the non-smokers, tumor tissues of current or ex-smokers demonstrated higher H-score of HER3 (p = 0.033), independent of tumor location. In contrast, in adjacent normal tissues, no significant differences of H-score of HER3 were observed depending on the smoking status. The H-score higher than 100 was defined as HER3-high group. Among oropharynx and hypopharynx tumors with positive correlation between H-score of HER3 and HPV status (n = 56), HER3-high group was significantly correlated with HPV positivity (p = 0.009) and good response to ICT (p = 0.035). Good clinical response was also strongly related to HPV-positivity (p = 0.001). In addition, there was a tendency for longer overall survival in HER3-high group than low HER3 expression group (93 vs. 70 months, p = 0.053). The paired tumor samples collected at pre- and post-ICT (n = 21) were analyzed. HER3 expressions in adjacent normal tissue were higher at post-ICT compared to pre-ICT (p = 0.003), and these results were more pronounced in the non-smoking group, but no differences were found according to HPV-status. Conclusions: Taken together, our results showed that H-score of HER3 was significantly higher in both HPV-positive and smoking group. Interestingly, high HER3 expression in oropharynx and hypopharynx tumors was associated with HPV positivity and good clinical response to ICT. Additional analysis of tumor immune microenvironment in tumor tissues are underway.

Abstract 1511: Origin of synchronous or metachronous multiple pancreatic cancers

Abstract Introduction: The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Moreover, synchronous multiple pancreatic cancers were often reported. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. In this study, we aimed to reveal the origin of multiple pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. Methods: Serially obtained formalin-fixed paraffin-embedded surgical specimens from 18 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. Results: We analyzed 18 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 14 metachronous, and 2 synchronous tumors. In metachronous cases, pathology for primary cancer were margin-negative in all patients, and the median interval between the initial and second surgery was 38.9 months (7 - 85 months). In addition, a total of 20 pancreatic intraepithelial neoplasia (PanIN) from 5 of these cases were analyzed. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=17), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. Conclusions: In conclusion, our study suggests that even early pancreatic cancer might be disseminated within the pancreas and contribute to synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Tomomi Nishimura, Toshihiko Masui, Kazuyuki Nagai, Takayuki Anazawa, Sachiko Minamigushi, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Atsuhiro Masuda, Takeshi Tanaka, Seishi Ogawa, Hiroko Tanaka, Satoru Miyano. Origin of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1511.

Abstract P5-13-04: Clonal evolution of mammary epithelial cells into breast cancers

