Heterogeneity In Squamous Cell Carcinoma Research Articles

CT assessment of tumor heterogeneity and the potential for the prediction of human papillomavirus status in oropharyngeal squamous cell carcinoma.

The aim of this study is to find a correlation between tumoral heterogeneity of squamous cell carcinoma of the oropharynx and human papillomavirus (HPV) status and to determine whether analysis of texture features of primary lesion on contrast-enhanced CT (CECT) images can be useful in predicting the HPV positivity. Fifty patients with diagnosis of oropharyngeal carcinoma and pre-treatment CECT were included; tumoral heterogeneity of each lesion was evaluated by extracting quantitative texture parameters of first and higher orders. T test and logistic regression were conducted to evaluate the effects of different textural characteristics. There were 35 HPV+ and 15 HPV- lesions. Statistically significant (p < 0.05) differences were seen in multiple higher-order extracted parameters. The logistic regression model correctly classified lesions with an accuracy of 95.2%. CT texture analysis of primary oropharyngeal cancer may be used as a tool for predicting the HPV status.

Intratumor genetic heterogeneity in squamous cell carcinoma of the oral cavity.

We sought to evaluate intratumor heterogeneity in squamous cell carcinoma of the oral cavity (OCC) and specifically determine the effect of physical separation and histologic differentiation within the same tumor. We performed whole exome sequencing on five biopsy sites-two from well-differentiated, two from poorly differentiated regions, and one from normal parenchyma-from five primary OCC specimens. We found high levels of intratumor heterogeneity and, in four primary tumors, identified only 0 to 2 identical mutations in all subsites. We found that the heterogeneity inversely correlated with physical separation and that pairs of well-differentiated samples were more similar to each other than analogous poorly differentiated specimens. Only TP53 mutations, but not other purported "driver mutations" in head and neck squamous cell carcinoma, were found in multiple biopsy sites. These data highlight the challenges to characterization of the mutational landscape of OCC with single site biopsy and have implications for personalized medicine.

Abstract CT170: A phase II, multicenter study to evaluate the efficacy and safety of autologous tumor infiltrating lymphocytes (LN-145) for the treatment of patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck

Abstract Adoptive cell therapy (ACT) may be effective in treating immunogenic tumors with high mutational load, such as melanoma and virally-associated tumors, like cervical cancer, with several patients in studies performed at various institutions achieving durable, complete responses for years. Despite the heterogeneity of squamous cell carcinomas of the head and neck (HNSCC), most tumors are either virally-associated (e.g., HPV in oropharyngeal) or carry high mutational load (e.g., tobacco-related) providing an increased diversity of potential targets ideal for the polyclonal nature of ACT. Furthermore, outcomes for patients with recurrent and/or metastatic HNSCC remain poor. Therefore, a clear rationale exists for the potential application of ACT in patients with HNSCC. Clinical trial C-145-03 (NCT03083873) is a prospective phase II multicenter, open-label study evaluating the efficacy of a single autologous tumor infiltrating lymphocyte infusion (LN-145) followed by IL-2 after a non-myeloablative lymphodepletion (NMA-LD) regimen in patients with recurrent and/or metastatic HNSCC. Study-related therapy begins with resection of a tumor lesion that is then shipped to a central GMP manufacturing facility where TIL are extracted, expanded, packaged, and shipped for administration (LN-145). One week prior to LN-145 infusion, patients undergo NMA-LD consisting of cyclophosphamide (60 mg/kg) daily x 2 days followed by fludarabine (25 mg/m2) daily x 5 days. LN-145 is infused 24 hours after the last dose of fludarabine followed by up to 6 doses of IL-2 (600,000 IU/kg) every 8-12 hours. The primary efficacy endpoints are the objective response rate per RECIST v1.1 and the safety summarization of treatment-emergent adverse events (AEs) including serious AEs, AEs leading to discontinuation, and clinical laboratory tests. Secondary efficacy endpoints include CR, DOR, PFS, and OS. Patients must have been treated with at least one systemic chemotherapy or immunotherapy treatment for recurrent and/or metastatic HNSCC and, in addition to the tumor targeted for excision and TIL manufacture, must have an additional measurable lesion for assessment of response. Additional eligibility criteria include amongst others: adequate bone marrow, liver, pulmonary, cardiac, and renal function; ECOG performance status of 0 or 1. Systemic steroids greater than 10 mg/day prednisone equivalents are prohibited as are a history of serious immunotherapy-related adverse events. Citation Format: Rom Leidner, Ammar Sukari, Christine Chung, James Ohr, Missak Haigentz, Ezra Cohen, Robert Brown, Sam Suzuki, Igor Gorbatchevsky, Maria Fardis, Robert L. Ferris. A phase II, multicenter study to evaluate the efficacy and safety of autologous tumor infiltrating lymphocytes (LN-145) for the treatment of patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT170.