Abstract [Introduction] Proliferative lesions in the breast have been implicated in the development of breast cancer. Previous studies showed that some proliferative lesions and adjacent breast cancers shared common genetic alterations, suggesting that these originated from the same ancestral cell. However, the clonal structure of normal epithelia and their clonal history during evolution to cancer are poorly understood. In this study, we analyzed genetic profiles of normal epithelia and proliferative lesions in the cancer-borne breast to illustrate the clonal evolution of cancer from a normal epithelial cell. [Methods] Single cell-derived organoids (n=47) were established from breast milk of 4 healthy women aged 22–36 and normal breast tissue of 15 breast cancer patients aged 29–83 to evaluate somatic mutation rate in normal epithelial cells. Multiple normal lobules and proliferative lesions together with cancer lesions were collected using laser-capture micro-dissection (LCM) from fresh frozen (n=3) or formalin-fixed paraffin-embedded (n=5) surgical specimens in 9 premenopausal breast cancer patients. Somatic mutations and copy number alterations were evaluated using whole-genome sequencing. [Results] The mutation profile of single cell-derived organoids suggests that somatic mutations accumulate in normal mammary epithelial cells at a constant rate of 19.4/genome/year before menopause, and the mutation rate decreases to 6.9/genome/year after menopause. Parity was negatively associated with mutation number (-49.3 per life birth). In total, we analyzed 143 LCM samples, including those from 72 normal lobules, 43 proliferative lesions, and 19 non-invasive and 9 invasive cancer samples. Five cases showed a large expansion of proliferative lesions sharing a substantial number of somatic mutations with cancer. These lesions expanded over a distance of 35-90 mm, sharing tens to hundreds of mutations including those in breast cancer-related driver genes, such as PIK3CA, AKT1, GATA3, CBFB and PTEN, while harboring private mutations or copy number alterations of their own. Of interest, the cancers in 4 out of these 5 cases was luminal-A type invasive ducal carcinoma or ER-positive HER2-negative ductal carcinoma in situ, and characterized in common by the presence of der(1;16), concurrent whole-arm 1q gain and 16q loss, in both cancer and proliferative lesions. Phylogenetic analysis adapted with the mutation rate in normal cells predicted that der(1;16) had been acquired between puberty and early 20’s, and the common ancestors of non-cancerous and cancerous lesions emerged by early 30’s, &amp;gt;10 years earlier than at the time of cancer diagnosis. By contrast, analysis of non-cancerous lobules unrelated to cancer showed that der(1;16)-negative non-cancer clones that had emerged after puberty stayed within a single lobule or spatially confined to adjacent lobules and rarely expanded to a large area as observed for those carrying der(1;16), even if the clones had acquired mutations in driver genes such as PIK3CA and PIK3R1, which highlighted the role of der(1;16) in wide clonal expansion. [Conclusions] Our results suggest that in some breast cancer cases, particularly in those with der(1;16), a highly recurrent translocation accounting for the major subset of Luminal A breast cancer, the clones with the funder driver alterations expanded macroscopically long before the onset of cancer, in which further clonal evolutions recursively occur multi-focally, giving rise to multiple proliferative lesions and ultimately, invasive cancers. Our findings provide new insight into the early development of breast cancer. Citation Format: Tomomi Nishimura, Nobuyuki Kakiuchi, Kenichi Yoshida, Takaki Sakurai, Tatsuki R. Kataoka, Eiji Kondoh, Yoshitsugu Chigusa, Masahiko Kawai, Morio Sawada, Takuya Inoue, Yasuhide Takeuchi, Hirona Maeda, Satoko Baba, Yusuke Shiozawa, Ryunosuke Saiki, Masahiro M. Nakagawa, Yasuhito Nannya, Yotaro Ochi, Tomonori Hirano, Yukiko Inagaki-Kawata, Kosuke Aoki, Masahiro Hirata, Eiji Suzuki, Masahiro Takada, Masahiro Kawashima, Kosuke Kawaguchi, Kenichi Chiba, Yuichi Shiraishi, Junko Takita, Satoru Miyano, Masaki Mandai, Kengo Takeuchi, Hironori Haga, Masakazu Toi, Seishi Ogawa. Clonal evolution of mammary epithelial cells into breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-04.

Abstract 6085: Clonal evolution of mammary epithelial cells into breast cancers

Abstract [Introduction] Previous studies suggested that breast cancers (BCs) and some adjacent atypical proliferative lesions evolved from the same ancestral cell. However, the clonal structure of normal epithelial cells and their clonal history during evolution to cancer are poorly understood. In this study, we analyzed genetic profiles of normal epithelia and proliferative lesions in the cancer-borne breast to illustrate the clonal evolution of cancer from a normal epithelial cell. [Methods] Single cell-derived normal epithelial organoids (n=47) were established from breast milk of 4 healthy women aged 22-36 and surgical specimens of 15 BC patients aged 29-83 to evaluate somatic mutation rate. Multiple normal lobules or proliferative lesions together with cancer cells were collected using laser-capture micro-dissection (LCM) from fresh frozen (n=3) or formalin-fixed paraffin-embedded (n=6) surgical specimens in 9 premenopausal BC patients. Somatic mutations and copy number alterations were evaluated using whole-genome sequencing. [Results] On the basis of single cell-derived mammary gland-derived organoids, somatic mutations increased in mammary gland cells at a constant rate of 19.4/genome/year before menopause, which then decreased to 6.9/genome/year after menopause. Parity was negatively associated with mutation number (-49.3 per life birth). In total, we analyzed 113 LCM samples, including those from 66 normal lobules, 28 proliferative lesions, and 14 non-invasive and 5 invasive cancer samples. Phylogenetic analysis showed that most of the neighboring normal lobules (53/66) from 3 BC patients shared no somatic mutations with each other and therefore were clonally independent. In other 13 normal lobules, shared mutations including a PIK3CA mutation were observed across multiple adjacent samples, although they were still confined to &amp;lt;10 mm regions. By contrast, we found a large expansion of proliferative lesions sharing a substantial number of somatic mutations with cancer in 5 out of the 6 remaining BC cases. They were expanded over a distance of 25-85 mm, sharing tens to hundreds of mutations including those in BC-related driver genes, such as PIK3CA, AKT1, GATA3, CBFB and PTEN. Of interest, Tumors in 4 out of these 5 cases was Luminal-A type and characterized in common by the presence of 1q gain and 16q loss (1q+/16q-) in both cancer and proliferative lesions. Phylogenetic analysis adapted with the mutation rate in normal cells predicted that 1q+/16q- had been acquired during adolescence to mid-20s, where the clonal expansion had occurred years to decades before the onset of cancer. [Conclusions] Our results suggest that in some BC cases, particularly in those with 1q+/16q-, the clones with the founder driver mutations expanded macroscopically long before the onset of cancer, in which further clonal evolutions recursively occur multi-focally, giving rise to multiple proliferative lesions and ultimately, invasive cancers. Citation Format: Tomomi Nishimura, Nobuyuki Kakiuchi, Kenichi Yoshida, Yasuhide Takeuchi, Hirona Maeda, Yusuke Shiozawa, Masahiro M. Nakagawa, Ryunosuke Saiki, Yotaro Ochi, Tomonori Hirano, Yukiko Kawata, Kosuke Aoki, Masahiro Hirata, Tatsuki R. Kataoka, Takaki Sakurai, Satoko Baba, Yuichi Shiraishi, Kenichi Chiba, Kengo Takeuchi, Hironori Haga, Satoru Miyano, Masakazu Toi, Seishi Ogawa. Clonal evolution of mammary epithelial cells into breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6085.

Abstract 6198: Genetic analysis of synchronous or metachronous multiple pancreatic cancers

Abstract [Introduction] The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Moreover, synchronous multiple pancreatic cancers were often reported. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. In this study, we aimed to reveal the origin of multiple pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 17 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] We analyzed 17 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 10 metachronous, and 2 synchronous tumors. In metachronous cases, pathology for primary cancer were margin-negative in all patients, and the median interval between the initial and second surgery was 37.7 months (12 - 85 months). In addition, we analyzed 5 adjacent pancreatic intraepithelial neoplasia (PanIN) in one of these cases. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=16), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. [Conclusions] In conclusion, our study suggests that even early pancreatic cancer might be disseminated within the pancreas and contribute to synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Takeuchi Yasuhide, Tomomi Nishimura, Toshihiko Masui, Kazuyuki Nagai, Takayuki Anazawa, Sachiko Minamiguchi, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Atsuhiro Masuda, Takeshi Tanaka, Yuichi Shiraishi, Satoru Miyano, Seishi Ogawa. Genetic analysis of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6198.

Primary endocrine resistance of ER+ breast cancer with ESR1 mutations interrogated by droplet digital PCR