Cytogenetic analysis of metaplastic squamous cell carcinoma of the breast inter- and intratumoral heterogeneity.

Squamous cell carcinoma (SCC) of the breast is a rare and generally aggressive disease that accounts for less than 0.1% of all breast carcinomas. Although SCCs have distinct morphological features, their origin and cytogenetic profile are not well understood. Five patients with SCC were studied. The tumor area that was predominantly composed of SCC components was macrodissected and DNA was extracted. In three cases, an invasive or noninvasive ductal carcinoma of no special type (NST) component was also present. NST-component DNA was also extracted. The tumor DNA was used for array comparative genomic hybridization analysis using a high-density oligonucleotide microarray. The cytogenetic profile of the SCC components was compared with each other and with the paired NST component in three of the five cases. The cytogenetic profile of the SCC components indicated large intertumoral heterogeneity. There were between 2 and 160 copy number alterations per case, and no common copy number alterations were identified. The cytogenetic profiles of the paired SCC and NST components were similar but not identical. Although, in one case, a larger number of copy number aberrant regions were detected in the SCC component than the NST component. In this case, all the NST component aberrations were present in the SCC component. This implies that the SCC component originated from the NST component. There were no common SCC component-specific aberrations in the three NST-component cases. Our results demonstrate the cytogenetic inter- and intratumoral heterogeneity of SCC of the breast. Our comparison of cytogenetic profiles indicated that the SCC component originated from the NST component in one case.

Single-cell analyses of transcriptional heterogeneity in squamous cell carcinoma of urinary bladder.

Cell-to-cell expression heterogeneity within a single tumor is a common phenotype among various cancer types including squamous cell carcinoma. To further study the fundamentals and importance of heterogeneity of cell functions and its potential mechanisms, we performed single-cell RNA-seq (scRNA-seq) on human squamous cell carcinoma of the bladder (SCCB) and its corresponding physiologically normal epithelia. Extensive differentially expressed genes were uncovered by comparing cancer and normal single cells, which were preferentially enriched in cancer-correlated pathways, such as p53 signaling and bladder cancer pathway. Furthermore, the most diversely expressed genes were particularly enriched in MAPK signaling pathway, such as CACNG4, CACNA1E and CACNA1H, which involve in cancer evolution and heterogeneity formation. Co-expression network and hub-gene analyses revealed several remarkable “hub genes” of each regulatory module. Some of them are cancer related, such as POU2F3, NKD1 and CYP2C8, while LINC00189, GCC2 and OR9Q1 genes are rarely reported in human diseases. The genes within an interesting module are highly correlated with others, which could be treated as potential targets for SCCB patients. Our findings have fundamental implications for SCCB biology and therapeutic strategies.