We investigated the patterns of recurrence and primary endocrine resistance according to estrogen receptor (ER) alpha gene (ESR1) mutations, as assessed by digital droplet (dd) PCR, in patients with non-metastatic ER+ breast cancer. We collected 121 formalin-fixed paraffin-embedded (FFPE) surgical specimens from ER+ breast cancer patients who had relapsed after surgery. Genomic DNA was extracted from the FFPE samples and ESR1 mutations were evaluated using ddPCR. ESR1 mutations were detected in 9 (7.4%) of 121 primary breast cancer specimens. The median recurrence-free interval and overall survival were significantly lower in patients with ESR1 mutations than in those without. Of the patients treated with ET (N = 98), eight had ESR1 mutations. Of these, six (75.0%) had primary endocrine resistance and two (25.0%) had secondary endocrine resistance. By contrast, only 22 of 90 (24.4%) patients without ESR1 mutations had primary endocrine resistance. A multivariable model showed that an ESR1 mutation is a significant risk factor for primary endocrine resistance. Our findings provide clinical evidence that the presence of rare ESR1 mutant clones identified by ddPCR in primary tumors is associated with primary endocrine resistance in an adjuvant setting.

PD-L1 quantification across tumor types using the reverse phase protein microarray: implications for precision medicine

BackgroundAnti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely...

Genetic Analysis of Archived Tumor Specimens for Hereditary Colorectal Cancer Syndromes in the Cajuns of Louisiana, a US Founder Population.

INTRODUCTION:The Louisiana Acadian region (population 1.2 million), home of the Cajuns, has among the highest US colorectal cancer (CRC) rates. Although Cajuns are a known genetic founder population, studies assessing for hereditary CRC have not been performed.METHODS:A retrospective review of 2 hospital cancer registries was performed to identify young (<55) Cajun CRC patients in Lafayette, Louisiana (the Acadian region population center), diagnosed from 2003 to 2016. Men were studied because of the higher likelihoods of retaining Cajun surnames for ancestry identification compared with women. Immunohistochemistry for mismatch repair proteins associated with the Lynch syndrome (LS) was performed on tumors. Germline sequencing was performed on adjacent normal tissue of these archived formalin-fixed paraffin-embedded surgical resection specimens for pathogenic variants underlying CRC-associated syndromes, including LS, familial adenomatous polyposis, and others.RESULTS:Of 9 young Cajuns, a germline analysis revealed LS in 2 (MLH1 frameshift, MLH1 missense pathogenic variants). Both had immunohistochemistry-deficient MLH1. Two others had the same adenomatous polyposis coli variant of unknown significance (2 algorithms predicting deleterious and probably damaging change), making this a potential familial adenomatous polyposis founder effect candidate.DISCUSSION:This is the first study assessing for hereditary CRC in a large US regional founder population. This small study did not identify clear Cajun founder pathogenic variants. However, larger studies are warranted, which could also help clarify the clinical significance of the adenomatous polyposis coli variant of unknown significance. This study is important because it demonstrates that a retrospective tumor analysis can be used to ascertain the prevalence of genetic susceptibility in specific populations.

Abstract 2185: Genetic analysis of metachronous pancreatic cancers

Abstract [Introduction] Early detection of pancreatic cancer is a key to curable surgery. However, many patients are diagnosed with an advanced disease and even in those who have an early stage tumor successfully resected, the development of metachronous cancer in the residual pancreas prevents a long-term survival. Thus, it is important to control metachronous tumors to improve clinical outcomes, whose pathogenesis, however, is poorly understood. In this study, we aimed to reveal the origin of metachronous tumors using an unbiased detection of somatic mutations in primary and metachronous tumors as well as adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 12 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for enrichment of tumor and precancerous components, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] Pathology for resected primary cancer was margin-negative in all patients. The median interval between the initial and second surgery was 34.6 months (12 - 65.1 months). Paired primary and metachronous cancers were analyzed in all 12 patients. An additional 5 pancreatic intraepithelial neoplasia (PanIN) samples were also analyzed in one case. We identified a median of 86 (range: 40-145) and 20 (14-42) somatic mutations using WES in cancers and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In each patient, all driver mutations and many passenger mutations were shared between primary and metachronous cancer samples, suggesting that those metachronous tumors are evolutionally closely related to the primary cancer, despite long years before recurrence. Negative pathology of the margins at the initial surgery suggested that those metachronous lesions originated from distant dissemination or metastasis, rather than contiguous, intraductal invasion. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor lesions and cancer samples. Despite multiple independent clones in the precancerous lesion, all metachronous tumors originated from the primary lesions in this study. [Conclusions] Our study suggests that even early pancreatic cancer might be disseminated within the pancreas and give rise to metachronous cancers, suggesting the importance of close monitoring of recurrence in the residual pancreas. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Tomomi Nishimura, Toshihiko Masui, Sachiko Minamiguhi, Hironori Haga, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Uza Norimitsu, Yuzo Kodama, Hiroshi Seno, Seishi Ogawa. Genetic analysis of metachronous pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2185.

A Distinct Innate Immune Signature of Early Onset Colorectal Cancer.

Despite a decrease in the prevalence of colorectal cancer (CRC) over the last 40 y, the prevalence of CRC in people under 50 y old is increasing around the globe. Early onset (≤50 y old) and late onset (≥65 y old) CRC appear to have differences in their clinicopathological and genetic features, but it is unclear if there are differences in the tumor microenvironment. We hypothesized that the immune microenvironment of early onset CRC is distinct from late onset CRC and promotes tumor progression. We used NanoString immune profiling to analyze mRNA expression of immune genes in formalin-fixed paraffin-embedded surgical specimens from patients with early (n = 40) and late onset (n = 39) CRC. We found three genes, SAA1, C7, and CFD, have increased expression in early onset CRC and distinct immune signatures based on the tumor location. After adjusting for clinicopathological features, increased expression of CFD and SAA1 were associated with worse progression-free survival, and increased expression of C7 was associated with worse overall survival. We also performed gain-of-function experiments with CFD and SAA1 in s.c. tumor models and found that CFD is associated with higher tumor volumes, impacted several immune genes, and impacted three genes in mice that were also found to be differentially expressed in early onset CRC (EGR1, PSMB9, and CXCL9). Our data demonstrate that the immune microenvironment, characterized by a distinct innate immune response signature in early onset CRC, is unique, location dependent, and might contribute to worse outcomes.

Comparison of breast cancer surrogate subtyping using a closed-system RT-qPCR breast cancer assay and immunohistochemistry on 100 core needle biopsies with matching surgical specimens

BackgroundRoutine clinical management of breast cancer (BC) currently depends on surrogate subtypes according to estrogen- (ER) and progesterone (PR) receptor, Ki-67, and HER2-status. However, there has been growing demand for reduced immunohistochemistry (IHC) turnaround times. The Xpert® Breast Cancer STRAT4* Assay (STRAT4)*, a standardized test for ESR1/PGR/MKi67/ERBB2 mRNA biomarker assessment, takes less than 2 hours. Here, we compared the concordance between the STRAT4 and IHC/SISH, thereby evaluating the effect of method choice on surrogate subtype assessment and adjuvant treatment decisions.MethodsIn total, 100 formalin-fixed paraffin-embedded core needle biopsy (CNB) samples and matching surgical specimens for 98 patients with primary invasive BC were evaluated using the STRAT4 assay. The concordance between STRAT4 and IHC was calculated for individual markers for the CNB and surgical specimens. In addition, we investigated whether changes in surrogate BC subtyping based on the STRAT4 results would change adjuvant treatment recommendations.ResultsThe overall percent agreement (OPA) between STRAT4 and IHC/SISH ranged between 76 and 99% for the different biomarkers. Concordance for all four biomarkers in the surgical specimens and CNBs was only 66 and 57%, respectively. In total, 74% of surgical specimens were concordant for subtype, regardless of the method used. IHC- and STRAT4-based subtyping for the surgical specimen were shown to be discordant for 25/98 patients and 18/25 patients would theoretically have been recommended a different adjuvant treatment, primarily receiving more chemotherapy and trastuzumab.ConclusionsA comparison of data from IHC/in situ hybridization and STRAT4 demonstrated that subsequent changes in surrogate subtyping for the surgical specimen may theoretically result in more adjuvant treatment given, primarily with chemotherapy and trastuzumab.