Abstract P6-07-10: Cytogenetic analysis of squamous cell carcinoma of the breast reveals inter- and intra-tumoral heterogeneity

Abstract Background: Squamous cell carcinoma (SCC) of the breast is a rare and generally aggressive disease constituting less than 0.1 % of all breast carcinomas. Although they have distinct morphological features, the origin and cytogenetic profiles of SCCs are still not well understood. In this study, five cases of SCC of the breast, three of which had an SCC component and an invasive or noninvasive ductal carcinoma of no special type (NST) component, were analyzed to evaluate their cytogenetic inter- and intra-tumoral heterogeneity. Methods: Using a pathological database of approximately 3,000 patients with breast cancer, five patients with SCC were identified. Their medical records were retrospectively reviewed to obtain clinicopathological, radiological, therapeutic and prognostic information. The area largely consisting of the SCC component was macro-dissected from five 10 μm-thick sections and tumor DNA was extracted using the QIAmp DNA Mini Kit (Qiagen). Three cases contained a component of invasive or noninvasive ductal carcinoma of NST as well as an SCC component, and tumor DNA from NST components were also extracted as described above. Tumor DNA from each case were used for array comparative genomic hybridization (aCGH) analysis using a high-density oligonucleotide microarray (Agilent® SurePrint G3 8x60k microarray) and the cytogenetic profiles of SCC components were compared with each other and in three of the five cases with their paired NST components. Results: Sufficient amounts of DNA were obtained for aCGH analysis with an average of 0.78 μg (0.39–1.35 μg). The quality of the aCGH was acceptable, as judged by the mean derivative log ratio spread (DLRSpread) of 0.45 (0.20–0.55), which estimates the log ratio noise by calculating the spread of log ratio differences between consecutive probes along all chromosomes. The cytogenetic profiles of SCC components showed large inter-tumoral heterogeneity with between 2 and 160 copy number alterations per case and no common copy number alterations were found among cases. Meanwhile, cytogenetic profiles were almost identical between paired SCC and NST components,. However, in one case, a large number of copy number aberrant (CNA) regions were detected in the SCC component compared with the NST component and all aberrations in the NST component were also present in the SCC component, which implies that the SCC component originated from the NST component. There were no common SCC-component-specific aberrations in the three cases with NST components. Conclusion: These results demonstrate the degree of cytogenetic inter- and intra-tumoral heterogeneity in SCC of the breast. The comparison of cytogenetic profiles in one case indicated that the SCC component originated from the NST component. Citation Format: Oikawa M, Igawa A, Ishida M, Nakamura Y, Nishimura S, Koga C, Akiyoshi S, Koi Y, Taguchi K, Ohno S, Tokunaga E. Cytogenetic analysis of squamous cell carcinoma of the breast reveals inter- and intra-tumoral heterogeneity. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-10.

P027 The analysis of cytogenetic intra-tumoral heterogeneity in squamous cell carcinoma of the breast

P027 The analysis of cytogenetic intra-tumoral heterogeneity in squamous cell carcinoma of the breast

P026 Mechanical ventilation promotes lung metastasis in 4T1 breast cancer lung-metastasized models