Association of Mutation Profiles with Postoperative Survival in Patients with Non–Small Cell Lung Cancer

Simple SummaryIn this study, we comprehensively and synthetically analyzed mutations in lung cancer based on the next generation sequencing data of lung tumors surgically removed from the patients, and identified the mutation-related factors that can affect clinical outcomes. Detailed understanding of the genomic landscape of lung cancers will establish the ideal model for best surgical outcomes in the era of “precision medicine”.Findings on mutations, associated with lung cancer, have led to advancements in mutation-based precision medicine. This study aimed to comprehensively and synthetically analyze mutations in lung cancer, based on the next generation sequencing data of surgically removed lung tumors, and identify the mutation-related factors that can affect clinical outcomes. Targeted sequencing was performed on formalin-fixed paraffin-embedded surgical specimens obtained from 172 patients with lung cancer who underwent surgery in our hospital. The clinical and genomic databases of the hospital were combined to determine correlations between clinical factors and mutation profiles in lung cancer. Multivariate analyses of mutation-related factors that may affect the prognosis were also performed. Based on histology, TP53 was the driver gene in 70.0% of the cases of squamous cell carcinoma. In adenocarcinoma cases, driver mutations were detected in TP53 (26.0%), KRAS (25.0%), and epidermal growth factor receptor (EGFR) (23.1%). According to multivariate analysis, the number of pathogenic mutations (≥3), presence of a TP53 mutation, and TP53 allele fraction >60 were poor prognostic mutational factors. The TP53 allele fraction tended to be high in caudally and dorsally located tumors. Moreover, TP53-mutated lung cancers located in segments 9 and 10 were associated with significantly poorer prognosis than those located in segments 1–8. This study has identified mutation-related factors that affect the postoperative prognosis of lung cancer. To our knowledge, this is the first study to demonstrate that the TP53 mutation profile varies with the site of lung tumor, and that postoperative prognosis varies accordingly.

Neoadjuvant chemotherapy promotes the expression of HER3 in patients with ovarian cancer.

HER3 (erbB3) signaling serves an important role in the development and chemoresistance of ovarian cancer, and is activated by chemotherapy. To evaluate the influence of neoadjuvant chemotherapy and other clinical factors on the expression of HER3, as well as to examine its role as a prognostic marker, the present study evaluated archived tissues from patients who underwent surgery for ovarian cancer between 2011 and 2018 at our hospital. Immunohistochemical staining for HER3 was performed using formalin-fixed paraffin-embedded surgical specimens and biopsy samples. In total, data from 111 patients with sufficient surgically resected tumor samples were extracted. A total of 28 patients with histology type high-grade serous carcinoma (HGSC) had specimens available from both pre-chemotherapy biopsies and post-chemotherapy surgery. High HER3 expression (HER3-high) was observed in 64 patients (58%), whereas low HER3 expression (HER3-low) was observed in 47 patients (42%). Multivariate logistic regression analysis identified neoadjuvant chemotherapy [odds ratio (OR), 7.49; 95% confidence interval (CI), 2.48–22.64; P<0.001) and non-HGSC histology (OR, 5.42; 95% CI, 1.99–14.78; P<0.001) as significant predictive factors for HER3-high. In pre-chemotherapy biopsy specimens, 15 patients were HER3-high and 13 were HER3-low. After chemotherapy, eight of 13 patients with HER3-low exhibited a change in status to HER3-high, with a trend toward poorer progression-free survival compared to that of patients whose status remained HER3-low. In conclusion, HER3 overexpression was revealed to be common among patients with ovarian cancer, especially in those with non-HGSC histology. In addition, HER3 expression may be promoted by chemotherapy. These findings suggested that patients with ovarian cancer are good candidates for emerging HER3-targeting therapies.

Abstract 5877: Genetic analysis of metachronous pancreatic cancers

Abstract [Introduction] Early detection of pancreatic cancer is a key to curable surgery. However, many patients are diagnosed in an advanced stage and even in those cases who had a surgical resection of early stage tumors, metachronous pancreatic cancer in residual pancreas prevents cure. Thus, it is important to control metachronous tumors to improve clinical outcomes, whose pathogenesis is poorly understood. In this study, we aimed to reveal the origin of metachronous pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 8 patient who had undergone curative surgery for an early stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and PanIN lesions, from which DNA was extracted and analyzed for somatic mutations of each lesion by whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] Pathology for primary cancer were margin-negative in all patients. The median interval between the initial and second surgery was 29.8 months (23 - 64.4 months). We analyzed a median of two cancer and one PanIN samples (0-5), from which we identified a median number of 45(range: 26-69) and 20 (14-42) somatic mutations using WES, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In each patient, all driver mutations and more than half of passenger mutations were shared between primary and metachronous cancer samples, suggesting that those metachronous tumors are evolutionally closely related to the primary cancer. Negative pathology of the margins at the initial surgery suggested that those metachronous lesions originated from distant dissemination or metastasis, rather than contiguous, intraductal invasion. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor lesions and cancer samples. Despite multiple independent clones in the precancerous lesion, all metachronous tumors originated from the primary lesions in this study. [Conclusions] Our study suggests that even early pancreatic cancer might be disseminated within the pancreas and contribute to metachronous cancers, suggesting the importance of close monitoring of the recurrence in the residual pancreas. Citation Format: Hirano Tomonori, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Yoshikage Inoue, Tomomi Nishimura, Yoichi Fujii, Akira Yokoyama, Hideki Makishima, Toshihiko Masui, Shinji Uemoto, Sachiko Minamiguchi, Hironori Haga, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Norimitsu Uza, Yuzo Kodama, Hiroshi Seno, Seishi Ogawa. Genetic analysis of metachronous pancreatic cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5877.

HER3 protein expression as a risk factor for post-operative recurrence in patients with early-stage adenocarcinoma and adenosquamous carcinoma of the cervix.

Patients with cervical adenocarcinoma (AC) and adenosquamous carcinoma (ASC) have a poorer prognosis than those with squamous cell carcinoma (SCC). Erb-b2 receptor tyrosine kinase 3 (HER3) is a member of the epidermal growth factor receptor family and its expression is associated with unfavorable prognosis in several cancer types, including SCC of the cervix. As there is limited information on the prognostic value of HER3 for AC and ASC of the cervix, the present study aimed to evaluate the expression of HER3 and its impact on post-operative recurrence in patients with AC and ASC of the cervix. This retrospective study included 39 patients with early-stage AC and ASC who underwent primary surgery between January 1997 and December 2017. Immunohistochemical staining for HER3 was performed on formalin-fixed paraffin-embedded surgical specimens. The possible influence of HER3 expression on disease-free survival (DFS) was studied by using multivariate Cox regression with adjustment for established risk factors of post-operative recurrence. High expression of HER3 (HER3-high) was detected in 85.1% of cases of AC (23/27) and in 58.3% of cases of ASC (7/12). The median follow-up duration was 63.1 months and Kaplan-Meier analysis indicated that the 5-year DFS rates of patients with AC and ASC of the cervix were 56.7% in patients with HER3-high and 77.8% in patients with HER3-low (log rank, P=0.20). On multivariate analysis, HER3-high [hazard ratio (HR)=6.32, 95% CI: 1.10–36.26, P=0.039), pelvic lymph node metastasis (HR=7.61, 95% CI: 2.07–28.00, P=0.002) and vascular invasion (HR=4.28, 95% CI: 1.12–16.31, P=0.033) were indicated to be independent predictors of DFS. To date, the present study is the most comprehensive analysis to evaluate the expression of HER3 in patients with early-stage AC and ASC of the cervix. The results suggested that HER3 overexpression may be an independent risk factor for post-operative recurrence. However, these results and the prognostic value of HER3 should be confirmed in a larger sample.