Goals: Recent evidence suggests that general anaesthesia and perioperative factors significantly increased risk of cancer recurrence compared to locoregional anaesthesia in patients undergoing surgery, however, their effects on behavior of cancer cells are not yet known. The endothelial cell and epithelial disruption was observed during mechanical ventilation, general anaesthesia in animalmodels. This studywas to test the hypothesis thatmechanical ventilation during cancer surgery induces lung stroma/tissue milieu changes, creating a favorable microenvironment for postoperative lung metastatic tumor establishment. Methods: Female BALB/c mice were anesthetized by intraperitoneal pentobarbital administration and divided into a mechanical ventilation group [mice ventilated with low pressures and low tidal volumes, 60min; n =8) and a control group (no ventilation; n =8). All the mice were subjected to intravenous injection of murine mammary adenocarcinoma 4T1-green fluorescent protein (GFP) 10 cells during anaesthesia. 24 hr later, sections from the lung tissue were examined and 4T1 celler were counted by direct GFP fluorescence observation, hematoxylineosin histology and Ep-CAM immunohistochemistry. To closely mimic human cancer surgical and anaesthetic manipulation, A clinically relevant animal model of spontaneous breast cancer lung metastasis with surgical resection of primary tumor was used to investigate the purified mechanical ventilation event that dictate lung metastasis post-operation. Female BALB/c mice were inoculated with metastatic murine mammary adenocarcinoma 4T1 cells in the mammary fat pad (3×105/mouse). After 14 day growth, mice were anesthetized by intraperitoneal pentobarbital administration and divided into a mechanical ventilation group [mice ventilated with low pressures and low tidal volumes, 60min; n =6) and a control group (no ventilation; n =6). All the mice were subjected to flank tumor resection during anaesthesia. Two weeks later, Metastatic tumor burden was assessed by both macroscopic metastatic nodule count and hematoxylin-eosin histology. Results: Mechanical ventilation was associated with increased circulating breast tumor cells arrest at microvasculature of lung (P = 0.01). The histology analysis data indicated that mechanical ventilation induced endothelial-epithelium abnormalities, inflammatory reaction and significantly upregulated the expression of epithelial adhesion molecules (Ep-CAM). Postoperative metastases exhibited significantly increase in mechanical ventilated groups in comparison with those in non-ventilated group (P < 0.05). Conclusion: Mechanical ventilation led to a progressive phenotype of lung metastases that exhibited a microenvironment for cancer cell adhesion, invasion, survive and growth within lungs. Disclosure of Interest: No significant relationships. P027 The analysis of cytogenetic intra-tumoral heterogeneity in squamous cell carcinoma of the breast M. Oikawa *, A. Igawa, M. Ishida, Y. Nakamura, S. Nishimura, C. Koga, S. Akiyoshi, Y. Koi, K. Taguchi, S. Ohno. Breast Oncology, National Kyushu Cancer Center, Fukuoka, Japan, Pathology, National Kyushu Cancer Center, Fukuoka, Japan

Abstract B76: Novel human 3D in vitro invasion model identifies tumor and stroma heterogeneity in squamous cell carcinoma

Abstract 3D in vitro skin models are valuable research tools to investigate normal keratinocyte as well as skin carcinoma cell interaction with dermal fibroblasts. Currently, the models lack either a structured vital human extracellular matrix or long-term stability and longevity. Therefore, we aimed at a new 3D in vitro skin model for normal human epidermis and tumorigenic stages of skin cancer. This model is based on dermal equivalents containing a highly structured cell-derived matrix (CDM) produced by human dermal fibroblasts. First, we show that the CDM dermal equivalent supports a high structural integrity of the skin model and maintains a vital epidermis for six months. The epidermis is well-stratifying and differentiates in a regular orthokeratotic pattern. Transmission electron microscopy reveals ultrastructural entities as basement membranes, hemidesmosomes, desmosomes, keratohyalin granulae and lamellar bodies. In line, the molecular differentiation markers are properly distributed. Second, five non-tumorigenic and tumorigenic HaCaT keratinocyte variants are tested for their proliferation, differentiation and invasion capacities. After four weeks of co-cultivation the tumorigenic HaCaT variants show hyperproliferative epithelia and lose the capability of an ordered stratification and differentiation. Furthermore, they destroy the basement membrane as well as the extracellular matrix and invade the dermal equivalent. Moreover, seven squamous cell carcinoma (SCC) cell lines were co-cultivated with the CDM dermal equivalent. All SCC cells form hyperproliferative epithelia that heavily invade the extracellular matrix. Remarkably, the epithelia are individually distinct for their invasion and differentiation capacity. Thus, the CDM-based skin cancer model is suitable for demonstrating the heterogeneity of SCC tumors and their interaction with the microenvironment. In conclusion, the CDM model for normal skin and tumorigenic keratinocytes provides a novel tool to investigate keratinocyte-fibroblast interactions and microenvironment modulations during SCC development. Therefore, this system will provide further insights into mechanistic processes and the interaction of SCCs with their tumor microenvironment. Citation Format: Manuel Berning, Petra Boukamp. Novel human 3D in vitro invasion model identifies tumor and stroma heterogeneity in squamous cell carcinoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B76. doi:10.1158/1538-7445.CHTME14-B76

Heterogeneity of squamous cell carcinomas of the head and neck in relation to clinicopathological variables