Abstract 5126: Genetic analysis of metachronous pancreatic cancers

Abstract [Introduction] Early detection of pancreatic cancer is a key to curable surgery, although many are diagnosed in advanced stage. However, even in those cases in which cancer was detected early enough for curative resection, metachronous pancreatic cancer may occur in residual pancreas, whose pathogenesis, including its relationship with primary cancer, has been poorly understood. In this study, we aimed to reveal the origin of metachronous pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. [Methods] Samples were obtained from longitudinal sampling from above lesions using laser microdissection from formalin-fixed paraffin-embedded surgical specimens. DNA was extracted and analyzed for somatic mutations of each lesion by whole-exome sequencing with matched normal DNA. On the basis of shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] Four patients were enrolled who underwent curative surgery for early pancreatic cancer and subsequently for metachronous tumors in the residual pancreas. Pathology from surgery for primary cancer were margin-negative in all patients. The median interval between the initial and second surgery was 29.8 months (22.8 - 38.3 months). The number of precursor lesions analyzed was from 0 to 5 per patient. The median number of somatic mutations per sample was 78 (range: 41-92) in cancers and 20 (14-42) in precursor lesions. None of the patients have known pathogenic germline mutations. With a median of 78 and 20 mutations per sample, all samples had one or more driver mutations. In each case, all driver mutations that were detected in cancers were shared between primary and metachronous cancer. It indicated that metachronous cancer branched off from primary cancers at later stage of carcinogenesis. And negative margin at initial surgery suggested that metachronous cancer was formed by dissemination or metastasizing rather than Intraductal progression. By contrast, none of the mutations other than a hotspot KRAS mutations were shared between precursor lesions and cancers, suggesting multiple independent clonal growth of precancerous cells in the cancer bearing pancreas. [Conclusions] Our study shows that metachronous pancreatic cancers are derived from the same origin of initial cancer. Therefore, even in the case of early pancreatic cancer careful checkup for recurrence in the residual pancreas is important. Citation Format: Hirano Tomonori, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Yoshikage Inoue, Tomomi Nishimura, Yoichi Fujii, Akira Yokoyama, Hideki Makishima, Toshihiko Masui, Shinji Uemoto, Sachiko Minamiguchi, Hironori Haga, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Norimitsu Uza, Yuzo Kodama, Hiroshi Seno, Tsutomu Chiba, Seishi Ogawa. Genetic analysis of metachronous pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5126.

Association of CDX2 expression and overall survival and clinicopathologic disease manifestations in patients with gastric cancer in Northwest of Iran

Introduction: In the northwest of Iran, gastric cancer is the first and third common form of malignancy in men and women, respectively. Homeobox protein CDX2, was proposed as a prognostic biomarker in gastric cancer. Objectives: This study aimed to assess the association of CDX2 expression overall survival as well as clinicopathologic variables in patients with gastric cancer. Materials and Methods: CDX2 expression was examined by the immunohistochemistry method in the formalin-fixed paraffin-embedded surgical specimens of gastric cancer. The association between CDX2 expression and overall survival, stage of the disease, metastasis status, pathology type of the tumor, and serum levels of tumor markers serum levels of tumor markers carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), cancer antigen 19-9 (CA 19-9) was then evaluated. Results: Ninety-two patients with primary gastric cancer were recruited. CDX2 expression was positive in 72 (78%) of the subjects. Overall survival was not significantly different between two groups of patients (CDX2 positive versus negative). However, the risk of death was 21% lower in CDX2 positive patients, after adjusting for age and gender (P=0.565). Disease stage and metastasis rate were significantly lower in CDX2 positive patients (P=0.027, and P=0.021, respectively). Additionally, 71% of the CDX2 positive subjects had intestinal pathology type of the disease (P=0.025). Accordingly, a significant lower level of CA19-9 was found in CDX2 positive patients as well (P=0.002). Conclusion: There was not any significant association between CDX2 expression and overall survival in patients with gastric cancer, although the risk of death was lower in CDX2 positive subjects. However, there was a less invasive pattern of gastric carcinoma in this group of patients.