We analysed 32 primary, 8 recurrent and 16 metastatic squamous cell carcinomas of the head and neck by flow cytometry to assess tumoural heterogeneity. Intratumoural heterogeneity was found in 9 of 30 primary tumours (30%). All heterogeneous tumours had diploid and aneuploid cell lines. DNA indices were different from those of the primary tumours in 3 of the 8 recurrent lesions (38%) and in 8 of the 12 metastatic lesions (67%). The results indicate that new cell lines evolve during the process of recurrence and metastasis. The incidence of recurrence, metastasis, and heterogeneity increased with the size of the tumour. Histologically, there were tumours in which the degree of differentiation and mode of invasion of primary lesions were not consistent with the results of flow cytometry in both homogeneous and heterogeneous tumours. In conclusion, multiple sampling for flow cytometric analysis is essential for better characterization of oral carcinomas.

Immunohistochemical detection of p21 WAF1/CIP1 and p53 proteins in formalin-fixed paraffin-embedded tissue sections of squamous cell carcinoma of the skin

Thirty-two cases of squamous cell carcinoma (SCC) of the skin were investigated as to the expression of p53 and p21 WAF1/CIP1 using an immunohistochemical method. These cases were surgically resected or biopsied, tissue samples were then fixed in formalin and embedded in paraffin in the conventional way. Microwave heating was used for antigen retrieval. The primary monoclonal anti-p21 antibody and the monoclonal antibody against p53 were employed. The labeled-streptoavidin-biotin-peroxidase method was used for immunohistochemical staining. Of these, 30 cases showed overexpression of p53 staining, but normal epidermal cells were free of stain. p21 positive cells were detected faintly in the middle layer cells of normal epidermis. Of these, 30 cases showed overexpression of p21 staining. The staining pattern of p53 and p21 showed intratumoral heterogeneity in SCC. In general, there was the inverse relationship between p21 and p53 staining in tumors, namely p53 positive cells were p21 negative and vice versa. However, some of the tumor cells expressed both genes simultaneously. This study supports a hypothesis that p21 expression is regulated by p53, and that it is also regulated by an additional pathway(s) in SCC.

Heterogeneity of squamous cell carcinomas of the head and neck--analysis of tumor biologic factors and proliferation rates.

Specimens obtained from five different tumor regions in 12 patients who underwent surgery for squamous cell carcinoma (SCC) of the oropharynx were examined. The evaluation of each biopsy included quantitative DNA measurements based on image analysis, immunohistochemical assessment of proliferations markers (i.e., Ki67-MIB1, proliferating cell nuclear antigen [PCNA]), and morphological tumor-front grading. From single cell measurements, several DNA indices were derived which are known to reflect tumor aneuploidy. The results revealed a marked variation of proliferation and cellular differentiation in different regions of tumors and a wide intraindividual variation between particular tumors for all markers examined. There was good correlation between DNA data and proliferative cell fractions (Ki67 score, PCNA score). With the use of diagrams, three-dimensional distribution of proliferation rates and markers reflecting tumor aggressiveness within each tumor was obtained. The results confirmed previous clinical and histological observations that SCCs of the oropharynx are heterogeneous tumors. One might expect that the regions with increased proliferation and aggressiveness may predict the location of possible tumor recurrence.

Heterogeneity of Squamous Cell Carcinomas of the Oral Cavity Studied by Flow Cytometry

The DNA pattern was determined by flow cytometry in 76 samples from 16 squamous cell carcinomas of the oral cavity to assess intratumour DNA heterogeneity. Heterogeneous DNA content was found in 2 tumours (12%); both containing DNA diploid and DNA aneuploid cell clones. The remaining 14 tumours showed a homogeneous DNA distribution in the different specimens; 9 (56%) were diploid, 3 (19%) aneuploid and 2 (12%) were polyploid. The DNA non-diploid tumours were clinically more advanced than the DNA diploid ones (p less than 0.05). The tumour proliferation rate (fraction of cells in S-phase) was higher in aneuploid tumours than in diploid ones (p less than 0.01).